June 22, 2010 -- People with diabetes can be extra sensitive to high temperatures, and many don't take enough precautions early enough, according to a new survey.
The results weren't all bad. "Most patients incorporated appropriate personal protective measures such as staying indoors, drinking additional fluids on schedule, applying sunscreen, and wearing protective clothing," says Adrienne A. Nassar, MD, a third year resident at Mayo Clinic Arizona, who presented the findings? at ENDO 2010 in San Diego, the annual meeting of the Endocrine Society.
But the respondents fell short in other ways. For instance, one in five waited until temperatures were over 100 degrees before taking precautions, Nassar said at a news conference? And 23% began drinking fluids when they got thirsty -- typically too late to prevent dehydration effectively. Many left their medications and monitoring equipment at home during a heat wave.
Nassar and her colleagues analyzed responses of 152 people with diabetes living in Phoenix, where the average July temperature is 107 degrees.
On average, the patients were 64 years old, 85% had type 2 diabetes, and 77% were on insulin injections or pumps. Their blood glucose test results on the hemoglobin A1c tests were on average 7.9%, although the goal for those with diabetes is 7%.
While many respondents protected their medication in the heat by carrying it in a cooler, 37% left medication or supplies at home.
"This is quite concerning,'' Nassar says, "because they would not have the means to check their blood sugar" if they became faint, for instance.
While most respondents, 72%, knew about the effect of heat on insulin, just 40% said they had gotten information from their health care providers about the ill effects of high temperature on oral medications, 41% on glucose monitors, and 38% on glucose monitoring strips.
The point at which respondents said they would take protective measures varies. Nassar can't pinpoint an exact temperature at which protective measures should begin since it depends not only on temperature but on such factors as humidity.
Only 55% of the survey respondents knew the definition of ''heat index," she found. Heat index is a measure of how hot it feels when the relative humidity is added to the air temperature. For instance, if the temperature is 108 degrees but the humidity is 45%, the heat index is 137.
According to the National Weather Service, those in high risk groups should begin taking precautions when the heat index reaches 80 or 90 to avoid heat stroke, sunstroke, and other problems.
Nassar found that 68% of respondents limited heat exposure to less than an hour, but that many people delayed protective measures too long. ''Overall, we found many patients expose themselves to high temperatures before taking protective measures," Nassar says.
Sept. 23, 2009 (Berlin) -- Tumors in soft tissues such as muscle, fat, and nerves around the joints are much less likely to come back if they are heated at the time patients receive chemotherapy, a study shows.
There's also a suggestion that patients who receive targeted heat therapy may live longer, says the study's leader, Rolf Issels, MD, a professor of medical oncology at Klinikum Grosshadern Medical Center at the University of Munich.
"These findings provide a new standard treatment option and we believe they are likely to change the way many specialists treat these tumors," he tells WebMD.
The results were presented at a joint meeting of the European Cancer Organization and the European Society of Medical Oncology.
Soft tissue sarcomas involve tumors that start in the soft, supporting tissues of the body, such as muscle, fat, blood vessels, nerves, tendons, tissue around the joints, and deep layers of the skin. They are relatively rare, accounting for about 3% of all cancers, but are more common in children and young adults.
Surgery is the primary treatment, but the tumors are difficult to remove, so radiation and/or chemotherapy are often given to kill lingering cancer cells.
However, the benefits of chemotherapy have been shown to be limited, Issels says, and high-risk patients often relapse within two or three years.
That's where targeted heat therapy comes in. The technique, known as regional hyperthermia, uses focused electromagnetic energy to warm the tissue in and around the tumor to between 104 and 109.4 degrees Fahrenheit.
The heat packs a four-pronged attack against the tumor, says Gerard C. van Rhoon, PhD, head of the hyperthermia unit at Erasmus University Medical Center in Rotterdam, Netherlands.
First, it directly kills cancer cells, he says. It also improves blood flow, which allows more chemotherapy to get through to the area of the tumor.
The improved blood flow also brings more oxygen to the tumor, which makes it more sensitive to radiation. Finally, the heat interferes with cancer cells' repair mechanisms and they die off, van Rhoon tells WebMD.
The new study involved 341 patients being treated for locally advanced soft tissue sarcomas that were at high risk of recurrence and spread.
All patients were given chemotherapy before and after surgery and radiation treatment afterward. Half were randomly assigned to targeted heat treatment along with the chemotherapy. Then they were followed for an average of nearly three years.
"Patients receiving the targeted therapy fared better on all the outcome measures," Issels says. Among the findings:
1 From the Thermal and Mountain Medicine Division, US Army Research Institute of Environmental Medicine, Natick, MA.
2 Presented at the workshop Role of Dietary Supplements for Physically Active People, held in Bethesda, MD, June 3–4, 1996. 3 The views, opinions, and findings contained in this report are those of the authors and should not be construed as an official Department of Army position or decision, unless so designated by other official documentation. 4 Address reprint requests to MN Sawka, Thermal and Mountain Medicine Division, US Army Research Institute of Environmental Medicine, Kansas Street, Natick, MA 01760-5007.
ABSTRACT
During exercise in the heat, sweat output often exceeds water intake, resulting in a body water deficit (hypohydration) and electrolyte losses. Because daily water losses can be substantial, persons need to emphasize drinking during exercise as well as at meals. For persons consuming a normal diet, electrolyte supplementation is not warranted except perhaps during the first few days of heat exposure. Aerobic exercise is likely to be adversely affected by heat stress and hypohydration; the warmer the climate the greater the potential for performance decrements. Hypohydration increases heat storage and reduces a person's ability to tolerate heat strain. The increased heat storage is mediated by a lower sweating rate (evaporative heat loss) and reduced skin blood flow (dry heat loss) for a given core temperature. Heat-acclimated persons need to pay particular attention to fluid replacement because heat acclimation increases sweat losses, and hypohydration negates the thermoregulatory advantages conferred by acclimation. It has been suggested that hyperhydration (increased total body water) may reduce physiologic strain during exercise heat stress, but data supporting that notion are not robust. Research is recommended for 3 populations with fluid and electrolyte balance problems: older adults, cystic fibrosis patients, and persons with spinal cord injuries.
Key Words: Skin blood flow • cystic fibrosis • dehydration • fluid redistribution • hypohydration • hyperhydration • older adults • spinal cord injury • sweating • thermoregulation
INTRODUCTION
Water and electrolyte balance are critical for the function of all organs and, indeed, for maintaining health in general (1, 2). Water provides the medium for biochemical reactions within cell tissues and is essential for maintaining an adequate blood volume and thus the integrity of the cardiovascular system. The ability of the body to redistribute water within its fluid compartments provides a reservoir to minimize the effects of water deficit. Each body water compartment contains electrolytes, the concentration and composition of which are critical for moving fluid between intracellular and extracellular compartments and for maintaining membrane electrochemical potentials.
Physical exercise and heat stress cause both fluid and electrolyte imbalances that need to be corrected (3–6). Generally, persons dehydrate during exercise in the heat because of the unavailability of fluids or a mismatch between thirst and water requirements (7, 8). In these instances, the person is euhydrated (normally hydrated) at the beginning of exercise but incurs hyphohydration (a body water deficit) over a prolonged period. Hypohydrated persons who exercise in the heat will incur significant adverse effects (9). Hypohydration increases physiologic strain, decreases exercise performance, and negates the thermoregulatory advantages conferred by high aerobic fitness (10, 11) and heat acclimation (10, 12). If strenuous exercise is performed by hypohydrated persons, the medical consequences can be devastating (13, 14).
We review human fluid and electrolyte balance relative to their effects on temperature regulation and exercise performance in the heat. In addition, needs for research on fluids and electrolytes will be discussed for 3 special populations: cystic fibrosis patients, older persons, and persons with spinal cord injuries.
FLUID AND ELECTROLYTE BALANCE
To support the contraction of skeletal muscles, physical exercise routinely increases total body metabolism to 5–15 times the resting rate. Approximately 70–90% of this energy is released as heat, which needs to be dissipated to achieve body heat balance. The relative contributions of evaporative and dry (radiative and conductive) heat exchange to total heat loss vary according to climatic conditions (15). In hot climates, a substantial volume of body water may be lost via sweating to enable evaporative cooling (7).
In addition to climatic conditions, clothing and exercise intensity influence the sweating rate (15, 16). Residents of desert climates often have sweating rates of 0.3–1.2 L/h while performing occupational activities (17). Clothing may be a major concern; persons wearing protective garments often have sweating rates of 1–2 L/h while performing light-intensity exercise (18). For athletes performing high-intensity exercise in the heat, sweating rates of 1.0–2.5 L/h are common. Expected sweating rates from running in different climatic conditions are shown in Figure 1 (7). The influence of climate and amount of physical activity on daily fluid requirements is shown in Figure 2 (19, 20). Daily requirements for sedentary to very active persons range from 2–4 L/d in temperate climates and from 4–10 L/d in hot climates.
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FIGURE 1. . An approximation of hourly sweating rates as a function of climate and running speed (7).
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FIGURE 2. . Influence of climatic temperature and daily metabolic rate on daily fluid requirements (19, 20).
Sodium chloride is the primary electrolyte in sweat, with potassium, calcium, and magnesium present in smaller amounts. The sodium concentration in sweat averages 35 mmol/L (range: 10–70 mmol/L) and varies by diet, sweating rate, hydration, and degree of heat acclimation (21, 22). Sweat glands reabsorb sodium by active transport, but the ability to reabsorb sweat sodium does not increase with the sweating rate; thus, at high sweating rates the concentration of sodium increases (15). Because heat acclimation improves the ability to reabsorb sodium, acclimated persons have lower sodium concentrations in sweat (>50% reduction) for any specific sweating rate (22). The potassium concentration in sweat averages 5 mmol/L (range: 3–15 mmol/L); that of calcium, 1 mmol/L (range 0.3–2 mmol/L); and that of magnesium, 0.8 mmol/L (range 0.2–1.5 mmol/L) (21). Sex, maturation, and aging do not appear to affect sweat electrolyte concentrations markedly (23, 24). Except for the first several days of heat exposure, electrolyte supplementation is not necessary because normal dietary sodium intake will cover the sweat losses (3, 5). Although sweat contains vitamins, their concentrations are low; thus, vitamin supplementation is not needed (25).
During exercise in the heat, hypohydration must be avoided by matching fluid consumption with sweat loss. This is difficult because thirst is not a good indicator of body water requirements (17, 26, 27). In general, thirst is not perceived until a person has incurred a water deficit of 2% body weight loss (BWL) (17, 26, 28). Correspondingly, ad libitum water intake during exercise in the heat results in an incomplete replacement of body water losses (Figure 3; 17). As a result, unless forced hydration is stressed, some dehydration is likely to occur during exercise in the heat. Humans will usually fully rehydrate at meals, when taking fluids is stimulated by consuming food (4, 17). Thus, active persons need to stress drinking at meals to avoid persistent hypohydration.
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FIGURE 3. . Relation between sweating rate and voluntary dehydration (water deficit) during ad libitum drinking by heat-acclimated persons in the desert (17).
Hypohydration reduces the gastric emptying rate of ingested fluids during exercise in the heat (29, 30). Neufer et al (29), for example, found a reduction of 20–25% in the gastric emptying rate when their subjects were hypohydrated (5% body weight) that was related to increased core temperature. Thus, beginning fluid intake during the early stages of exercise heat stress is important not only to minimize hypohydration but also to maximize the bioavailability of the ingested fluids.
During situations of stress and prolonged high sweat loss, adults will dehydrate by 2–8% BWL. Water constitutes 45–70% of body weight (1); the average male (75 kg) has 45 L of water (about 60% of body weight). Because adipose tissue is 10% water but muscle tissue is 75% water, a person's total body water varies by body composition (1). In addition, muscle water and glycogen content parallel each other, probably because of the osmotic pressure exerted by glycogen granules within the muscle's sarcoplasm (31). As a result, trained athletes have relatively greater total body water than their sedentary counterparts by virtue of a smaller percentage body fat and a higher concentration of skeletal muscle glycogen.
The water contained in body tissues is distributed between the intracellular and extracellular fluid spaces. Because there is free fluid exchange, hypohydration mediated by sweating will influence each fluid space. Nose et al (32) determined the distribution in the rat of body water loss among the fluid spaces as well as among different body organs; they thermally dehydrated rats by 10% BWL and after the animals regained their normal core temperature they measured the animals' body water. Forty-one percent of the water deficit was intracellular and 59% was extracellular. In terms of organs, 40% of the water deficit was from muscle, 30% from skin, 14% from viscera, and 14% from bone. Neither the brain nor liver lost significant water. These researchers concluded that hypohydration redistributes water largely from the intracellular and extracellular spaces of muscle and skin as a way of maintaining blood volume.
Resting plasma volume and osmolality values for heat-acclimated persons hypohydrated to various degrees are shown in Figure 4 (33). Sweat-induced hypohydration will decrease plasma volume and increase plasma osmotic pressure in proportion to the amount of fluid loss. Plasma volume decreases because it provides the precursor fluid for sweat, and osmolality increases because sweat is ordinarily hypotonic relative to plasma. Sodium and chloride are primarily responsible for the elevated plasma osmolality (34, 35), which mobilizes fluid from the intracellular to the extracellular space to enable defense of plasma volume in hypohydrated persons. This concept is illustrated by heat-acclimated persons, who have a smaller reduction in plasma volume than do unacclimated persons for a given body water deficit (36). By virtue of having more dilute sweat, heat-acclimated persons have additional solutes remaining within the extracellular space to exert an osmotic pressure and redistribute fluid from the intracellular space.
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FIGURE 4. . Body water loss effects on plasma osmolality and plasma volume (PV) in heat-acclimated persons (33). TBW, total body water.
Persons who use diuretics for medical purposes or to reduce their body weight are at increased risk of hypohydration because diuretics increase urine formation and generally result in the loss of solutes. Commonly used diuretics include thiazides (eg, Diuril; Merck & Co, Inc, West Point, PA), carbonic anhydrase inhibitors (eg, Diamox Parenteral; Lederle Laboratories, Philadelphia), and furosemide (eg, Lasix; Hoechst-Roussel, Somerville, NJ). Diuretic-induced hypohydration generally results in an isoosmotic hypovolemia, with a much greater ratio of plasma loss to body water loss than in either exercise- or heat-induced hypohydration. Relatively less intracellular fluid is lost after diuretic administration because there is no extracellular solute excess to stimulate redistribution of body water.
EXERCISE PERFORMANCE
Numerous studies have examined the influence of hypohydration on maximal aerobic power and physical work capacity (33). In temperate climates, maximal aerobic power reportedly decreases when hypohydration reaches or exceeds 3% BWL (10, 37, 38). In hot climates, however, water deficits of 2–4% BWL were reported by Craig and Cummings (39) to cause a large reduction in maximal aerobic power. Data from this study indicate a disproportionately larger decrease in maximal aerobic power with an increased magnitude of body water deficit.
The physical work capacity for aerobic exercise of progressive intensity is decreased when a person is hypohydrated (33). Physical work capacity has been shown to be decreased by marginal (1–2% BWL) water deficits that do not alter maximal aerobic power (37, 40), and the reduction is larger with increasing water deficits. Hypohyration resulted in much larger decrements of physical work capacity in hot than in temperate climates. It appears that the thermoregulatory system, perhaps via increased body temperatures, plays an important role in the reduced exercise performance mediated by a body water deficit.
A reduced maximal cardiac output might be the physiologic mechanism by which hypohydration decreases a person's maximal aerobic power and physical work capacity for progressive-intensity exercise. Hypohydration is associated with decreased blood (plasma) volume during both rest and exercise (41, 42); decreased blood volume increases blood viscosity and can reduce venous return. During maximal exercise, viscosity-mediated increased resistance and reduced cardiac filling could decrease both stroke volume and cardiac output. Several investigators reported a tendency for reduced cardiac output when subjects are hypohydrated during short-term, moderate-intensity exercise (43–45).
It is not surprising that climatic heat stress potentiates the hypohydration-mediated reduction in maximal aerobic power and physical work capacity for progressive-intensity exercise. For euhydrated persons, climatic heat stress alone decreases maximal aerobic power by 7% (46). In the heat, the superficial skin veins reflexively dilate to increase cutaneous blood flow and volume. The redirection of blood flow to the cutaneous vasculature could decrease maximal aerobic power by 1) reducing the portion of cardiac output that perfuses contracting muscles or 2) decreasing the effective central blood volume and central venous pressure, thereby reducing venous return and cardiac output. Persons who are hypohydrated and encounter environmental heat stress would be hypovolemic and still have to perfuse simultaneously the cutaneous vasculature and contracting skeletal muscles. A substantial volume of blood could be redirected to the skin and thus be removed from the effective central circulation and be unavailable to perfuse the skeletal muscles (47, 48). Thus, both climatic heat stress and hypohydration can act independently to limit cardiac output and therefore oxygen delivery during maximal exercise.
The effects of hypohydration and air temperature on submaximal work output are illustrated in Figure 5, which draws on research conducted >50 y ago in the California desert by Adolph et al (17). A metabolic rate of 650 W (which represents "hard work" for occupational tasks but "moderate work" for athletes) was assumed, with an air temperature of 43°C and low humidity (49). Climatic heat stress reduced submaximal work output at all hydration levels. In addition, the work output decrements from heat stress and hypohydration were additive (17). For example, exposure to 43°C reduced work output by 25% (compared with temperate conditions), and a 2.5% BWL (compared with euhydration) reduced work output by the same amount; if these events were experienced together, work output was decreased by 50%.
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FIGURE 5. . Effects of hydration level and climatic temperature on submaximal work output (17). BWL, body weight loss from dehydration.
Hypohydration also impairs endurance performance in athletes. Armstrong et al (40) had athletes compete in 1500-, 5000-, and 10000-m races when euhydrated and when hypohydrated. Hypohydration (2% BWL) was achieved by diuretic administration (furosemide), which decreased plasma volume by 11%. Running performance was impaired at all distances but more in the longer races (5% for the 5000 and 10000 m) than in the shortest race (3% for the 1500 m). Burge et al (50), who examined simulated 2000-m rowing performance, found that it took the rowers an average of 22 s longer to complete the task when they were hypohydrated than when they were euhydrated. In addition, hypohydration reduced average power by 5%.
Surprisingly, few investigators have documented the effects of hypohydration on physiologic tolerance to submaximal exercise in the heat. In research conducted by Adolph et al (17), soldiers attempted endurance (2–23 h) walks in the California desert at 4–6.5 km/h [ambient temperature (Ta) 38°C] and either drank water ad libitum or refrained from drinking. These investigators reported that 1 of 59 (2%) soldiers who drank and 11 of 70 (16%) who did not drink suffered exhaustion from heat strain. In subsequent experiments, they reported that 1 of 59 drinking subjects (2%) and 15 of 70 nondrinking subjects (21%) suffered exhaustion from heat strain during an attempted 8-h desert walk. The magnitude of hypohydration was not provided in either set of experiments.
About a decade later, Ladell (51) had subjects attempt a 140-min walk in a hot (Ta = 38°C) environment while ingesting different combinations of salt and water. Exhaustion from heat strain occurred in 9 of 12 (75%) experiments when subjects received neither water nor salt and 3 of 41 (7%) experiments when subjects received only water. More recently, Sawka et al (41) had subjects attempt lengthy treadmill walks (25% of maximal oxygen uptake for 140 min) in a hot-dry (Ta = 49°C, relative humidity = 20%) environment when euhydrated and when hypohydrated by 3%, 5%, and 7% BWL. All 8 subjects completed the euhydration and 3% hypohydration experiments, and 7 of 8 completed the 5% hypohydration experiments. For the 7% hypohydration experiments, 6 subjects discontinued after completing only 64 min (mean). Clearly, hypohydration increases the incidence of exhaustion from heat strain.
To address whether hypohydration alters physiologic tolerance to heat strain, Sawka et al (52) had subjects walk to exhaustion in either a euhydrated or hypohydrated (8% of total body water) state. The experiments were designed so that the combined environment (Ta = 49°C, relative humidity = 20%) and exercise intensity (47% of maximal oxygen uptake) would not allow thermal equilibrium, and heat exhaustion would eventually occur. Hypohydration reduced tolerance time from 121 to 55 min, but more important, it reduced the core temperature a person could tolerate (the core temperature at heat exhaustion was 0.4 degrees lower in the hypohydrated state). These findings suggest that hypohydration not only impairs exercise performance but also reduces physiologic tolerance to heat strain.
TEMPERATURE REGULATION
Hypohydration increases core temperature responses during exercise in temperate (11, 53) and hot (12, 41) climates. A critical deficit of 1% of body weight elevates core temperature during exercise (54). As the magnitude of water deficit increases, there is a concomitant graded elevation of core temperature during exercise heat stress (41, 55). Relations between body water loss and core temperature elevations reported by studies that examined several water deficits are shown in Figure 6 (33). The magnitude of core temperature elevation ranges from 0.1 to 0.23°C for every percentage point of body weight lost (17, 41, 55, 56).
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FIGURE 6. . Relation between the elevation in core temperature (above euhydration) and hypohydration (measured as percentage body weight loss) during exercise heat stress (33). 
Hypohydration not only elevates core temperature responses but also negates the core temperature advantages conferred by high aerobic fitness and heat acclimation (10–12). The effects of hypohydration (5% BWL) on core temperature responses in the same persons when unacclimated and when acclimated are shown in Figure 7 (12). Acclimation lowered core temperature responses when subjects were euhydrated; when they were hypohydrated, similar core temperature responses were observed for both acclimation states. Thus, the core temperature penalty induced by hypohydration was greater in heat-acclimated than in unacclimated persons.
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FIGURE 7. . Core temperature responses during exercise heat stress in euhydrated (EU) and hypohydrated (hypo; 5% body weight loss) persons both before (UA) and after (HA) being heat acclimated.
Hypohydration impairs both dry and evaporative heat loss (or, if the air is warmer than the skin, dehydration aggravates dry heat gain) (6, 7, 9, 57). In Figure 8, the local sweating response (58) and skin blood flow responses (59) to hypohydration (5% BWL) during exercise in the heat are illustrated. As shown, hypohydration reduces both effector heat loss responses for a given core temperature (36). In addition, hypohydration is usually associated with either reduced or unchanged whole-body sweating rates at a given metabolic rate in the heat (60). However, even when hypohydration is associated with no change in sweating rate, the core temperature is usually elevated, so that the sweating rate for a given core temperature is lower when a person is hypohydrated (60).
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FIGURE 8. . Local sweating rate and forearm skin blood flow (FBF) response data for euhydrated (Eu) () and hypohydrated () [5% body weight loss (BWL)] persons during exercise heat stress.
Both the singular and combined effects of plasma hyperosmolality and hypovolemia have been suggested as mediating the reduced heat loss response during exercise heat stress (36). Changes in plasma osmolality may relate to tonicity changes in the extracellular fluid that bathes the hypothalamic neurons (61–63). Silva and Boulant (64) showed that in rat brain slices, preoptic-anterior hypothalamic neurons are present that are both thermosensitive and osmosensitive. Such data suggest a central interaction between thermoregulation and body water regulation (65).
Isotonic hypohydration alone can impair heat loss and increase core temperature during exercise heat exposure (66–68). Isotonic hypohydration reduces skin blood flow for a given core temperature and thus the potential for dry heat exchange (67, 69). Fortney et al (69) provided a rationale as to why an isoosmotic hypohydration might reduce skin blood flow and sweating rate, theorizing that hypovolemia might reduce cardiac preload and alter the activity of atrial baroreceptors, which have afferent input to the hypothalamus. Thus, a reduced atrial filling pressure might modify neural information to the hypothalamic thermoregulatory centers, which control skin blood flow and sweating. Gonzalez-Alonso et al (70) showed that hypohydration-mediated hypovolemia increases plasma norepinephrine, which is associated with increased cutaneous vascular resistance and reduced blood flow during exercise heat stress. Other studies reported that acute unloading of atrial baroreceptors during exercise with periods of lower-body negative pressure (physiologic technique to unload cardiopulmonary baroreceptors) impairs heat loss and increases core temperature (71, 72).
The effects of hypohydration on cardiovascular responses to exercise have been investigated by several researchers (70, 73–75). During submaximal exercise with little heat strain, hypohydration elicits an increase in heart rate and decrease in stroke volume, but cardiac output does not usually change from what is seen in a euhydrated state (43–45). Apparently, during hypohydration a decreased blood volume reduces central venous pressure (73) and cardiac filling, which reduces stroke volume and requires a compensatory increase in heart rate (75). The combination of exercise with heat strain results in competition between the central and peripheral circulations for a limited blood volume (47, 48). As body temperature increases during exercise, cutaneous vasodilation occurs and the superficial veins become more compliant, thus decreasing venous resistance and pressure (15). As a result of decreased blood volume and blood displacement to cutaneous vascular beds, central venous pressure, venous return, and thus cardiac output decrease below euhydration values.
HYPERHYDRATION
Hyperhydration (greater than normal body water) has been suggested to improve thermoregulation and exercise heat performance above that achieved with hypohydration. The idea that hyperhydration might benefit exercise performance arose from observation of the adverse consequences of hypohydration. The theory was that expanding body water might reduce the cardiovascular and heat strain of exercise by expanding blood volume and reducing blood tonicity, thereby improving exercise performance.
Studies in which blood volume was directly expanded (eg, by infusion) have usually reported decreased cardiovascular strain (76–78) during exercise but have yielded disparate results on heat dissipation (77–79) and exercise heat performance (79, 80). Studies that attenuated plasma hyperosmolality during exercise heat stress generally have reported improved heat dissipation (58, 81–83) but have not addressed exercise performance.
Many studies have examined the effects of hyperhydration on thermoregulation in the heat, but most suffer from serious design problems (eg, the control condition represented hypohydration rather than euhydration) (33). Some investigators reported lower core temperatures during exercise after hyperhydration (83–87), but other researchers did not (75, 88, 89). Also, several studies (83, 84, 90) reported higher sweating rates with hyperhydration. In all studies, heart rate was lower during exercise with hyperhydration (33). Together, these findings support the notion that hyperhydration might reduce the thermal and cardiovascular strain of exercise.
The mechanisms responsible for the lower exercise core temperatures in the hyperhydrated state remain unclear. Several studies reported that overdrinking before exercise lowers body core temperature before exercise (84, 85), but this is likely due to the energy cost of warming the ingested fluid. In these studies, exercise per se did not exacerbate the difference that existed before exercise. Hyperhydration, therefore, apparently did not improve heat dissipation during the exercise period. Other studies, however, reported greater exercise sweating rates when subjects were hyperhydrated (84, 85, 90). In addition, Grucza et al (87) found that sweating began earlier when subjects were hyperhydrated. The findings of these last 4 studies suggest that hyperhydration may improve heat dissipation during exercise heat stress.
Although many studies have tried to induce hyperhydration by having subjects overdrink water or take water-electrolyte solutions, these approaches have resulted in only transients expansions of body water. Often, much of the fluid overload is rapidly excreted (91). Evidence has accrued that greater fluid retention can be achieved with an aqueous solution containing glycerol (91, 92). Riedesel et al (92) were the first to report that after hyperhydration with a glycerol solution, subjects excreted significantly less of the water load than did those consuming water alone.
Lyons et al (90) studied whether glycerol-mediated hyperhydration improves thermoregulatory responses to exercise heat stress. Subjects completed 3 trials in which they exercised in a hot (42°C) climate. For one trial, fluid ingestion was restricted to 5.4 mL/kg body wt, and in the other 2 trials subjects ingested water (21.4 mL/kg) with or without a bolus of glycerol (1 g/kg). Ninety minutes after this hyperhydration period, the subjects began exercise. Compared with drinking water alone, glycerol ingestion increased fluid retention by 30%. In addition, during exercise, glycerol hyperhydration produced a higher sweating rate and substantially lower core temperature (0.7°C) than did control conditions and water hyperhydration. Research from our own laboratory has failed to show any thermoregulatory advantages of either water or glycerol hyperhydration during exercise heat stress (93, 94).
Few studies have examined whether hyperhydration improves exercise performance or heat tolerance. In a study by Blyth and Burt (95), the first to report the effects of hyperhydration on performance during exercise heat stress, subjects ran to exhaustion in a hot climate (49°C) when normally hydrated and when hyperhydrated by drinking 2 L of fluid 30 min before exercise. Thirteen of 18 subjects ran longer when hyperhydrated, but the difference in average time to exhaustion (17.3 compared with 16.9 min) was not statistically significant. More recently, Luetkemeier and Thomas (80), who examined whether hypervolemia improved cycling performance, reported that expansion of blood volume (by 450–500 mL) increased simulated time trial performance by 10% (81 compared with 90 min).
RESEARCH NEEDS
Older adults, persons with cystic fibrosis, and persons who have suffered a spinal cord injury all have unique problems associated with fluid and electrolyte balance during exercise heat stress; additional research is needed on all 3 of these groups. For example, older adults (>55 y) have reduced thirst sensation, less ability to concentrate urine, and reduced potential to dissipate body heat (96–98). The reduced thirst sensation older persons experience when fluid homeostasis is challenged (96) is illustrated in Figure 9 from the research of Mack et al (98). Those investigators compared younger (18–28 y) and older (65–78 y) persons for thirst sensation and fluid intake during exercise heat stress. After this perturbation, both groups were hypohydrated by 2.5% BWL, and thirst sensation and fluid intakes were lower in the older group. However, when matched fluid intakes were plotted against thirst sensation, the 2 groups drank the same amounts. Thus, older persons have a reduced thirst sensation, but for a given thirst sensation, they have the same drinking behavior. This observation is important in light of the reduced ability of older persons to conserve water and electrolytes in their kidneys when challenged by body water deficits. Research issues include the testing of strategies [eg, adding solute to stimulate thirst (2)] to optimize fluid and electrolyte replacement in these populations and the effects of hypohydration on thermoregulation in this group.
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FIGURE 9. . Subjective thirst sensation of younger (•) and older () men during exercise heat stress and the relation between drinking behavior and thirst sensation.
Persons with cystic fibrosis, a multisystem disorder that alters sweat gland function, suffer a high incidence of heat injury or illness, probably because of fluid or electrolyte imbalances (99, 100). They have high sweating rates and very high sweat sodium chloride losses that are not abated by heat acclimation (100). In addition, cystic fibrosis patients have suppressed thirst sensations because of the high sweat solute losses, which reduce the osmotic drive for thirst (2, 8, 99). Together, these factors cause large body water deficits during exercise heat stress (99). If cystic fibrosis patients are forced to drink, however, they tolerate the consumed fluid as well as do their healthy counterparts (99). Research should focus on developing fluid-electrolyte strategies to stimulate thirst and minimize voluntary dehydration in this population.
Persons with spinal cord injury have an impaired ability to thermoregulate during heat stress (101–104). The magnitude of impairment is related to the level and completeness of the lesion; the higher and more complete the cord injury, the greater the thermoregulatory impairment (105). The consequences of spinal cord injury are loss of sympathetic control of heat loss (via vasomotor and sudomotor adjustments) over large areas of skin (106) and the isolation of thermal receptors throughout the body (107). During heat exposure, some sweating can occur over the insensate skin, but it is sparse and not synchronous with sensate skin sweating. Together, these factors lead to a reduced ability to thermoregulate during exercise heat stress (105). Research has not addressed water and electrolyte requirements, thirst sensation, or thermoregulation during hypohydration for this population.
A final research need is the development of rapid, noninvasive technologies to measure total body water and hydration status (108). Valid methods are needed that have enough resolution to measure deficits as small as 5% of total body water.
SUMMARY
During exercise, sweat output often exceeds water intake, producing a body water deficit (hypohydration). The water deficit lowers both intracellular and extracellular volume and also results in plasma hypertonicity and hypovolemia. Aerobic exercise tasks are likely to be adversely affected by hypohydration (109), potentially more so at higher exercise intensities in warm environments (51). Hypohydration increases heat storage and reduces a person's ability to tolerate heat strain. The increased heat storage is mediated by a reduced sweating rate and lower skin blood flow for a given core temperature. Hyperhydration may reduce thermal strain during exercise in the heat, but data supporting that notion are not robust (110).
REFERENCES
【摘要】
Objective- Thromboembolization and subsequent microvascular perfusion failure is implicated in the pathology of a variety of diseases, including transient ischemic attack (TIA), stroke, and myocardial infarction, and also for the complications after interventional and microsurgical procedures in coronary heart disease and peripheral arterial occlusive disease. In vitro heat shock priming has been suggested to induce plasminogen activators, which are the major upregulators of the fibrinolytic system. Herein, we determined whether local heat shock priming endogenously upregulates plasminogen activators also in vivo, and whether this promotes recanalization of thromboembolized microvasculature.
Methods and Results- To induce thromboembolization, a suspension of preformed microthrombi (maximum diameter: 40 µm) was injected via the femoral artery into the left hindlimbs of anesthetized rats. Local heat shock priming (42.5°C, 30 minutes) was performed 24 hours before embolization and resulted in a significant increase of endothelium-derived plasminogen activator expression. The study of the microcirculation by intravital microscopy revealed in all tissues analyzed (muscle, periosteum, subcutis, and skin) that heat shock priming significantly ( P <0.05) accelerates recanalization of the thromboembolized microvasculature when compared with nonprimed and sham-primed controls. Importantly, the addition of plasminogen activator inhibitor-1 to the microthrombi suspension completely blunted the heat shock-induced acceleration of microvascular recanalization.
Conclusions- Heat shock induces endogenous hyperfibrinolysis by upregulation of plasminogen activators that promote recanalization of thromboembolized microvasculature.
Using a rat thromboembolization model and intravital fluorescence microscopy, this study demonstrates that heat shock priming induces endothelium-derived plasminogen activator expression in vivo, and accelerates spontaneous recanalization of thromboembolized microvasculature. This was completely blunted by application of plasminogen activator inhibitor-1. Thus, heat shock induces endogenous hyperfibrinolysis, which promotes recanalization of thromboembolized microvasculature.
【关键词】 heat shock intravital microscopy microcirculation plasminogen activator inhibitor thromboembolization urokinase plasminogen activator
Introduction
In myocardial ischemia and stroke, thromboembolization of the microvasculature essentially contributes to infarction and necrosis. 1 In addition, vascular interventional procedures, such as PTA 2 and PTCA, 3 but also coronary bypass surgery 4 and peripheral reconstructive microsurgery, 5 may be complicated by downstream microvascular thromboembolization with the consequence of nutritive capillary perfusion failure.
Recanalization of thromboembolized microvasculature can be achieved by urokinase therapy; however, this bears some risk for bleeding and aggravation of organ dysfunction, particularly if a surgical procedure is involved. 6 In contrast, endogenous induction of hyperfibrinolysis would represent a more elegant approach; however, little information is available as to whether this may be capable of successfully promoting recanalization of thromboembolized microvasculature.
Preconditioning by heat shock requires the exposure of the tissue to a supraphysiological but sublethal temperature, which results in a transient change of cellular biosynthesis with an accelerated induction of only a few distinct proteins, including heat shock proteins. 7
The serine proteases urokinase plasminogen activator(uPA) and tissue-type plasminogen activator (tPA) initiate the endogenously mediated lysis of platelet arterial emboli. Both enzymes derive from endothelial cells and convert plasminogen to the fibrinolytic protease plasmin. 8 Various types of stimuli are known to directly modulate plasminogen activator synthesis and release from endothelial cells. 9,10 In vitro, stress conditioning by heat shock has been shown to induce plasminogen activators in human umbilical vein endothelial cells. 11 With the use of a rat hindlimb microcirculation model, we herein demonstrate for the first time to our knowledge that local heat shock priming induces upregulation of plasminogen activators also in vivo, and that this promotes endogenous hyperfibrinolysis, which results in accelerated recanalization of thromboembolized microvasculature.
Methods
Animals
Experiments were performed in 72 Sprague-Dawley rats with a body weight of 280 to 350 grams. The study complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and was approved by the local animal care committee.
Surgical Procedure
Under pentobarbital anesthesia (50 mg/kg intraperitoneal; Abbott, Chicago, Ill), the animals underwent tracheotomies and were placed on a heating pad to guarantee 37°C body temperature. Polyethylene catheters were inserted into the right carotid artery and left jugular vein. The catheters allowed monitoring of blood pressure, continuous infusion of saline (1 mL/100 g per hour), withdrawal of blood, and injection of fluorescent dyes for intravital microscopy.
The left hindlimb preparation for microcirculatory analysis was performed according to the technique described previously in detail. 12 The preparation exposed tibial periosteum, gracilis and semitendinosus muscles, subcutis and skin, and was supplied by the femoral vessels. All branching vessels were ligated up to the superficial epigastric artery, in which a catheter was inserted with the tip directed toward the femoral artery.
Microvascular Thromboembolization
Arterial platelet-rich thrombi were preformed in vitro in a moving high-pressure closed compartment system. 13 For each experiment a 1-mL insulin syringe was filled with thrombin (60 U/mL; T6634; Sigma-Aldrich, Taufkirchen, Germany). In a second syringe, 400 µL blood was drawn from the carotid artery. After 20 seconds, the 2 syringes were interconnected and the suspension containing blood and thrombin (4:1) was moved during 3 minutes &70 times from one syringe to the other. In additional experiments, a rat-specific plasminogen activator inhibitor (PAI)-1 (30 pg/mL; #102; American Diagnostica, Pfungstadt, Germany), the principal physiological inhibitor of both tPA and uPA 14 was added to the microthrombi suspension. The syringes were left standing for 30 minutes until embolization. 40 µm, the thrombi suspension was filtered through a nylon strainer (2340; Falcon; Becton Dickinson, Heidelberg, Germany). For thromboembolization of the microvasculature in vivo, 30 µL of the filtered thrombi suspension was gently injected into the femoral artery. 15
Intravital Fluorescence Microscopy
The tissues were positioned on a micromanipulator-adjusted stage and covered with a glass slide to prevent drying and exposure to ambient air. The microcirculation was analyzed with a blue 520 nm) after intravenous injection of 5% fluorescein isothiocyanate (FITC)-labeled dextran (MW 150 000; Sigma-Aldrich) using a modified epi-illumination Axiotech microscope (Zeiss, Jena, Germany). The contrast enhancement achieved guarantees high-resolution imaging of the microcirculation. 16
Video Analysis
Microscopic images were recorded by a charge-coupled device video camera (FK-6990, COHU, Prospective Measurements, San Diego, Calif) and transferred to a video system. Microcirculatory parameters were quantified off-line using a computer-assisted image analysis system (Capimage, Zeintl, Heidelberg, Germany). After thromboembolization, microvascular recanalization was analyzed by studying the re-onset of capillary perfusion. In addition, a 4-hour follow-up assessment included the determination of: (1) the fraction of perfused capillaries (%), defined as percentage of the length of red blood cell (RBC) perfused capillaries relative to the total length of all capillaries per unit area; (2) capillary V RBC (mm/s); and (3) capillary diameters (µm). 16 Volumetric capillary blood flow (CBF) (pL/s) was calculated for each microvessel as CBF= *(D/2) 2 *V RBC.
Stress Conditioning
For stress conditioning, left hindlimbs of anesthetized rats were heated in a waterbath 24 hours before thromboembolization. 17 During local heating, muscle temperature was increased to 42.5°C and was kept constant for 30 minutes. Animals undergoing a sham heating procedure were also anesthetized 24 hours before thromboembolization, and the hindlimb was exposed in a waterbath to 30°C for 30 minutes. In these animals, the muscle temperature was kept at &36°C to 37°C. Muscle temperature was monitored with a needle thermo-probe (LICOX-System; GMS, Kiel-Mielkendorf, Germany).
Immunohistochemistry
After elimination of endogenous peroxidase activity and nonspecific protein binding, specimens were incubated overnight at 4°C with either anti-uPA-antibody (1:200; #1191; American Diagnostica), anti-tPA-antibody (1:200; kindly provided by J.J. Emeis; Leiden, Netherlands) or anti-PAI-1-antibody (1:200; #1062; American Diagnostica), which react specifically with rat uPA, rat tPA and rat PAI-1, respectively. As secondary antibody, either a biotinylated donkey-anti-chicken-IgG (Dianova, Hamburg, Germany) or goat-anti-rabbit-IgG antibody (DAKO-Cytomation, Hamburg, Germany) was used. Thereafter, streptavidin-horseradish peroxidase complex was added for 30 minutes at 20°C, followed by 5 minutes of treatment with 3,3'diaminobenzidine.
Slides were counterstained with hematoxylin. For negative controls, slides were treated similarly but without the primary antibody. All control stainings were negative.
The staining intensity was evaluated blindly according to Page et al 18 A "0 to 4" score was applied, depending on color intensity and extent, ie, 0 indicates negative, comparable to control section; 1, very weakly positive; 2, weakly positive; 3, positive; and 4, strongly positive.
Western Blot Analysis of uPA, tPA, and PAI-1
Muscle and skin tissue (n=4 per group) of control and heat shocked animals was harvested after 24 hours and homogenized in lysis buffer (10 mmol/L Tris pH 7.5, 10 mmol/L NaCl, 0.1 mmol/L EDTA, 0.5% Triton-X-100, 0.02% NaN 3, 0.2 mmol/L phenyl-methyl-sulfonyl-fluoride), incubated for 30 minutes on ice, and centrifugated for another 30 minutes at 16 000 g and 4°C. Equal amounts of protein per lane (90 µg) were separated discontinuously on 10% sodium dodecyl sulfate polyacrylamide gels under denaturing conditions and transferred to a polyvinyldifluoride membrane (BioRad, Munich, Germany). After blockade of nonspecific binding sites, membranes were incubated for 2 hours with a chicken-anti-rodent-uPA (1:50; American Diagnostica), a goat-anti-rat-tPA (1:50; Santa Cruz Biotechnology) or a rabbit-anti-rat-PAI-1 antibody (1:50; American Diagnostica) followed by the secondary horseradish peroxidase-conjugated bovine-anti-chicken (1:5000; Santa Cruz Biotechnology), rabbit-anti-goat (1:2000; R&D, Wiesbaden, Germany), and donkey-anti-rabbit (1:2500; Amersham Biosciences, Freiburg, Germany) IgG antibodies, respectively. Protein expression was visualized using luminol-enhanced chemiluminescence and exposure of membranes to blue light-sensitive autoradiography film. Signals were densitometrically assessed and normalized to ß-actin signals to correct unequal loading.
Experimental Groups
In a first group of animals (n=8), stress conditioning by local heat shock priming was performed 24 hours before thromboembolization. Animals undergoing a sham procedure of local heat shock priming (n=4) and animals without stress conditioning (n=8) served as controls. In an additional control group, the normal microcirculation was studied without thromboembolization and without treatment (n=8), and a further group of animals was designed to study the effect of heat shock preconditioning alone without thromboembolization (n=8). To study the role of plasminogen activators, further heat shock-primed (n=8) and nonheat shock-primed animals (n=8) underwent thromboembolization with PAI-1-supplemented microthrombi suspensions. The microcirculation of muscle, periosteum, subcutis and skin was analyzed before and at 30 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes after thromboembolization. Tissue samples for immunohistochemistry were obtained at thromboembolization (additional animals) and 4 hours recanalization.
Statistical Analysis
Results are expressed as means±SEM. Differences between groups were assessed by 1-way ANOVA, differences within each group were analyzed by 1-way repeated measures ANOVA. To isolate overall differences, appropriate Student-Newman-Keuls or Dunn post-hoc tests were performed. Differences were considered significant at P <0.05.
Results
Systemic Circulatory Parameters 100 mm Hg) and heart rate (340 to 400 minutes -1 ) were in normal range without significant differences between the groups studied.
Expression of uPA, tPA, and PAI-1
In nonheat shock-primed controls, immunohistochemistry revealed almost lack of expression of uPA and an only slight expression of tPA, which was found predominantly localized in arteriolar endothelial cells ( Figure 1 ). After heat shock, expression of uPA and tPA was significantly enhanced ( Table ), predominantly in endothelial and smooth muscle cells ( Figure 1 ). PAI-1 increased slightly on heat shock-priming but was less pronounced compared with uPA and tPA ( Table; Figure 1 ). In all experiments, uPA, tPA, and PAI-1 expression was not affected by adding PAI-1 to the microthrombi suspension.
Figure 1. Expression of uPA (A,B,C), tPA (D,E,F), and PAI-1 (G,H,I) in subcutaneous arterioles 4 hours after thromboembolization without heat shock priming (A,D,G), after heat shock priming (B,E,H), and after heat shock priming and PAI-1 application (C,F,I). Note that after heat shock priming, uPA (B,C) is predominantly expressed in endothelial cells (arrowheads), smooth muscle cells (arrows), and fibroblasts (asterisk), and tPA (E,F) mainly in endothelial cells (arrowheads). Nonheat shock-primed controls reveal almost lack of uPA expression (A), and only weak expression of tPA in endothelial cells (D). PAI-1 was only slightly increased after heat shock in endothelial cells (arrowheads) compared with nonheat shock-primed controls. Magnifications x 160 (A,B,C,G,H,I) and x 230 (D,E,F).
Immunohistochemistry of uPA, tPA, and PAI-1 in Arterioles of Muscle and Subcutis at Thromboembolization and 4-Hour Recanalization
Quantitative Western blot analysis confirmed a significant upregulation of uPA and tPA at 24 hours after heat shock preconditioning, whereas PAI-1 expression was only slightly increased in muscle and not affected in skin compared with nonheat shocked controls ( Figure 2 ).
Figure 2. Western blot analysis of PAI-1, uPA, and tPA in muscle (A,B) and skin (C) at 24 hours after heat shock preconditioning (hs) (n=4) and in nonheat shock-primed controls (con) (n=4). Representative examples from muscle (A) and quantitative analysis from muscle (B) and skin (C). Note the significant increase of uPA and tPA after heat shock priming, whereas PAI-1 was only slightly increased. Mean±SEM; * P <0.05 vs nonheat shock-primed controls.
Microvascular Thromboembolization
Directly after injection, intravital microscopy revealed that the microthrombi were arrested in the downstream microcirculation ( Figure 3 ). This resulted in a complete shutdown of microvascular perfusion in all tissues analyzed.
Figure 3. Intravital microscopy of thrombolysis in a transverse arteriole. The thrombus (arrows) is visualized by negative contrast using FITC-dextran plasma staining (A) and directly by rhodamine-staining of platelets (B). C and D, Identical arteriole after onset of recanalization. Magnification x 80. E and F, Rate and time course of recanalization of microthrombi (E, n=8) and microthrombi containing PAI-1 (F, n=8). Note the acceleration of recanalization by heat shock preconditioning (closed circles) compared with nonpreconditioned (open circles) and sham-preconditioned (open diamonds) controls.
Microvascular Recanalization
During the first 30 minutes after thromboembolization, nonheat shock-primed and sham heat shock-primed controls showed almost complete lack of recanalization. Only 1 of 8 and 1 of 4 preparations revealed signs of recanalization ( Figure 3 ). During the second 30 minutes after thromboembolization, all preparations developed recanalization ( Figure 3 ), but with different quality of microvascular reperfusion. After 30 minutes only 10% of the initially perfused capillaries were found reperfused ( Figure 4 ). Recanalization improved during the next 2 hours, as indicated by an increase of perfused capillaries to &50% of baseline, but without further recovery during the 4-hour observation period ( Figure 4 ). This was associated with a markedly lowered volumetric CBF ( P <0.05; Figure 5 ). There were no differences in recanalization between muscle, periosteum, subcutis and skin, and no significant differences between nonheat shock-primed and sham heat shock-primed animals ( Figure 4 ).
Figure 4. Fraction of perfused capillaries in nonheat shock-primed (open circles), sham heat shock-primed (open diamonds) and heat shock-primed (closed circles) muscle (A,B,C), skin (D,E,F), subcutis (G,H,I), and periosteum (J,K,L) before and after thromboembolization with microthrombi (B,E,H,K) and microthrombi containing PAI-1 (C,F,I,L). Preparations without thomboembolization served as controls (A,D,G,J). Mean±SEM; * P <0.05 vs nonheat shock-primed controls, # P <0.05 vs baseline.
Figure 5. Volumetric CBF in nonheat shock-primed (open circles), sham heat shock-primed (open diamonds) and heat shock-primed (closed circles) muscle (A,B,C), skin (D,E,F), subcutis (G,H,I), and periosteum (J,K,L) before and after thromboembolization with microthrombi (B,E,H,K) and microthrombi containing PAI-1 (C,F,I,L). Preparations without thromboembolization served as controls (A,D,G,J). Mean±SEM; * P <0.05 vs nonheat shock-primed controls, # P <0.05 vs baseline.
Heat shock priming effectively accelerated initial recanalization of the obstructed microvasculature. Within the first 15 minutes after thromboembolization, all 8 tissue preparations showed recanalization ( Figure 3 ). At 30 minutes, already 60% of the initially perfused capillaries were found reperfused in either of the tissues analyzed ( P <0.05; Figure 4 ). Recanalization over the subsequent 3.5-hours further improved capillary perfusion to &80% of baseline, which was significantly higher than that of nonheat shock-primed and sham-heat shock-primed controls ( P <0.05; Figure 4 ).
Importantly, heat shock-primed tissues showed already at baseline a significantly higher volumetric CBF ( P <0.05) than nonconditioned tissues ( Figure 5 ). Further, heat shock produced reactive hyperemia during initial recanalization and preserved CBF over the entire post-thromboembolization period.
Addition of PAI-1 to the thrombi resulted in prolonged failure of recanalization ( P <0.05). Until 2 hours after thromboembolization, zero and, at 4 hours only 2, of 8 preparations showed recanalization ( Figure 3 ). This was associated with a significantly ( P <0.05) lowered fraction of perfused capillaries ( Figure 4 ) and a marked ( P <0.05) compromise of CBF ( Figure 5 ) compared with nontreated controls. Heat shock priming accelerated the onset of recanalization and improved the microcirculation ( Figures 4 and 5; P <0.05), although the addition of PAI-1 was associated with a reduction of capillary density and CBF when compared with heat shock-conditioned but non-PAI-1-treated controls ( Figures 4 and 5; P <0.05).
Microvascular Response to Heat Shock Priming
In animals without any treatment, analysis of capillary perfusion over the 4-hour observation period showed &100% of the capillaries perfused without significant changes of CBF ( Figures 4 and 5 ). Heat shock priming without thromboembolization did not affect capillary density ( Figure 4 ) and confirmed the increased CBF compared with nonheat-shocked controls ( Figure 5 ).
Discussion
The major novel findings of the present study are that heat shock priming: (1) upregulates plasminogen activators in vivo; (2) accelerates recanalization of thromboembolized microvasculature; and (3) counteracts the function of PAI-1. Thus, local upregulation of heat shock proteins induces endogenous hyperfibrinolysis.
Thromboembolization significantly contributes to the shutdown of microvascular perfusion, infarction, and necrosis. 1,3 The microemboli may clot primarily arteriolar and capillary segments of the microvasculature, and thus produce initially a negative angiogram. 19 Over time, obstruction of the vasculature may aggravate, producing relevant infarction, or may dissolve because of spontaneous recanalization. 20,21
The study of the mechanisms of thromboembolization requires an adequate experimental model, which should consider the development of both infarction and spontaneous recanalization. O?Shaughnessy et al 22 introduced a thromboembolization model, demonstrating that platelet emboli, originating from the site of arterial vessel repair, pass downstream, and block the microcirculation. 22 This confirms the particular risk of interventional and microsurgical procedures for thromboembolism. Herein, we have not chosen this model, because the development of thromboemboli is heterogeneous, and the cremaster muscle allows only a 2-hour study period, which is too short to analyze spontaneous recanalization. We have used the rat hindlimb model, because this allows repetitive intravital microscopy of the microcirculation of muscle, periosteum, subcutis, and skin for a period of 6 hours. 12 Further, we have injected in vitro produced microthrombi, mimicking platelet-rich arterial thrombi, which adequately standardizes the experiments. 15
The process of platelet-rich arterial thrombus formation can be partitioned into platelet adhesion, coagulation factor activation and thrombus propagation. 23 Accordingly, in the model used thrombi were generated by addition of thrombin (coagulation factor activation). Because a low pressure in vitro system can produce large "white" thrombi, however, with a "red" tail, 24 we have chosen a moving high-pressure closed compartment system, 13 which generates arterial "white" thrombi, rich in platelets and fibrin, intermingled with only a few erythrocytes and leukocytes. 13 The moving high-pressure closed compartment system mimics conditions of pressure and turbulences as known in arterial thrombus formation in vivo. The platelet-rich thrombi were filtered through a 40-µm 40 µm. Only small thrombi of <40 µm were used for the experiment to guarantee that embolization takes place within terminal arterioles. This microembolization mimics the clinical situation of ischemic stroke and flap tissue failure. 5,13,22
Because terminal arterioles are not regularly visible by intravital microscopy, 15 recanalization was not assessed by direct visualization of the lysis of arrested microthrombi, but indirectly by evaluation of the re-onset of capillary perfusion within the downstream microcirculation. This can reliably be performed, because downstream capillary perfusion failure, which additionally indicates tissue viability, has been shown to correlate with embolization-induced arteriolar vessel obstruction. 25
Previous studies have demonstrated that heat shock increases capillary perfusion in flap tissue. 17 Thus, the increased baseline CBF may have contributed to the accelerated recanalization. However, although baseline skin CBF did not differ significantly between heat shock preconditioning versus non-preconditioned controls, heat shock significantly accelerated recanalization, similarly as observed in the other tissues, in which baseline CBF was different between the 2 groups. Thus, the heat shock-mediated increase in basal CBF may not be considered a primary determinant for recanalization.
Beside acceleration of recanalization and improvement of capillary perfusion, heat shock increased CBF above baseline levels initially during recanalization. This reactive hyperemia, which classically is produced only after short ischemia periods, may be caused by the rapid lysis-associated reduction of ischemia time by heat shock priming.
Because anesthesia and immersion of the hindlimb may also represent a stress conditioning, we have exposed additional animals to a sham procedure, which was not associated with tissue heating. Of interest, these animals did not show acceleration of recanalization and improvement of capillary perfusion. This supports that the protective effect on the microcirculation is caused by heat induction rather than anesthesia or hindlimb manipulation-associated stress.
By converting plasminogen to plasmin, plasminogen activators are the main upregulators of the fibrinolytic system. 8 The serine proteases uPA 26 and tPA 27 are synthesized by endothelial cells. In vivo, tPA is the principal plasminogen activator, whereas uPA serves as an amplifier of the tPA-mediated fibrinolysis after its activation by tPA-mediated generation of plasmin. 28 A procoagulant condition induced by thrombin is the predominant physiological stimulus for expression and release of both plasminogen activators. 29
In vitro studies have demonstrated that prolonged hyperthermia 8 hours downregulates plasminogen activators and stimulates PAI-1 expression in human umbilical vein endothelial cells. 30,31 However, hyperthermic exposure times of <8 hour indicated that both uPA and tPA are upregulated by heat shock in endothelial cells. 11 The present study extends this observation, demonstrating for the first time to our knowledge that 30 minutes of heat shock upregulates uPA and tPA also in vivo, and that the expression is found primarily in arteriolar endothelial cells. Thus, an enhanced lysis of fibrin in platelet emboli by uPA and tPA may be the cause for the observed acceleration of recanalization after heat shock priming.
Fibrinolysis is inhibited by endothelium-derived PAI-1, which binds irreversibly to the active site of uPA and tPA. 27,32 The observed abrogation of recanalization by PAI-1 is in line with these previous reports, 32,33 and confirms the causative role of uPA and tPA in spontaneous recanalization of thromboembolized microvasculature. Our PAI-1 experiments further indicate that the heat shock-mediated upregulation of endogenous uPA and tPA does not only counteract the function of endogenous but also exogenously applied PAI-1.
The increase of endogenously expressed PAI-1 on heat shock induction was markedly less pronounced compared with the increase of uPA and tPA. This is in line with previous observations, demonstrating a stronger expression of uPA and tPA compared with PAI-1 in response to hyperthermia. 34 Although PAI-1 is a major stress-regulated gene, it is only weakly expressed in endothelial cells, 35 and may be further reduced in endothelial cells located close to fibrin clots. 34
The endogenous induction of a local pro-thrombolytic state by heat shock priming offers some advantages compared with the systemic application of plasminogen activators. Plasminogen activators are rapidly cleared from the systemic circulation, mainly by the function of the liver. 36 Additionally, the thrombolytic properties after local heat shock are restricted to the endangered microcirculation. Thus, systemic side effects, such as the assumed neurotoxicity of tPA, 37 adverse bleeding complications, 38 or anaphylactoid reactions related to recombinant tPA, 39 may be avoided.
Heat shock priming may represent a promising tool also in the clinical setting. Of interest, fever in septic conditions has been shown to induce heat shock proteins and to result in better respiratory function, lower blood lactate concentrations, and prolonged survival times. 40 These experimental results support clinical studies, demonstrating protective functions of intracellular heat shock protein-70 expression in patients with severe sepsis. 41 However, the effect of sepsis-associated fever on blood coagulation and thrombus formation remains to be determined.
In conclusion, we herein demonstrate that local heat shock priming induces endogenous hyperfibrinolysis by upregulation of plasminogen activators and consequently results in an accelerated and improved recanalization of thromboembolized microvasculature. Thus, the efficacy of local heat shock preconditioning should be further evaluated experimentally as a novel treatment strategy in disease states associated with microthromboembolization and infarction.
Acknowledgments
We appreciate the work of Klaus Saueressig and Janine Becker.
Source of Funding
This study was supported by the Deutsche Forschungsgemeinschaft (Me 900/1-3 and 1-4).
Disclosures
None.
【参考文献】
Topol EJ, Yadav JS. Recognition of the importance of embolization in atherosclerotic vascular disease. Circulation. 2000; 101: 570-580.
Reimers B, Corvaja N, Moshiri S, Sacca S, Albiero R, Di Mario C, Pascotto P, Colombo A. Cerebral protection with filter devices during carotid artery stenting. Circulation. 2001; 104: 12-15.
Baim DS, Wahr D, George B, Leon MB, Greenberg J, Cutlip DE, Kaya U, Popma JJ, Ho KK, Kuntz RE. Randomized trial of a distal embolic protection device during percutaneous intervention of saphenous vein aorto-coronary bypass grafts. Circulation. 2002; 105: 1285-1290.
Borger MA, Ivanov J, Weisel RD, Rao V, Peniston CM. Stroke during coronary bypass surgery: principal role of cerebral macroemboli. Eur J Cardiothorac Surg. 2001; 19: 627-632.
Andresen DM, O?Shaughnessy M, Acland RD, Anderson GL, Schuscke D, Banis J Jr, Barker JH. Direct visualization and measurement of microsurgically induced thromboembolism. Microsurgery. 1994; 15: 413-420.
Sobel BE. Intracranial bleeding, fibrinolysis, and anticoagulation. Causal connections and clinical implications. Circulation. 1994; 90: 2147-2152.
De Maio A. The heat shock response. New Horizons. 1995; 3: 198-207.
Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood. 1991; 78: 3114-3124.
Iba T, Shin T, Sonoda T, Rosales O, Sumpio BE. Stimulation of endothelial secretion of tissue-type plasminogen activator by repetitive stretch. J Surg Res. 1991; 50: 457-460.
Levin EG, Santell L, Salijooque F. Hyperosmotic stress stimulates tissue plasminogen activator expression by a PKC-independent pathway. Am J Physiol. 1993; 265: C387-C396.
Fukao H, Hagiya Y, Ueshima S, Okada K, Takaishi T, Izaki S, Matsuo O. Effect of heat shock on the expression of urokinase-type plasminogen activator receptor in human umbilical vein endothelial cells. Thromb Haemost. 1996; 75: 352-358.
Rücker M, Roesken F, Vollmar B, Menger MD. A novel approach for comparative study of periosteum, muscle, subcutis, and skin microcirculation by intravital fluorescence microscopy. Microvasc Res. 1998; 56: 30-42.
Overgaard K, Sereghy T, Boysen G, Pedersen H, Hoyer S, Diemer NH. A rat model reproducible cerebral infarction using thrombotic blood clot emboli. J Cereb Blood Flow Metab. 1992; 12: 484-490.
Loskutoff DJ, Samad F. The adipocyte and hemostatic balance in obesity studies of PAI-1. Arterioscler Thromb Vasc Biol. 1998; 18: 1-6.
Rücker M, Schäfer T, Stamm A, Saueressig K, Vollmar B, Spitzer WJ, Menger MD. New model for in vivo quantification of microvascular embolization, thrombus formation, and recanalization in composite flaps. J Surg Res. 2002; 108: 129-137.
Rücker M, Strobel O, Vollmar B, Roesken F, Menger MD. Vasomotion in critically perfused muscle protects adjacent tissues from capillary perfusion failure. Am J Physiol. 2000; 279: H550-H558.
Rücker M, Schäfer T, Roesken F, Spitzer WJ, Bauer M, Menger MD. Local heat shock priming-induced improvement in microvascular perfusion in osteomyocutaneous flaps is mediated by heat shock protein 32. Br J Surg. 2001; 88: 450-457.
Page C, Rose M, Yacoub M, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol. 1992; 141: 673-683.
Derex L, Tomsick TA, Brott TG, Lewandowski CA, Frankel MR, Clark W, Starkman S, Spilker J, Udsten GJ, Khoury J, Grotta JC, Broderick JP. Outcome of stroke patients without angiographically revealed arterial occlusion within four hours of symptom onset. Am J Neuroradiol. 2001; 22: 685-690.
Kassem-Moussa H, Graffagnino C. Nonocclusion and spontaneous recanalization rates in acute ischemic stroke: a review of cerebral angiography studies. Arch Neurol. 2002; 59: 1870-1873.
Gargiulo NJ 3rd, Veith FJ, Lipsitz EC, Ohki T, Suggs WD, Cayne NS, Dadian N, Wain RA. Spontaneous recanalization of arterial occlusions: an unusual mechanism for symptomatic improvement. J Vasc Surg. 2002; 36: 1161-1166.
O?Shaughnessy M, Gu JM, Wyllie F, Acland RD, Anderson GL, Banis JC Jr, Barker JH. Microcirculatory consequences of microvascular surgery. Microsurgery. 1994; 15: 405-412.
Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992; 326: 310-318.
Chandler AW. In vitro thrombotic coagulation of the blood. Lab Invest. 1958; 7: 110-114.
Krapohl BD, Siemionow M, Piza P, Pieramici SF, Zins JE. Thrombogenic stimulus causes long-term decrease of muscle flap perfusion. Ann Plast Surg. 1999; 42: 299-305.
Barnathan ES, Kuo A, Kariko K, Rosenfeld L, Murray SC, Behrendt N, Ronne E, Weiner D, Henkin J, Cines DB. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA. Blood. 1990; 76: 1795-1806.
Emeis JJ. Perfused rat hindlegs. A model to study plasminogen activator release. Thromb Res. 1983; 30: 195-203.
Munkvad S. Fibrinolysis in patients with acute ischaemic heart disease. Dan Med Bull. 1993; 40: 383-408.
van Hinsbergh VWM, Sprengers ED, Kooistra T. Effect of thrombin on the production of plasminogen activators and PA-inhibitor-1 by human foreskin microvascular endothelial cells. Thromb Haemost. 1987; 57: 148-153.
Wojta J, Holzer M, Hufnagl P, Christ G, Hoover RL, Binder BR. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro. Am J Pathol. 1991; 139: 911-919.
Ang C, Dawes J. The effects of hyperthermia on human endothelial monolayers: modulation of thrombotic potential and permeability. Blood Coagul Fibrinolysis. 1994; 5: 193-199.
Torr-Brown SR, Sobel BE. Attenuation of thrombolysis by release of plasminogen activator inhibitor type-1 from platelets. Thromb Res. 1993; 72: 413-421.
Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation. 1998; 97: 1002-1008.
Fukao H, Matsuo O. Antithrombotic regulation in human endothelial cells by fibrinolytic factors. Semin Thromb Hemost. 2000; 26: 33-38.
Yamamoto K, Takeshita K, Shimokawa T, Yi H, Isobe K, Loskutoff DJ, Saito H. Plasminogen activator inhibitor-1 is a major stress-regulated gene: Implications for stress-induced thrombosis in aged individuals. PNAS. 2002; 99: 890-895.
Bounameaux H, Stassen JM, Seghers C, Collen D. Influence of fibrin and liver blood flow on the turnover and the systemic fibrinogenolytic effects of recombinant human tissue-type plasminogen activator in rabbits. Blood. 1986; 67: 1493-1497.
Vivien D, Buison A. Serine protease inhibitors: novel therapeutic targets for stroke? J Cereb Blood Flow Metab. 2000; 20: 755-764.
Nowak-Gottl U, Auberger K, Halimeh S, Junker R, Klinge J, Kreuz WD, Ries M, Schlegel N. Thrombolysis in newborns and infants. Thromb Haemost. 1999; 82: 112-116.
Hill MD, Barber PA, Takahashi J, Demchuk AM, Feasby TE, Buchan AM. Anaphylactoid reactions and angioedema during alteplase treatment of acute ischemic stroke. CMAJ. 2000; 162: 1281-1284.
Su F, Nguyen ND, Wang Z, Cai Y, Rogiers P, Vincent JL. Fever control in septic shock: beneficial or harmful? Shock. 2005; 23: 516-520.
Bruemmer-Smith S, Stuber F, Schroeder S. Protective functions of intracellular heat shock protein (HSP) 70-expression in patients with severe sepsis. Intensive Care Med. 2001; 27: 1835-1841.
作者单位：Institute for Clinical & Experimental Surgery (M.R., T.S., C.S., Y.H., M.M.), University of Saarland, Homburg/Saar; the Department of Oral and Maxillofacial Surgery (M.R.), Hannover Medical School, Hannover; and the Department of Experimental Surgery (B.V.), University of Rostock, Rostock, Germa
Nov. 14, 2008 -- Does health care reform still have a chance for quick action?
Several lawmakers, fearing that it may not get the immediate attention of President-elect Barack Obama because of the economic crisis, are working to keep the subject front and center.
One of them, Sen. Max Baucus, D-Mont., chairman of the influential Senate Finance Committee, introduced his plan for health care reform this week. Another, Sen. Ted Kennedy, D-Mass., has set up a task force on health care reform and is working behind the scenes with major stakeholders on the issue.
But lobbyists, politicians, and analysts are waiting to see how hard and how fast Obama will move on health reform plans after he is sworn in on Jan. 20, 2009.
Leading Democrats in Congress have already said they would like to act quickly next year on a bill expanding the State Children's Health Insurance Program (SCHIP). House Speaker Nancy Pelosi, D-Calif., said last week that a bill expanding SCHIP "would probably be one of the first bills we would put on President Obama's desk."
A bill adding 4 million uninsured children to the program passed Congress last year but was vetoed by President Bush.
But quick action on children's health insurance -- in addition to possible early action to broaden funding for embryonic stem cell research -- raises questions of when the White House and Congress will choose to move on the wider, more difficult question of lowering health care costs and making insurance accessible to some 47 million Americans now without coverage.
"There are two discussions going on now. Do all or nothing, or do SCHIP first and come back later" to debate bigger health reform issues, says Dean Rosen, a health care lobbyist and one-time aide to former Senate Majority Leader Bill Frist, R-Tenn.
Obama has not yet named a team that would advise him on domestic affairs like health policy, nor has he chosen a nominee for Secretary of Health and Human Services.
"I don't think the Obama senior people have really made any decisions," Rosen tells WebMD.
A plan put forward by Obama during the presidential campaign called for mandatory coverage for all children as well as expanded government subsidies and tax credits to help lower coverage costs.
Earlier this week, Baucus issued a health reform "white paper" calling for insurance "exchanges" similar to those in Obama's plan, as well as a program allowing adults between 55 and 65 to buy into Medicare.
Obama has said that shoring up the economy will be his first priority as president. Baucus is among those lawmakers arguing that relieving businesses and families from rising health care costs is a key part of the effort.
【摘要】
Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, ClC, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR.
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Irradiation (IR) delivery to head and neck is a common treatment modality for malignancies, and the radiation field frequently includes salivary glands. Salivary glands in the IR field are severely damaged, consequently resulting in marked salivary hypofunction in 80% of patients.1-3 Patients experiencing reduced salivary flow suffer from considerable morbidity including dental caries, mucosal infections, dysphagia, and extensive discomfort. Although the effects of IR have been recognized as a significant clinical problem for more than 90 years, the mechanism of these effects remains unknown, and no adequate prevention or treatment is yet available. Saliva is essential for maintaining the health of the oral cavity.4 Although salivary glands should be considered to be radioresistant because of their highly differentiated cellular state,5 their function is rapidly affected when they are exposed to ionizing radiation.6 Serous cells are considered to be more radiosensitive than mucous cells because serous secretory granules are rich in transition metals such as Zn2+, Fe2+, and Mn2+, which may leak into the cytoplasm and cause autolysis and cell death.7 At later stages changes in salivary gland and reduction in salivary function are observed within 3 months to 1 year.2,3
Aquaporins (AQPs) are specific water channels that allow rapid transcellular movement of water in response to osmotic/hydrostatic pressure gradients.8 AQP5, cloned from submandibular glands, is present in the water-transporting epithelia of lacrimal gland, trachea, eye, lung, and salivary glands of rat.9 In human salivary glands, AQP5 is anatomically localized in the apical membranes of acinar cells, but not in those of ductal cells,10 and functions to stimulate the outflow of water into the acinar lumen. In fact, a reduction of salivary gland secretion has been shown in mice harboring a mutant AQP5 channel.11
Heat shock proteins (HSPs) are a group of highly conserved proteins originally identified as proteins that are up-regulated in response to elevated temperatures; however, they are now shown to be induced by a wide range of noxious or stressful stimuli, including heat, hypoxia, ischemia, and heavy metals.12-14 A number of studies have shown that a mild stress induces rapid synthesis of HSP27, -70, and -90, which then confer cells with some protection against further and more severe cellular insult.15-18 Although it is likely that the various HSPs function in a coordinated manner to protect the cell after stress, overexpression of a single species (HSP27, -70, or -90) can also confer cells with resistance to various stimuli.19-22 HSP25 (or HSP27) has been suggested to protect cells against apoptotic cell death triggered by hyperthermia, ionizing radiation, oxidative stress, Fas ligand, and cytotoxic drugs,1-3,23,24 and several mechanisms have been proposed to account for HSP27-mediated apoptotic protection. For example, its specific interaction with cytochrome c released from mitochondria into the cytosol prevents apoptosome formation,25,26 and elimination of unfolded protein through extralysosomal, energy-dependent, ubiquitin-proteasome degradation pathway contributes to protection of cells from stressful stimuli.7 HSP27 binds to polyubiquitin chains as well as 26S proteasomes, and the ubiquitin-proteasome pathway is involved in the activation of transcription factor nuclear factor (NF)-B by degrading its main inhibitor I-kB.7 Moreover, phosphorylated HSP27 has been shown to bind the adaptor protein Daxx and then to inhibit Fas-mediated apoptosis.4 Interaction between HSP27 and Akt is necessary for Akt activation, which is then followed by dissociation of phosphorylated HSP27 from Akt.5 We recently reported that the radioprotective effect of HSP25 involves delayed cell growth27,28 and HSP25-mediated MnSOD gene expression.29,30 HSP25 overexpression down-regulates ERK1/2 expression28,30 and also inhibits radiation-induced PKC-mediated production of reactive oxygen species and cell death.31 However, a radioprotective effecting an in vivo system has not yet been evaluated.
The aim of this study was to evaluate the potential of exogenous HSP25 expression, delivered by adenoviral vectors, to protect rats from radiation-induced salivary gland damage. We showed that overexpression of HSP25 in salivary glands could significantly inhibit radiation-induced cell death, saliva fluid loss, and alteration of saliva chemistry and AQP5 expression.
【关键词】 radioprotective submandibular
Materials and Methods
Description of the Genes
Mouse cDNAs for HSP25 and inducible HSP70 (HSP70i) were cloned by polymerase chain reaction (PCR) into pGFP3 vector to make pGFP3-HSP25 and pGFP3-HSP70i fusion constructs. Because only the coding sequence was cloned, no flanking sequence was added into the vector. GFP (green fluorescent protein) tag was attached at the C terminus of the HSPs to monitor the efficiency of the protein expression during DNA construction. The following primers were used in PCR to prepare those constructs: for pGFP3-HSP25 cloning, forward primer 5'-CGGAATTCATGACCGAGCGCCGCGTGCCCTT-3', reverse primer 5'-CTAGTCTAGATTACTTGGCTCCAGACTGTTCA GA-3' and for pGFP3-HSP70i cloning, forward primer 5'-CCGGAATTCGCCAAAGCCGCGGCGATCGGC-3' and reverse primer 5'-CGCGGATCCATCTACCTCCTCAATGGTGGGGCC-3'.
Construction of Recombinant Viral Vectors
To make viral vectors that contained mouse HSP25 and HSP70i cDNA, the sequences in pGFP3-HSP25 or pGFP3-HSP70i were removed by digesting the DNA with XhoI and XbaI. Isolated inserts were ligated to corresponding restriction sites of pShuttleCMV vector to make pShuttleCMV-HSP25 or pShuttleCMV-HSP70i, respectively.32 These constructs were recombined with pAdEAsy-1 to make recombinant viral vectors that contained mouse HSP25 or HSP70i, respectively. Expression of HSPs in mouse was detected by using antibody to HSPs. As a control, we used a recombinant adenovirus that expressed luciferase for convenient detection of expression in the cell.33 The final plasmids were linearized with Pacl and transfected into 293A cells34 using WelFect-Q transfection reagent (WelGENE Inc., Daegu, Korea). Recombinant viruses were harvested by viral plaques 7 to 10 days after transfection. For large scale-production, 30 15-cm plates of 293A cells were infected with recombinant viruses. Two days after infection, when a clear CPE was visible, cells were harvested by low-speed centrifugation. The cell pellets resuspended with Dulbecco??s modified Eagle??s medium were repeatedly frozen at C80??C and thawed in a 37??C water bath for a total of four cycles. The samples were spun at 12,000 rpm for 10 minutes, and viral supernatant was stored at C80??C. The recombinant virus particles were purified by cesium chloride gradient ultracentrifugation.35 Virus titers were determined by tissue culture infectious dose 50 (TCID50). Typical virus titers were 109 to 1010 pfu/ml.
Amifostine was obtained from Sigma-Aldrich Co. (St. Louis, MO). A solution of 100 mg/ml was prepared in 0.9% NaCl and stored at 4??C.
Wistar male rats (250 to 300 g body weight) were purchased from SLC (Hamamatsu, Japan) and kept in polycarbonate cages under an alternating 12-hour light/dark cycle. Animals were maintained at animal care facilities, and food and water were supplied ad libitum. Studies were conducted under guidelines for the use and care of laboratory animals and were approved by the Institutional Animal Care and Use Committee of the Korea Institute Radiological and Medical Sciences.
Experimental Design
Rats were divided into nine groups, and each group consisted of three rats. Each animal was treated as described. Group I (normal control) received no pretreatment, group II (radiation control) was irradiated only, group III (vector control) was control adenoviral vector transferred, group IV was adenovirus with HSP25 gene transferred, group V was adenovirus with HSP70i gene transferred, group VI was viral vector transferred and irradiated, group VII was HSP25 gene transferred and irradiated, group VIII was HSP70i gene transferred and irradiated, and group IX was pretreated with amifostine (100 mg/kg per body weight, i.v.) and irradiated.
IR of Salivary Glands
All rats were subjected to 17.5 Gy of radiation directed to the submandibular glands. Animals were first anesthetized with a combination of ketamine chloride (6 mg/kg) and xylazine (0.6 mg/kg) injected intraperitoneally. Head and neck regions of animals, including bilateral submandibular glands, were exposed to a single dose of 17.5 Gy at a dose rate of 190 cGy/minute. Size of the IR field was controlled (Theratron 780; AECL, Ontario, ON, Canada), and the radiation field was 4 x 34 cm and an 80-cm source-to-skin distance. Radiation was given to six rats at a time with ventral surface being exposed to the source. Control animals were sham-irradiated; ie, anesthetized and placed in the irradiator.
In Vivo Gene Transfer to Submandibular Glands
Adenoviral vectors were suspended in Dulbecco??s modified Eagle??s medium media and delivered to both submandibular glands by direct injection. Individual rats were randomly assigned to receive either control adenoviral vector or adenovirus with HSP25 or HSP70i gene (1 x 108 pfu/gland). All gene transfers were performed 1 day before 17.5-Gy IR. Animals were first anesthetized and 0.5 cm of neck ventral skin was incised. Viral vector was directly injected by a syringe, and the incision was then sutured with silk. Animals received 1 mg of dexamethasone (intramuscularly) at the time of gene transfer to suppress inflammation due to adenoviral infection. On the following day after gene transfer, distribution of viral vector was monitored with an IVIS ImagingSystem (Xenogen, Alameda, CA).
Collection of Saliva and Harvesting of Salivary Glands
Forty and 90 days after IR, animals were weighed and then anesthetized with a combination of ketamine and xylazine. Saliva flow rates were measured for both right and left submandibular gland of each rat. The orifices of main excretory duct of the submandibular glands were identified intraorally and cannulated with polyethylene tubing (PE-10; Becton Dickinson and Company, Mountain View, CA). The total saliva was collected for 30 minutes after subscapular injection with pilocarpine hydrochloride (Sigma-Aldrich Inc., Steinheim, Germany). Saliva flow rates were expressed as the volume of saliva secreted per 100 g of body weight. Submandibular glands were harvested immediately after saliva collection and weighed after carefully trimming fat and connective tissue. Body and gland weights were obtained for all animals in which flow measurements were performed.
Sialochemical Analysis
Salivary constituents of the submandibular glands were analyzed. Concentrations of sodium, potassium, chloride, and total protein were determined with a Hitachi Clinical Analyzer 7180 (Histachi High-Technologies Co., Tokyo, Japan), and ionized calcium was measured with AVL 9180 (Diamond Diagnostics, Holliston, MA). Amylase was determined with Fuji DRI Chemiclinical Chemistry Analyzer 3500 (Fuji Photo Film Co., Tokyo, Japan).
Immunohistochemistry and Light Microscopic Examination
The rat submandibular glands were removed, postfixed for 1 hour in the same fixative solution using perfusion, dehydrated in ethanol followed by xylene, and then finally embedded in paraffin. Sections were cut at 3-µm thickness on a rotary microtome (Leica, Wetzlar, Germany), dewaxed, and then rehydrated. For immunoperoxidase labeling, endogenous peroxidase was blocked by 0.3% H2O2 in absolute methanol for 15 minutes at room temperature. For antigen retrieval, sections were placed in citrate buffer (pH 6.0) and heated in a microwave oven for 10 minutes. Nonspecific binding for immunoglobulin was prevented by incubating the sections in blocking solution (Cap-Plus detection kit; Zymed Laboratories, San Francisco, CA) for 20 minutes. Sections were incubated overnight at 4??C with primary antibody diluted in antibody diluent (Zymed Laboratories), followed by three washes (5 minutes each) with phosphate-buffered saline (PBS) containing 0.05% Triton X-100. Incubation with corresponding secondary antibody and the peroxidase-anti-peroxidase complex was performed for 30 minutes at 22??C. Immunoreactive sites were visualized using 3,3'-diaminobenzidine 0.1% (w/v) and 0.03% (v/v) hydrogen peroxide solution.
Antibodies and Chemicals
The following primary antibodies were used: goat-polyclonal IgG HSP25 (M-20; Santa Cruz Biotechnology, Santa Cruz, CA), goat-polyclonal IgG AQP5 (C-19, Santa Cruz Biotechnology), mouse monoclonal anti-HSP70 (SPA-810; Stressgen Biotechniques Inc., San Diego, CA), mouse monoclonal anti-proliferating cell nuclear antigen (PC-10; DAKO, Kyoto, Japan), anti-cleaved-caspase3 (Asp175) (5A1; Cell Signaling Technology Inc., Beverly, MA), and anti-poly(ADP-ribose) polymerase (PARP) (no. 9542; Cell Signaling Technology Inc.) antibodies. Anti-mouse and anti-rabbit secondary antibodies and the corresponding peroxidase-anti-peroxidase complexes used were from Cap-Plus detection kit (Zymed). Anti-goat secondary antibody was N-His-tofine Simple Stain MAX PO, Universal Immuno-Peroxidase Polymer (anti-goat), H0502 (Nicheirei Bioscience Inc., Kyoto, Japan). Triton X-100, hydrogen peroxide, PBS, and other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO).
Terminal dUTP Nick-End Labeling (TUNEL) Assay
Apoptotic cells in salivary gland were visualized, using the In Situ Cell Death Detection kit (Roche Diagnostic GmbH, Mannheim, Germany), by the indirect TUNEL method following the manufacturer??s protocol. The paraffin-embedded tissue sections were hydrated and incubated with the TUNEL reaction mixture containing TdT and fluorescein-dUTP without proteinase K pretreatment. The reactions were terminated by three washes with PBS. Anti-fluorescein-peroxidase antibody was applied, and the reaction was visualized by 3,3'-diaminobenzidine. Sections were counterstained with autohematoxylin. Negative control sections were incubated with distilled water in the absence of TdT.
Whole submandibular gland sections (1 mm thick) were homogenized with a PRO200 homogenizer (PROScientific, Oxford, UK) in lysis buffer (PRO-PREP, Sungnam, Korea). The protein concentration was determined by the Bradford method (Bio-Rad, Richmond, CA). For polyacrylamide gel electrophoresis and Western blot, proteins were solubilized with lysis buffer , the samples were boiled for 5 minutes, and equal amounts of protein (80 µg/well) were analyzed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred onto a nitrocellulose membrane and processed for immunoblotting. Blocking was performed by incubation with 5% nonfat dry milk in PBS-0.1% Tween 20 (PBS-T) for 2 hours at room temperature. After blocking, membranes were probed with the corresponding antibody for 18 hours at 4??C and washed with PBS-T. Blots were further incubated with horseradish peroxidase-conjugated secondary antibody, diluted at 1:5000, and specific bands were visualized by enhanced chemiluminescence (Amersham International, Buckinghamshire, UK). Autoradiographs were recorded onto X-Omat duplicating films (Eastman Kodak Co., Rochester, NY).
For the quantification of TUNEL-positive signals, five sections were randomly chosen for each animal. In each section, 500 acinar cells, granular convoluted cells, and intercalated ductal cells were randomly counted at a magnification of 400, and the percentage of TUNEL-positive cells was calculated. For the quantification of HSP25 or HSP70i expression, we randomly chose five fields of each slide (three slides in each group) and took pictures using microscopy with digital camera (Leica DM IRBE; Leica Microsystems GmbH). The positive signals were measured by image analyzing software (Leica QWin) in the same area of each field; field size was 200 x 200 µm at a magnification of x200. Percentage of positive signal was obtained in the unit area of submandibular gland, and the results were compared on days 1 (after gene transfer), 40, and 90 (after IR). For the detection of AQP5 expression, we used similar methods for HSP25 or HSP70i, but the field size was 60 x 50 µm and the numbers of fields were 10 in each slide at x1000 magnification. We obtained positive signal ratio of AQP5 against normal control at 40 and 90 days after IR. The labeling incidences of each animal were obtained by averaging the percentages of sections or fields, and then mean and SD were determined at each time point for three experimental animals. Comparison between experimental and control data at each time point was made by one-way analysis of variance followed by the Student??s t-test, with P < 0.05 as statistically significant.
Transfer of HSP25 and HSP70i to Rat Submandibular Gland Using Adenoviral Vector System
The distribution of virus after transfer of HSP25 to submandibular gland was first investigated, using a virus encoding firefly luciferase (Fluc) that was administered at a titer equivalent to that used for HSP25-expressing viruses (1 x 108 pfu/ml). Bioluminescence imaging for luciferase was performed by image analyzer on the next day after the transfer, and active luminescence was detected in submandibular glands (Figure 1A) , indicating that localization of transferred virus was the submandibular glands. Immunohistochemical analysis revealed that HSP25 was overexpressed mainly in acinar cells, whereas HSP70i was in granular convoluted cells. Some HSP70i was expressed also in ductal cells. As seen in Figure 1B , in irradiated submandibular glands, radiation induced loss of HSP25 and HSP70i at 40 and 90 days; however, HSP25-transferred glands were found to have more HSP25 protein than other groups. It should also be noted that radiation induced a slight reduction of endogenous HSP25 or HSP70i in irradiated normal control group, and this was probably attributable to radiation-induced death of some cells containing HSPs. There were no differences detected between irradiated and nonirradiated control rats. In the case of amifostine-treated rats, no induction of HSP25 or HSP70i was detected (Figure 1, B and C) .
Figure 1. Bioluminescence imaging (BLI) and immunolocalization of HSP25 and HSP70i in HSP25 and HSP70i transferred rat. A: BLI of luciferase expression in a living rat at 24 hours after gene transfer into salivary gland. Active luminescence was detected in bilateral submandibular glands of the rat. B: Expression of HSP25 and HSP70i during the days after IR. Representative photographs show expression changes of HSP25 and HSP70i in rats after transfer of control adenoviral vector (ACF) and HSP70i-expressing (GCL) and HSP25-expressing (MCR) adenovirus and pretreatment with amifostine (SCX). HSP25 was expressed in acinar cells and HSP70i in granular convoluted cells. C: Distributions of HSP25 and HSP70i during experimental periods. The graphs indicate the percentage (mean ?? SD) of HSP25- or HSP70i-positive cells against the unit area (200 x 200 µm2, x200) of submandibular gland at 24 hours after gene transfer and 40 and 90 days after 17.5 Gy IR using image analyzer. Each group consisted of three rats, and five fields were obtained from each animal. Original magnifications, x200.
Effects on Radiation-Induced Glandular Weight Loss
When body weights were compared at 40 and 90 days after radiation, 17.5-Gy radiation was found to significantly reduce body weights, and HSP25 and HSP70i transfer could not prevent radiation-induced body weight loss. When treated with amifostine, which has radioprotective effects and has been approved by the United States Food and Drug Administration, the loss of body weight by radiation was significantly prevented compared with the level of nonirradiated control rats (Figure 2A) . Glandular weights were also decreased by radiation; however, the decrease was significantly attenuated when HSP25 was transferred to submandibular glands. Amifostine treatment 30 minutes before radiation restored the glandular weights inhibited (Figure 2B) . On the other hand, HSP70i did not show any protection from radiation-induced glandular weight loss. These results indicate that HSP25 protected the animal from the radiation-induced loss of glandular weight.
Figure 2. Total body weight and submandibular gland weight of rats. Measurements were obtained 40 and 90 days after 17.5-Gy IR of head and neck. A: Changes of total body weight during experimental periods. Radiation significantly reduced body weight of animals, and amifostine significantly prevented the loss of body weight induced by IR. B: The weights of submandibular glands. HSP25 and amifostine significantly attenuated the loss of glandular weight compared with each controls. Differences of body weight and glandular weight, compared with controls, are indicated by •, compared with normal control; ; compared with vector transferred control; , compared with vector transferred IR control; and , compared with radiation control (P < 0.05).
Effects on Pilocarpine-Stimulated Saliva Flow Rate and Chemistry
Salivary secretion, represented as salivary flow rate (µl/30 minutes/100 g body weight), was significantly reduced by radiation compared with nonirradiated control rats. Transfer of control vector alone also affected salivary flow rate, when examined at 90 days of virus transfer. Combined treatment of HSP25, HSP70i, or amifostine with radiation significantly ameliorated the damage of salivary flow rate (Table 1) , when examined 40 and 90 days after IR. Chemical constituents of saliva such as amylase, total protein, Ca2+, Na+, and ClC, were decreased by radiation, whereas the content of K+ was increased by radiation and lasted for 90 days after radiation. Transfer of HSP25 before IR significantly inhibited these radiation effects; however, no effect was observed by HSP25 on the K+ content. The effect of HSP25 was much stronger at 40 days after radiation than 90 days. HSP70i transfer showed protective effect only on the content of protein at 40 days of radiation, but amifostine showed protective effect on all salivary constituents examined until 90 days of radiation (Table 2) .
Table 1. Salivary Flow Rate Stimulated by Pilocarpine of the Different Groups of Rats
Table 2. Sialochemistry at 40 and 90 Days after Irradiation
Histopathology of Submandibular Gland
Histopathological analysis revealed vacuolization of acinar cells and pyknotic nuclei in irradiated rats at 40 days of radiation. These finding were more apparent at 90 days of radiation, when strong vacuolization of almost all acinar cells, many pyknotic nuclei, and lysis of acini were observed. However, transfer of HSP25 or HSP70i genes to submandibular gland and treatment with amifostine diminished vacuolization of acinar cells and pyknotic nuclei; the lobular structure and the cell membranes appeared to be remarkably normal, and the duct system was also relatively unaffected. Further, radiation-induced fibrosis in periductal connective tissue was decreased by HSP25 or HSP70i transfer and amifostine treatment. A similar protective effect was observed among the groups treated with HSP25, HSP70i, or amifostine. Treatment with viral vector alone did not affect histology of submandibular gland, and transfer of HSP25 or HSP70i alone did not affect either (Figure 3) .
Figure 3. Histopathological analysis of parenchymal changes in damaged salivary gland by radiation. H&E staining of nonirradiated control (ACD), 40 days (ECH) and 90 days (ICL) after 17.5-Gy IR. Nonirradiated control groups are composed of acinar (a), intercalated duct (i), granular convoluted duct (g), and secretory duct (d). At 40 days after 17.5-Gy IR in the vector control group, severe vacuolization (squares), some pyknotic nuclei (arrows), and lysis of acinar or granular convoluted ducts (l) are seen. At 90 days after IR, most of the parenchymal structures in the vector control group are destroyed, with severe fibrosis and some inflammatory infiltration. During the days after IR, HSP25 (F, J)-, HSP70i (G, K)-, and amifostine (H, L)-pretreated salivary glands show clearer lobular structures, more acinar and granular convoluted cells, and fewer vacuoles than vector-transferred IR control (E and I). It was more severe at 90 days than at 40 days after IR. Original magnifications, x400.
Apoptosis of Submandibular Gland
In all gland compartments such as acinar cells, granular convoluted cells, and intercalated ductal cells, apoptotic activity was seen to increase with radiation, when TUNEL assay was performed at 1, 40, and 90 days after radiation. Peak induction of apoptosis was observed at 1 day after radiation and was found to decrease at 40 and 90 days of radiation. Vector control-transferred rats showed more apoptosis by radiation than that of the irradiated normal control group. Transfer of HSP25 or HSP70i to submandibular gland before IR significantly reduced radiation-induced apoptosis, and the degree of reduction was higher in acinar cells than granular convoluted cells and intercalated ductal cells. In addition, the effect of HSP25 was more evident than HSP70i. When treated with amifostine, effects similar to HSP25-transferred rats were found, but to a lesser extent (Figure 4A) . Rats treated with viral vector, HSP25, or HSP70i alone without radiation did not induce any TUNEL-positive cells. Western blotting revealed that rats treated with HSP25 and HSP70i as well as amifostine dramatically reduced radiation-induced cleavage of caspase-3 and PARP when detected at 1 day after radiation (Figure 4B) .
Figure 4. Quantitative analysis of apoptotic cell, active caspase-3, and cleaved PARP. A: Apoptotic index of different cell types (mean ?? SD) in control and irradiated submandibular gland at 1 day, 40 days, and 90 days after IR. •, compared with normal control; , compared with vector transferred control; , compared with vector transferred IR control; and , compared with radiation control denote statistical significance of P < 0.05. B: Immunoblot of active caspase-3 and cleaved PARP in damaged submandibular gland at 1 day after 17.5-Gy IR. Activation of caspase-3 and PARP by radiation was inhibited in HSP25-, HSP70i-, and amifostine-pretreated salivary gland. Representative image of two independent animals from each group is shown.
AQP5 Expression in Submandibular Gland
AQP5 is topographically localized in the apical membranes of acinar cells and stimulates the outflow of water into the acinar lumen. As seen in Figure 5 , AQP5 was well expressed at apical and lateral sides of the plasma membrane without radiation, and treatment with HSP25, HSP70i, and amifostine alone did not affect AQP5 expression. Radiation of 17.5 Gy significantly reduced these expressions from 40 days of radiation, and almost no AQP5 was present at 90 days. However, HSP25 or HSP70i transfer dramatically inhibited AQP5 reduction. The effect of HSP25 was a peak at 40 days of radiation, and the effect of HSP70i was more dominant at 90 days than at 40 days after IR. Amifostine treatment also prevented radiation-induced reduction of AQP5 expression (Figure 5) .
Figure 5. Immunolocalization of AQP5 in irradiated rat salivary gland. A: Immunohistochemical analysis for AQP5 was performed at 40 and 90 days after IR. Nuclei were counterstained with autohematoxylin. ACD: AQP5 is located at apical membrane of secretory cells, and AQP5-positive cells are abundant in nonirradiated salivary glands. E and I: In vector transferred IR control, most of AQP5 activity has disappeared. J: HSP25-transferred submandibular glands have AQP5-positive cells until 90 days after IR. B: Distribution of AQP5 in salivary gland at 40 and 90 days after IR. The graph indicates the positive signal ratio of AQP5 against normal submandibular gland. There were 10 fields (60 x 50 µm2, x1000) per rat in each group. *Significance compared with control group (P < 0.05). Original magnifications, x1000.
Because salivary glands are frequently included in the radiotherapy field for the treatment of head and neck malignancies, their function is rapidly impaired.36,37 Using a rat model in this study, we showed that HSP25 and HSP70i protected radiation-induced submandibular damage and that this protective effect was attributable to inhibition of cell death and restoration of saliva fluid.
HSP25 (or HSP27) has been suggested to protect cells against apoptotic cell death triggered by heat, ionizing radiation, oxidative stress, Fas ligand, and cytotoxic drugs.1-3,23,24 To evaluate the potential of exogenous HSP25 expression, as delivered by adenoviral vectors, a rat salivary gland model with overexpression of HSP25 was used. The histopathological manifestation of radiation damage in the exposed areas of the gland starts relatively late, and no early loss of cells is shown in the major salivary gland. However, early impairment of cellular function (reduction in saliva flow) has been observed and attributed to radiation-induced distortion of signal transduction in the plasma membrane of the secretary cells. In the present study, gene transfer of HSP25 and HSP70i was shown to dramatically prevent radiation-induced damage in the submandibular region. Also, strong vacuolization of acinar cells, many pyknotic nuclei, and lysis of acini by radiation were ameliorated by the transfer of HSP25 or HSP70i, whereas the lobular structure and the cell membranes remained remarkably unchanged, and the duct system was also relatively unaffected by these molecules. Radiation-induced fibrosis in periductal connective tissue was decreased by HSP25 or HSP70i transfer (Figure 3) . To elucidate the mechanisms of this protection, induction of apoptosis was examined. Transfer of HSP25 or HSP70i to the submandibular region dramatically reduced radiation-induced apoptosis of acinar cells, granular convoluted cells, and intercalated ductal cells, and reduction of apoptosis by HSP25 or HSP70i was more robust in acinar cells, suggesting specific protective effect of these molecules on proliferating cells. In fact, cleavages of capase-3 and PARP in submandibular tissue was dramatically reduced by these molecules (Figure 4) . These data showed that HSP25 and HSP70i have a protective effect on radiation-induced submandibular gland damage and that its mechanism involves direct inhibition of radiation-induced apoptosis, especially inhibition of apoptosis of proliferating acinar cells. When proliferating cell nuclear antigen-positive cells were examined by immunohistochemistry, no evidence of increased proliferation by HSP25 or HSP70i was seen (data not shown), indicating that HSP25 and HSP70i did not affect cell proliferation but inhibited radiation-induced apoptosis.
Salivary glands of rat are quite similar to human salivary glands in which salivary flow is rapidly reduced after IR.38 Fluid transport in salivary glands is thought to be osmotically driven in response to transepithelial salt gradients that are generated by ion transport systems localized in the apical and basolateral membranes of the secretary cells. According to the classical two-stage hypothesis,39,40 a primary fluid containing plasma-like electrolyte concentrate is generated by the acinar cells, and the fluid is subsequently modified by solute reabsorption and secretion as it passes along the ductal system, resulting in the final hypotonic solution that enters the oral cavity. Therefore, the principal site of water transport is likely to be the acini with relatively little transepithelial water movement occurring in the ducts. Serous cells are considered to be more radiosensitive than mucous cells because serous secretary granules are rich in transition metals such as Zn2+, Fe2+, and Mn2+, which may leak into the cytoplasm, causing autolysis and cell death.7 In the present study, changes in salivary function were observed at later stages, such as 40 and 90 days after radiation; however, HSP25 or HSP70i transfer before radiation helped maintain the saliva flow rate and saliva chemistry. HSP25 transfer maintained amylase, total protein, Ca2+, Na+, and ClC, contents at the level of nonirradiated control rats, suggesting that HSP25 also affects quality of saliva content. Total salivary protein levels were much higher in all of the groups at 90 days of IR because of increased body weights (1.5- or 2-fold increase of body weight at 90 days when compared with that of the beginning of experiment). We do not know exactly why HSP70i could not protect saliva chemistry; only protein content was protected by HSP70i at 40 days of radiation. Because protective tendency in other factors such as content of amylase, Ca2+, ClC, and Na+ was not observed by HSP70i (Table 2) , protective potency of salivary gland appears to be stronger in HSP25-transfected cells than in HSP70i-transfected cells.
AQP water channels are expressed in a variety of fluid-transporting epithelia, and it is increasingly clear that they are likely to play a significant role in salivary secretion. Several rat exocrine glands have been shown to express AQP5 at the luminal surface of the acinar cells, and they include the lacrimal glands,41,42 the subepithelial glands of the upper airways, and the submandibular and parotid salivary glands. AQP5 is highly expressed in the apical plasma membrane,43 and it is suggested to play a significant role in saliva production on the basis of abundance of the channel. Furthermore, knockout mice lacking AQP5 show markedly depressed rates of salivary secretion.11,44 AQP5 is abundant in the apical domains of the serous acinar cells, secretary canaliculi, and intercalated ductal cells of rats but absent in the mucous acinar cells and striated duct cells.45,46 In our experiment, AQP5 expression was not diminished by radiation when HSP25 or HSP70i was transferred before radiation, suggesting that the protection of acinar cells by these proteins might also involve AQP5 expression (Figure 5) .
In conclusion, using a rat model of radiation-induced salivary hypofunction, we show that the transfer of HSP25 or HSP70i protects salivary gland from radiation. Protective effect of HSP25 was more predominant than HSP70i and was similar to the treatment of amifostine, which has been recently approved for use in prevention of xerostomia in head and neck cancer patients undergoing radiotherapy.23,24 However, controversy remains as to whether amifostine also confers tumor protection.25,26 Therefore, HSP25 may be a useful target for radiation protection, especially as an adjuvant to radiotherapy of head and neck tumors.
【参考文献】
Grdina DJ, Murley JS, Kataoka Y: Radioprotectants: current status and new directions. Oncology 2002, 63(Suppl 2):2-10
Nagler RM, Baum BJ: Prophylactic treatment reduces the severity of xerostomia following radiation therapy for oral cavity cancer. Arch Otolaryngol Head Neck Surg 2003, 129:247-250
Vissink A, Jansma J, Spijkervet FK, Burlage FR, Coppes RP: Oral sequelae of head and neck radiotherapy. Crit Rev Oral Biol Med 2003, 14:199-212
Takagi K, Yamaguchi K, Sakurai T, Asari T, Hashimoto K, Terakawa S: Secretion of saliva in X-irradiated rat submandibular glands. Radiat Res 2003, 159:351-360
Coppes RP, Zeilstra LJ, Vissink A, Konings AW: Sialogogue-related radioprotection of salivary gland function: the degranulation concept revisited. Radiat Res 1997, 148:240-247
Nagler RM: Short- and long-term functional vs morphometrical salivary effects of irradiation in a rodent model. Anticancer Res 1998, 18:315-320
Abok K, Brunk U, Jung B, Ericsson J: Morphologic and histochemical studies on the differing radiosensitivity of ductular and acinar cells of the rat submandibular gland. Virchows Arch B Cell Pathol Incl Mol Pathol 1984, 45:443-460
King LS, Yasui M: Aquaporins and disease: lessons from mice to humans. Trends Endocrinol Metab 2002, 13:355-360
Raina S, Preston GM, Guggino WB, Agre P: Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J Biol Chem 1995, 270:1908-1912
Agre P, Brown D, Nielsen S: Aquaporin water channels: unanswered questions and unresolved controversies. Curr Opin Cell Biol 1995, 7:472-483
Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS: Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J Biol Chem 1999, 274:20071-20074
Feder ME, Hofmann GE: Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol 1999, 61:243-282
Ehrnsperger M, Gaestel M, Buchner J: Analysis of chaperone properties of small Hsp??s. Methods Mol Biol 2000, 99:421-429
Benjamin IJ, McMillan DR: Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 1998, 83:117-132
Amin V, Cumming DV, Coffin RS, Latchman DS: The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress. Neurosci Lett 1995, 200:85-88
Mailhos C, Howard MK, Latchman DS: Heat shock proteins hsp90 and hsp70 protect neuronal cells from thermal stress but not from programmed cell death. J Neurochem 1994, 63:1787-1795
Mosser DD, Martin LH: Induced thermotolerance to apoptosis in a human T lymphocyte cell line. J Cell Physiol 1992, 151:561-570
Samali A, Cotter TG: Heat shock proteins increase resistance to apoptosis. Exp Cell Res 1996, 223:163-170
Amin V, Cumming DV, Latchman DS: Over-expression of heat shock protein 70 protects neuronal cells against both thermal and ischaemic stress but with different efficiencies. Neurosci Lett 1996, 206:45-48
Mailhos C, Howard MK, Latchman DS: Heat shock protects neuronal cells from programmed cell death by apoptosis. Neuroscience 1993, 55:621-627
Uney JB, Anderton BH, Thomas SM: Changes in heat shock protein 70 and ubiquitin mRNA levels in C1300 N2A mouse neuroblastoma cells following treatment with iron. J Neurochem 1993, 60:659-665
Wagstaff MJ, Collaco-Moraes Y, Smith J, de Belleroche JS, Coffin RS, Latchman DS: Protection of neuronal cells from apoptosis by Hsp27 delivered with a herpes simplex virus-based vector. J Biol Chem 1999, 274:5061-5069
Sodicoff M, Conger AD, Trepper P, Pratt NE: Short-term radioprotective effects of WR-2721 on the rat parotid glands. Radiat Res 1978, 75:317-326
Brizel DM, Wasserman TH, Henke M, Strnad V, Rudat V, Monnier A, Eschwege F, Zhang J, Russell L, Oster W, Sauer R: Phase III randomized trial of amifostine as a radioprotector in head and neck cancer. J Clin Oncol 2000, 18:3339-3345
Brizel DM, Overgaard J: Does amifostine have a role in chemoradiation treatment? Lancet Oncol 2003, 4:378-381
Schuchter L, Meropol NJ, Winer EP, Hensley ML, Somerfield MR: Amifostine and chemoradiation therapy: ASCO responds. Lancet Oncol 2003, 4:593
Park SH, Lee SJ, Chung HY, Kim TH, Cho CK, Yoo SY, Lee YS: Inducible heat-shock protein 70 is involved in the radioadaptive response. Radiat Res 2000, 153:318-326
Cho HN, Lee YJ, Cho CK, Lee SJ, Lee YS: Downregulation of ERK2 is essential for the inhibition of radiation-induced cell death in HSP25 overexpressed L929 cells. Cell Death Differ 2002, 9:448-456
Yi MJ, Park SH, Cho HN, Chung H Yong, Kim JI, Cho CK, Lee SJ, Lee YS: Heat-shock protein 25 (Hspb1) regulates manganese superoxide dismutase through activation of Nfkb (NF-kappaB). Radiat Res 2002, 158:641-649
Lee YJ, Cho HN, Jeoung DI, Soh JW, Cho CK, Bae S, Chung HY, Lee SJ, Lee YS: HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase. Free Radic Biol Med 2004, 36:429-444
Lee YJ, Lee DH, Cho CK, Bae S, Jhon GJ, Lee SJ, Soh JW, Lee YS: HSP25 inhibits protein kinase C delta-mediated cell death through direct interaction. J Biol Chem 2005, 280:18108-18119
Lee CT, Park KH, Yanagisawa K, Adachi Y, Ohm JE, Nadaf S, Dikov MM, Curiel DT, Carbone DP: Combination therapy with conditionally replicating adenovirus and replication defective adenovirus. Cancer Res 2004, 64:6660-6665
He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B: A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998, 95:2509-2514
Edholm D, Molin M, Bajak E, Akusjarvi G: Adenovirus vector designed for expression of toxic proteins. J Virol 2001, 75:9579-9584
Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, Newgard CB: Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 1994, 43:161-189
Dreizen S, Brown LR, Handler S, Levy BM: Radiation-induced xerostomia in cancer patients. Effect on salivary and serum electrolytes. Cancer 1976, 38:273-278
Nagler RM, Baum BJ, Fox PC: Acute effects of X irradiation on the function of rat salivary glands. Radiat Res 1993, 136:42-47
Nagler RM: The enigmatic mechanism of irradiation-induced damage to the major salivary glands. Oral Dis 2002, 8:141-146
Cook DI, Van Lennep EW, Roberts ML, Young JA: Secretion by the major salivary glands. Johson LR eds. Physiology of the Gastrointestinal Tract ed 3 1994:pp 1061-1117 Raven, New York
Winston DC, Hennigar RA, Spicer SS, Garrett JR, Schulte BA: Immunohistochemical localization of Na+, K+-ATPase in rodent and human salivary and lacrimal glands. J Histochem Cytochem 1988, 36:1139-1145
Ishida N, Hirai SI, Mita S: Immunolocalization of aquaporin homologs in mouse lacrimal glands. Biochem Biophys Res Commun 1997, 238:891-895
Matsuzaki T, Suzuki T, Koyama H, Tanaka S, Takata K: Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation. Cell Tissue Res 1999, 295:513-521
Poulsen JH, Bundgaard M: Quantitative estimation of the area of luminal and basolateral membranes of rat parotid acinar cells: some physiological applications. Pflueg Arch Eur J Physiol 1994, 429:240-244
Krane CM, Melvin JE, Nguyen HV, Richardson L, Towne JE, Doetschman T, Menon AG: Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation. J Biol Chem 2001, 276:23413-23420
Nielsen S, King LS, Christensen BM, Agre P: Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. Am J Physiol 1997, 273:C1549-C1561
Ishikawa Y, Eguchi T, Skowronski MT, Ishida H: Acetylcholine acts on M3 muscarinic receptors and induces the translocation of aquaporin5 water channel via cytosolic Ca2+ elevation in rat parotid glands. Biochem Biophys Res Commun 1998, 245:835-840
作者单位：From the Laboratories of Radiation Effect* and Molecular Cancer, and the Department of Radiation Oncology,¶ Korea Institute of Radiological and Medical Sciences, Seoul; and the College of Veterinary Medicine and the Department of Nuclear Medicine, Chonnam National University, Gwangju, Korea
【摘要】
Ischemic preconditioning protects steatotic livers against ischemia-reperfusion (I/R) injury, but just how this is achieved is poorly understood. Here, I/R or preconditioning plus I/R was induced in steatotic and nonsteatotic livers followed by investigating the effect of pharmacological treatments that modulate heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs). MAPKs, HSPs, protein kinase C, and transaminase levels were measured after reperfusion. We report that preconditioning increased HSP72 and heme-oxygenase-1 (HO-1) at 6 and 24 hours of reperfusion, respectively. Unlike nonsteatotic livers, steatotic livers benefited from HSP72 activators (geranylgeranylacetone) throughout reperfusion. This protection seemed attributable to HO-1 induction. In steatotic livers, preconditioning and geranylgeranylacetone treatment (which are responsible for HO-1 induction) increased protein kinase C activity. HO-1 activators (cobalt(III) protoporphyrin IX) protected both liver types. Preconditioning reduced p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in HSP72 induction though HO-1 remained unmodified. Like HSP72, both p38 and JNK appeared not to be crucial in preconditioning, and inhibitors of p38 (SB203580) and JNK (SP600125) were less effective against hepatic injury than HO-1 activators. These results provide new data regarding the mechanisms of preconditioning and may pave the way to the development of new pharmacological strategies in liver surgery.
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Steatotic livers tolerate ischemia-reperfusion (I/R) poorly.1,2 Results obtained under normothermic conditions indicate that heat shock preconditioning (whole body hyperthermia) induces a marked expression of heat shock protein 72 (HSP72) and heme-oxygenase-1 (HO-1) in steatotic livers, which protect against hepatic injury.3,4 The possible functions of HSPs in ischemic tissue include repair of damaged proteins, protection against oxidative stress, suppression of pro-inflammatory cytokines, and repair of the ion channel.5,6 However, despite the benefits of heat shock preconditioning, its application in the clinical setting is limited.
In common with heat shock preconditioning, ischemic preconditioning also involves the induction of organ stress to protect steatotic liver against I/R injury.7,8 There is some evidence in isolated hepatocytes and experimental models of perfused liver that mitogen-activated protein kinases (MAPKs) are involved in the protective mechanisms of ischemic preconditioning.9,10 Signaling pathways involved in HSP induction include MAPKs. HSP72 induction includes p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in heart in response to hypoxia.11 HO-1 overexpression by p38 and JNK has been reported in cultures of hepatocytes,12 isolated endothelial cells,13 and experimental models of lung I/R.14 The respective roles of HSPs and MAPKs in inducing the benefits of ischemic preconditioning on I/R injury in steatotic livers remain unclear.
Our experimental study aimed to evaluate whether 1) ischemic preconditioning, through HSP induction, protects steatotic livers against I/R injury and 2) HSP induction by preconditioning is related to MAPK activation. Ischemic preconditioning has been successfully used clinically under normothermic conditions for tumor hepatic resections.15-17 If ischemic preconditioning were understood at the molecular level, protective drugs that achieve similar protection could be developed, avoiding repeated vascular clamping and prolonged surgery.
【关键词】 proteins mitogen-activated steatotic undergoing ischemia-reperfusion
Materials and Methods
Homozygous (obese, Ob) and heterozygous (lean, Ln) Zucker rats (Iffa-Credo, L'Abresle, France), aged 16C18 weeks, were used in the experiments. The animals were anesthetized with ketamine (100 mg/kg) and xylazine (8 mg/kg).7,18 The study followed European Union regulations (Directive 86/609 EEC) on animal experiments. All animals were randomized into groups.
Experimental Design
Protocol 1. HSPs in Ischemic Preconditioning
Protocol 1 is as follows: Effect of preconditioning on HSPs: Group 1: Sham (n = 12). Hepatic helium vessels of Ln and Ob animals (six in each group) were dissected. Group 2: ischemia-reperfusion (I/R) (n = 24). Group 2.1: A group of animals (n = 12, 6 Ln and 6 Ob) were subjected to 60 minutes of partial (70%) hepatic ischemia followed by 6 hours of reperfusion. Group 2.2: A second group of animals (n = 12, 6 Ln and 6 Ob) were subjected to 60 minutes of partial (70%) hepatic ischemia followed by 24 hours of reperfusion.7,18 Group 3: PC (n = 24). Animals were treated as in group 2, but with previous preconditioning induced by 5 minutes ischemia followed by 10 minutes reperfusion.7,18
The Role of NO involved in preconditioning on HSPs was carried out as follows: Group 4: PC plus NAME (n = 24). As in group 3, but with N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma Chemical, St. Louis, MO.), nonspecific nitric oxide synthase (NOS) inhibitor (10 mg/kg i.v.), 5 minutes before preconditioning.7,18 Group 5: PC plus NAME plus Arg (n = 24). As in group 3, but with L-NAME (10 mg/kg i.v.) 5 minutes before preconditioning and L-arginine (Sigma Chemical) (100 mg/kg i.v.) immediately before L-NAME.7,18,19 Group 6: PC plus NNA (n = 24). As in group 3, but with N-nitro-L-arginine (NNA; Sigma Chemical), nonspecific NOS inhibitor (10 mg/kg i.v.), 5 minutes before preconditioning.20 Group 7: PC plus AG (n = 24). As in group 3, but treated with aminoguanidine hemisulfate (AG; Sigma Chemical), an inducible NOS inhibitor (10 mg/kg, i.v.) 5 minutes before preconditioning.18
The Role of HSP72 and HO-1 in hepatic injury was as follows: Group 8: PC plus HSP72inh (n = 24). As in group 3, but with Quercetin (Sigma Chemical), a Hsp72 inhibitor (100 mg/kg, i.p.), 2 hours before preconditioning.21,22 Group 9: PC plus HO-1inh (n = 24). As in group 3 but with zinc(II) protoporphyrin IX (ZnPP) (Oxisresearch, Portland, Oregon), a HO-1 inhibitor (20 mg/kg, i.p.), 24 hours before preconditioning.23 Group 10: I/R plus HSP72act (n = 24). As in group 2, but with geranylgeranylacetone (GGA; Eisai Co., Tokyo, Japan), a Hsp72 activator (200 mg/kg, orally), 24 hours before ischemia.24 Group 11: I/R plus HSP72act plus HO-1inh (n = 6). I/R was induced in Ob animals (as in group 2.2), but with GGA, a HSP72 activator (200 mg/kg, orally) and ZnPP, a HO-1 inhibitor (20 mg/kg, i.p.), 24 hours before ischemia.23,24 Group 12: I/R plus HO-1act (n = 24). As in group 2.2, but with cobalt(III) protoporphyrin IX (Alexis Biochemicals, Lausen, Switzerland), a HO-1 activator at dose of 5 mg/kg, i.p. (n = 12, 6 Ln and 6 Ob) and 10 mg/kg, i.p. (n = 12, 6 Ln and 6 Ob), 24 hours before ischemia.23
Following reperfusion, liver and plasma samples corresponding to protocol 1 were collected. In the sham group, liver and plasma samples were obtained 24 hours after the dissection of helium vessels. HSPs (HSP90, HSP72, and HO-1) and protein kinase C (PKC) were analyzed in liver. Hepatic injury was evaluated by determination of transaminases. Liver was also analyzed histologically.
Protocol 2. Relation between MAPKs and HSPs
Protocol 2 is as follows: Effect of preconditioning on MAPKs. Group 13: I/R30 (n = 12, 6 Ln and 6 Ob): Animals were subjected to 60 minutes of partial (70%) hepatic ischemia (as in group 2), and liver samples were obtained 30 minutes after reperfusion (as opposed to 6 and 24 hours) to evaluate p38 and JNK.25,26 Group 14: PC30 (n = 12): As in group 13, but with previous preconditioning induced by 5 minutes ischemia followed by 10 minutes reperfusion.
Effect of MAPKs on HSPs and hepatic injury: Group 15: PC plus p38/JNKactiv (n = 24): As in group 3, but with anisomycin (Sigma Chemical), an activator of both p38 and JNK (0.1 mg/kg, i.p.), 24 hours before preconditioning.27 Group 16: I/R plus p38inh (n = 24): As in group 2, but with SB203580 (Sigma Chemical), a p38 inhibitor (1 mg/kg i.p), 24 hours before ischemia27 Group 17: I/R plus JNK inh (n = 24): As in group 2, but with SP600125 (Sigma Chemical), a JNK inhibitor (6 mg/kg s.c.), 1 hour before ischemia.28 Following reperfusion, liver and plasma samples were collected. MAPKs (p38 and JNK) and HSPs (HSP72 and HO-1) in liver were analyzed. Transaminases in plasma were measured.
Biochemical Determinations
Transaminase Assay
Plasma alanine aminotransferase (ALT) was measured using standard procedures.
HSPs and MAPKs Analyses
Liver tissue was homogenized, and proteins were separated by sodium docecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.29 Western blotting was performed with primary antibodies against HSP72, HSP90, and HO-1 (BD Transduction Laboratories, San Jose, CA) and p38, JNK, phospho-p38, and phospho-JNK (Cell Signaling Technology Inc., Beverly, MA). Positive controls for HSPs and MAPKs were obtained from Stressgen Biotechnologies (Victoria, BC, Canada) and Cell Signaling Technology Inc., respectively. Signals were detected by enhanced chemiluminescence and quantified by scanning densitometry. All signals were standardized to the corresponding Ponceau S staining.
PKC Assay
Frozen liver samples were homogenized as described previously.30 Cytosolic and membrane extracts were then used in the biochemical assay of PKC activity and Western blotting. Total PKC activity in the membrane and cytosolic extracts were measured using an enzyme-linked immunosorbent assay kit from Stressgen Bioreagents. For Western blot assay, aliquots of the cytosol and membrane fractions were processed following a similar protocol to that described above for HSPs and MAPKs. Primary antibodies against PKC isozymes (, ß, , , and ) were obtained from BD Transduction Laboratories. Rat cerebrum lysate was used as a positive control (BD Transduction Laboratories).
To appraise the severity of hepatic injury, hematoxylin and eosin-stained sections were evaluated using a point-counting method on an ordinal scale.7
Data are expressed as means ?? SE and are compared statistically by variance analysis, followed by Student-Newman-Keuls. P < 0.05 was considered significant.
Role of HSPs in Inducing the Benefits of Preconditioning for Hepatic I/R Injury
Effect of Preconditioning on HSPs
HSP90 was unchanged in all groups (data not shown). However, at 6 hours of reperfusion, increased HSP72 levels were seen in both steatotic and nonsteatotic livers as a consequence of I/R (Figure 1A) , and similar results were observed at 24 hours (data not shown). Figure 1B shows HO-1 levels at 6 hours (I/R6 hours) and 24 hours (I/R24 hours) of reperfusion. HO-1 was only present at 24 hours of reperfusion (I/R 24 hours), but HO-1 levels were significantly higher in steatotic livers. Preconditioning (PC) increased HSP72 and HO-1 levels in both liver types more than in the I/R group (Figure 1, A and B) .
Figure 1. Effect of ischemic preconditioning on HSP72 (A) and HO-1 (B). Representative Western blots of three similar experiments at the top and densitometric analysis (area x density of the band) at the bottom. Steatotic livers had higher HO-1 levels compared with the nonsteatotic livers (#, P < 0.05). *, P < 0.05 versus sham; +, P < 0.05 versus I/R.
HSPs and Hepatic Injury
At 6 hours of reperfusion, HSP72 inhibition (PC plus HSP72inh) increased hepatic injury in both liver types but did not completely revert the effectiveness of preconditioning, because ALT levels did not rise as much as in the I/R group (Figure 2A) . HO-1 inhibition (PC plus HO-1inh) did not modify the benefits of preconditioning (data not shown). At 24 hours of reperfusion (Figure 2B) , HSP72 inhibition had no effect on the benefits of preconditioning. However, HO-1 inhibition (PC plus HO-1inh) abolished the protection conferred by preconditioning, resulting in transaminase levels comparable to those in the I/R group (see Figure 2B ). The histological results were consistent with the biochemical parameters of hepatic injury. At 24 hours of reperfusion, nonsteatotic livers corresponding to I/R and PC plus HO-1inh groups showed multifocal areas of moderate coagulative necrosis and neutrophil infiltration, randomly distributed throughout the parenchyma (Figure 3A) ; in steatotic livers, extensive and confluent areas of coagulative necrosis with neutrophil infiltration were found (Figure 3B) . By contrast, PC reduced the extent and number of necrosis areas in both liver types compared with the results obtained in the I/R group. In nonsteatotic livers (Figure 3C) , these areas were mainly of incipient necrosis; in steatotic livers (Figure 3D) , patchy areas of incipient hepatocyte necrosis and scattered multifocal areas of coagulative necrosis were observed. The percentage of grade 3 necrosis at 24 hours of reperfusion is shown in Figure 4B .
Figure 2. Role of HSP72 and HO-1 on hepatic I/R injury at 6 hours (A) and 24 hours (B) of reperfusion. *, P < 0.05 versus sham; +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
Figure 3. Histological lesions in liver after 24 hours of reperfusion. PC plus HO-1inh (A, Ln; B, Ob). A: Small area of coagulative hepatic necrosis (arrows) with neutrophil infiltration. B: Widespread coagulative hepatic necrosis with neutrophil infiltration (arrows). PC (C, Ln; D, Ob). C: Irregular area of incipient necrosis (asterisk). D: Small area of coagulative hepatic necrosis (arrows) with neutrophil infiltration. I/R plus HO-1act (E, Ln; F, Ob). Histological lesions similar to the PC group.
Figure 4. Percentage of grade 3 necrosis resulting from pharmacological modulation of HSPs and MAPKs. +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
Effect of NO on Hepatic Injury and HSPs
At 6 hours of reperfusion, NO synthesis inhibition with L-NAME (PC plus NAME) or L-NNA (PC plus NNA) abolished the benefits of preconditioning on the biochemical and histological parameters of hepatic injury (Figure 5, A and B , respectively) in both liver types. The effects of L-NAME were reverted with previous L-arginine addition (PC plus NAME plus Arg). Inducible NOS inhibition in the preconditioned group (PC plus AG) did not modify the benefits of preconditioning on transaminase and grade 3 necrosis (see Figure 5 ). Similar results were observed at 24 hours of reperfusion (data not shown). In regards to HSPs, NO synthesis inhibition with L-NAME in preconditioned group (PC plus NAME) had no effect on either HSP72 (Figure 1A) or HO-1 (data not shown). Similar results were obtained with the others NOS inhibitors used, L-NNA and AG. These results confirm the data previously reported18,31,32 on the role of NO (derived mainly from constitutive NOS) in the benefits of preconditioning on hepatic I/R injury. However, it appears unlike that the protective mechanisms of NO could be dependent on HSP.
Figure 5. Role of NO in steatotic livers undergoing I/R on ALT (A) and necrosis (B). +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
Effect of HSP Inductors on Hepatic Injury and HSPs
Treatment with HSP72 activators, such as GGA (I/R plus HSP72 act), protected against hepatic I/R injury in both liver types at 6 hours of reperfusion (Figures 2A and 4A) . However, at 24 hours of reperfusion (Figures 2B and 4B) , the benefits of GGA were only found in steatotic livers. We then evaluated the effect of GGA on HSP72 and HO-1. GGA pretreatment (I/R plus HSP72act) increased HSP72, but no significant differences in HSP72 levels were found between the two liver types (Figure 6A) . Interestingly, GGA pretreatment (I/R plus HSP72act) increased HO-1 levels in steatotic livers (Figure 6B) but not in nonsteatotic ones. HO-1 inhibition in steatotic livers pretreated with GGA (I/R plus HSP72act plus HO-1inh) abolished the benefits of GGA (I/R plus HSP72act) for hepatic I/R injury. Thus, at 24 hours of reperfusion ALT levels of 2398.3 ?? 150.4 and 570.9 ?? 52.5 were recorded in the I/R plus HSP72act plus HO-1inh and I/R plus HSP72act groups, respectively), which points to the importance of HO-1 in inducing benefits of GGA. Therefore, we evaluated the effect of HO-1 activators on hepatic I/R injury. In steatotic livers, pretreatment with HO-1 activators at a 5 mg/kg dose reduced transaminase levels; in nonsteatotic livers, a 10 mg/kg dose was needed to ameliorate the I/R injury (Figure 2B) . The administration of HO-1 activator (5 and 10 mg/kg in steatotic and nonsteatotic livers, respectively) (Figure 3, E and F) resulted in histological finding similar to those observed in the PC group (Figures 3, C and D) . The percentage of grade 3 necrosis is shown in Figure 4B .
Figure 6. Effect of HSP72 activator on HSP72 (A) and HO-1 (B). Representative Western blot of three similar experiments at the top and densitometric analysis (area x density of the band) at the bottom. Steatotic livers had higher HO-1 levels compared with the nonsteatotic livers (#, P < 0.05). +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
PKC and HO-1
HO-1 induction by PKC has been previously reported in various cell types.33,34 Our results indicate that both preconditioning and GGA were responsible for HO-1 induction in steatotic livers. We then sought to ascertain whether preconditioning and GGA might modify PKC activity. It has been widely reported that the translocation of the PKC isoenzymes from cytosol to the membrane fraction is a key step in the activation of PKC.35 Analysis of the specific PKC isoenzymes in both liver types revealed that, in liver preconditioned or pretreated with GGA, PKC isoforms (, ß, , and ) in membrane fraction (mPKC) remained unmodified (Figure 7A) and similar results were obtained for cytosolic fraction (cPKC) (data not shown). However, this was not the case for PKC. In steatotic livers, preconditioning and GGA pretreatment (which increased HO-1) did not modified PKC in cytosolic fraction (cPKC) but increased PKC in the membrane resulting in a higher PKC membrane/cytosol ratio (see Figure 7B ). PKC membrane/cytosol ratio has been considered as an index of PKC activation.36 Furthermore, both procedures (preconditioning and GGA) increased mPKC activity from steatotic livers (Figure 7B) . In nonsteatotic livers, preconditioning (which increased HO-1) resulted in high levels of mPKC activity, whereas in nonsteatotic livers, GGA pretreatment (which was unable to induce HO-1) had no effect on mPKC activity (see Figure 7B ). Cytosolic PKC activity remained unaltered in all groups (data not shown). As previously reported,30,36 in all of the groups of study, PKC activity in steatotic livers was higher than in nonsteatotic ones (Figure 7B) .
Figure 7. Effect of preconditioning and GGA (HSP72 activator) on PKC, -ß, -, and - (A) and PKC (B). A: Analysis of PKC, -ß, -, and - protein expression in membrane fraction (mPKC) by Western blot. B: Analysis of PKC in membrane and cytosolic fractions (mPKC and cPKC, respectively) by Western blot and densitometric analysis for mPKC (area x density of the band), relative densities of the PKC bands in the membrane fraction compared with the cytosolic fraction (PKC membrane/cytosol ratio) and biochemical assay of PKC activity (the data represent the -fold change versus sham). Western blot of three similar experiments at the top and densitometric analysis (area x density of the band) at the bottom. Steatotic livers had higher HO-1 levels compared with the nonsteatotic livers (#, P < 0.05). *, P < 0.05 versus sham. +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
Role of MAPKs in Inducing the Benefits of Preconditioning on HSP and Hepatic Injury
Effect of Preconditioning on MAPKs
MAPKs are activated by dual phosphorylation on tyrosine and threonine in response to extracellular stimuli.25 Both p38 and JNK (Figure 8, A and B , respectively) were notably phosphorylated (Pp38 and PJNK) at 30 minutes of reperfusion (I/R30 group). Preconditioning reduced Pp38 and PJNK in both liver types. Total p38 and JNK were unchanged in all groups (data not shown).
Figure 8. Effect of ischemic preconditioning on Pp38 (A) and PJNK (B). Representative Western blot of three similar experiments at the top and densitometric analysis (area x density of the band) of Pp38 and PJNK at the bottom. *, P < 0.05 versus sham; +, P < 0.05 versus I/R.
Effect of MAPKs on HSPs
The pretreatment with p38 or JNK inhibitors as well as p38/JNK activators modified HSP72 (see Figure 9A for the HSP72 results obtained with p38 inhibitor). Thus, HSP72 levels in I/R plus p38inh were significantly higher than those observed in I/R group. However, the various pharmacological treatments aimed at activating or inhibiting p38 and JNK had no effect on HO-1 (see Figure 9B for the HO-1 results obtained with JNK inhibitor). Thus, HO-1 levels in I/R plus JNKinh were similar to those observed in the I/R group.
Figure 9. Effect of MAPKs on HSP72 (A) and HO-1 (B). Representative Western blot of three similar experiments at the top and densitometric analysis (area x density of the band) at the bottom. Steatotic livers had higher HO-1 levels compared with the nonsteatotic livers (#, P < 0.05). +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
MAPKs and Hepatic Injury
Treatment with p38/JNK activators in the preconditioned group (PC plus p38/JNKact) increased transaminase levels (Figure 10) and grade 3 necrosis (Figure 4) but did not completely revert the benefits of preconditioning. MAPK inhibition (I/R plus p38inh and I/R plus JNKinh) reduced hepatic I/R injury in both liver types. In addition, the benefits resulting from MAPK inhibition were not as great as those obtained with HO-1 activators (Figures 2 and 10) .
Figure 10. Role of p38 and JNK on hepatic I/R injury. *, P < 0.05 versus sham; +, P < 0.05 versus I/R; ??, P < 0.05 versus PC.
Few studies have sought to evaluate the role of HSP72 in both steatotic and nonsteatotic livers undergoing I/R.3 The authors investigated the role of heat shock preconditioning under warm ischemia and revealed that, in steatotic livers, the maximum accumulation of HSP72 after heat exposure occurs earlier and is weaker than in nonsteatotic ones.3 Here, the induction of ischemic preconditioning resulted in the same response, in HSP72 levels, in both liver types. Our results indicate that the increase in HSP72 caused by preconditioning protected steatotic and nonsteatotic livers at 6 hours of reperfusion, whereas HO-1 induction seems essential for keeping both liver types protected throughout reperfusion. In addition, our results provide information concerning the action mechanisms of HSP activators in steatotic and nonsteatotic livers undergoing I/R and on their potential effectiveness in liver surgery.
The induction of HSP72 by GGA protected cultured gastric mucosal cells against ethanol-induced injury, and GGA has been widely used in clinical practice as an antiulcer drug for over 15 years.37-41 In liver surgery, GGA suppressed I/R injury in liver transplantation from nonsteatotic grafts.38,40,41 Our results indicate that, although pretreatment with HSP72 activators did protect nonsteatotic livers at 6 hours of reperfusion, this protection was transient. However, the pharmacological induction of HSP72 effectively protected steatotic livers against warm I/R injury for extended reperfusion periods.
Evidence from isolated hepatocytes suggests that GGA induces much higher levels of HSP72 in liver under stress conditions, such as ethanol or H2O2.37,40,42 Under our conditions, GGA induced similar HSP72 levels in both liver types. GGA increased HO-1 levels in steatotic livers but did not have the same effect in nonsteatotic livers. Thus, to determine whether the benefits of GGA in steatotic livers undergoing I/R might be explained by its effect on HO-1, we inhibited the action of HO-1 in steatotic livers pre-treated with GGA and found that, under these conditions the benefits of GGA were abolished. Similarly, it is reasonable to assume that the ineffectiveness of GGA in nonsteatotic livers is due to the fact that it fails to induce HO-1.
Our results reveal the potency of preconditioning and treatment with GGA in the activation of HO-1 and PKC in steatotic livers undergoing I/R. Our experiments based on the characterization of the PKC isoforms taking part in preconditioning and GGA signaling in steatotic livers have revealed that the classic PKCs (, ß, and ), which require Ca2+ for their activation,43 are not involved. However, it seems not to be the case for PKC (Ca2+-independent). Although further investigations on ischemic preconditioning and GGA pretreatment are required to ascertain whether PKC regulates HO-1 expression in steatotic livers, a potential relationship between PKC and HO-1 should not be ruled out. In fact, the activation of PKC has been reported as being involved in the signaling pathways of preconditioning in myocardium and isolated hepatocytes,44,45 and data obtained in isolated-perfused heart indicate that GGA activates PKC.46 PKC has been reported as being responsible for HO-1 induction in different cell types.33,34
Studies examining the benefits of HO-1 in hepatic I/R injury have focused primarily on nonsteatotic livers47,48 or steatotic livers.4,23 To the best of our knowledge, there are no studies in hepatic I/R investigating the role of HO-1 in both liver types. Our results indicate that HO-1 activators, such as cobalt(III) protoporphyrin IX, might protect both liver types against warm I/R injury and, consequently, reduce the inherent risk of surgery with steatotic liver. However, a lower dose of HO-1 activator was required to protect steatotic livers effectively. Given these results, the fact that steatotic livers undergoing I/R showed higher HO-1 levels than nonsteatotic livers and the capacity of GGA to induce HO-1 in steatotic liver, steatotic livers might well be more predisposed to overexpressing HO-1. This might be explained by the increased reactive oxygen species generated in steatotic livers due to I/R7,18 and/or the increased PKC activity observed in steatotic livers compared with nonsteatotic ones. In fact, the induction of HO-1 could be a response to oxidative stress,49,50 and HO-1 induction has been associated with high PKC activity.33,34 However, further studies are required to provide a full explanation.
Exogenous NO supplementation protects liver from hepatotoxicity and oxidative stress inducing HSP72 or HO-1 overexpression.51,52 However, our results suggest that endogenous NO generated by preconditioning is not responsible for increased HSP. In fact, the mechanisms involved in the beneficial effects of the two sources of NO might well be different.53 Given the benefits of MAPK activation caused by preconditioning in hepatocytes and experimental models of perfused liver,9,10 and the relationship between MAPKs and HSPs,11-14 we posited that the increased HSP levels caused by preconditioning were mediated by MAPK activation. However, our results obtained with the pharmacological modulation of MAPKs, are in line with those reported in a study in transplantation from steatotic liver grafts indicating that JNK activation might be harmful to hepatocytes after reperfusion.54 In addition, under the conditions reported here, the levels of both p38 and JNK fell in both liver types following preconditioning. These apparently contradictory data on MAPKs in liver preconditioning have also been reported for the heart.55 Thus, apart from species differences, or differences between in vivo and in vitro models, specific MAPK isoforms might play a role. For example, in the case of p38, studies in heart indicate that while p38 isoform activation is believed to accelerate the death pathway, the p38ß pathway may be antiapoptotic.55 Similarly, the differential activation of JNK isoenzymes could result in different functional roles.56 In addition, the importance of subcellular MAPKs should be considered, because ischemic preconditioning increased myocardial p38 activity in the cytosolic but not the nuclear fraction.56
To the best of our knowledge, no studies have established the effect of MAPKs on HO-1 induction in steatotic livers undergoing I/R. Our results suggest that HO-1 induction caused by preconditioning is not dependent on MAPKs. Indeed, preconditioning reduced MAPKs, which in turn increased HSP72 levels. Like HSP72, MAPKs do not seem to play a crucial role in the protective mechanisms of preconditioning. However, our results suggest that this does not exclude the potential benefits of MAPK inhibitor pretreatment.
Some of the inhibitors used in the present study, such as ZnPP and SB203580, were given 24 hours before ischemia, which may cast doubt on the efficacy of these drugs. However, in several experimental models of hepatic I/R,23,48,57 the pretreatment dose of ZnPP was given 24 hours before liver surgery, at a dose of 1.5 mg/kg48,57 or 20 mg/kg.23 Under our conditions, control experiments indicated that the effective dose of ZnPP (which inhibited HO-1) was 20 mg/kg. Although the exact half-life in the circulation of ZnPP is unknown,58 the plasma ZnPP concentration remained elevated for 2C3 days after a single dose of 25 mg/kg i.p.59 In regards to SB203580, the elimination half-life of this drug (2.1 mg/kg i.v.) was 3.5 hours.60 However, the data reported on SB203580, which have been mainly focused in myocardial I/R,27,61,62 indicate that the pretreatment dose was 24 hours at 1 mg/kg i.p.27,61 or 2.5 mg/kg.62 Under our conditions, SB203580 (1 mg/kg i.p. 24 hours before surgery) was enough to prevent phospho-p38 up-regulation. Moreover, the possibility that some of the effects ascribed to a particular inhibitor and pathway might be related to adaptation of other systems should not be ruled out. In addition to HO-1, ZnPP inhibits all HO activity mediated by HO-2 and HO-3.63,64 Similarly, protein kinase Raf and particular isoforms of JNK65,66 could be modified by SB20380, albeit less potently than p38.
The results of the present study point to a new pathway in the protective mechanisms of PC, which is not dependent on NO. The MAPKs-HSP72 pathway is less important than HO-1 in terms of the protection conferred by preconditioning throughout reperfusion. The results obtained with GGA, a HSP72 activator, indicate differences between the two liver types. GGA may be effective in the presence of steatosis, but its protection of nonsteatotic livers was transient. Both, preconditioning and GGA increased PKC activity in steatotic livers. Future investigations will be required to elucidate whether PKC is responsible for HO-1 induction in steatotic livers. Activators of HO-1 such as cobalt(III) protoporphyrin IX could well become an important pharmacological strategy for protecting both steatotic and nonsteatotic livers against I/R injury, but the effective dose may differ between the two liver types. Unlike HO-1 pretreatment, MAPK inhibitors (SB203580 and SP600125), at the same dose, protected against hepatic I/R injury, but their benefits in both liver types were not as great as those obtained with HO-1 activator pretreatment. These results may open the door to new pharmacological strategies for the effective protection of both steatotic and nonsteatotic livers from I/R injury.
We thank Robin Rycroft at the Language Advisory Service of the University of Barcelona for revising the English text. We are also grateful to Emma Puig-Oriol and Llorenç Quint?, of the Unit of Epidemiology and Biostatistics, University of Barcelona, for help and advice in conducting the statistical analyses and to Esai Co. for the generous gift of GGA.
【参考文献】
McCormack L, Clavien PA: Understanding the meaning of fat in the liver. Liver Transpl 2005, 11:137-139
Imber CJ, St Peter SD, Handa A, Friend PJ: Hepatic steatosis and its relationship to transplantation. Liver Transpl 2002, 8:415-423
Yamagami K, Yamamoto Y, Kume M, Kimoto S, Yamamoto H, Ozaki N, Yamamoto M, Shimahara Y, Toyokuni S, Yamaoka Y: Heat shock preconditioning ameliorates liver injury following normothermic ischemia-reperfusion in steatotic rat livers. J Surg Res 1998, 79:47-53
Mokuno Y, Berthiaume F, Tompkins RG, Balis UJ, Yarmush ML: Technique for expanding the donor liver pool: heat shock preconditioning in a rat fatty liver model. Liver Transplant 2004, 10:264-272
Morimoto RI, Santoro MG: Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nat Biotechnol 1998, 16:833-838
Chi NC, Karliner JS: Molecular determinants of responses to myocardial ischemia/reperfusion injury: focus on hypoxia-inducible and heat shock factors. Cardiovasc Res 2004, 61:437-447
Serafin A, Rosello-Catafau J, Prats N, Xaus C, Gelpi E, Peralta C: Ischemic preconditioning increases the tolerance of fatty liver to hepatic ischemia-reperfusion injury in the rat. Am J Pathol 2002, 161:587-601
Koti RS, Yang W, Glantzounis G, Quaglia A, Davidson BR, Seifalian AM: Effect of ischaemic preconditioning on hepatic oxygenation, microcirculation and function in a rat model of moderate hepatic steatosis. Clin Sci 2005, 108:55-63
Carini R, Grazia De, Cesaris M, Splendore R, Domenicotti C, Nitti MP, Pronzato MA, Albano E: Signal pathway responsible for hepatocyte preconditioning by nitric oxide. Free Radic Biol Med 2003, 34:1047-1055
Schauer RJ, Gerbes AL, Vonier D, op den Winkel M, Fraunberger P, Bilzer M: Induction of cellular resistance against Kupffer cell-derived oxidant stress: a novel concept of hepatoprotection by ischemic preconditioning. Hepatology 2003, 37:286-295
Rafiee P, Shi Y, Pritchard KA, Jr, Ogawa H, Eis AL, Komorowski RA, Fitzpatrick C, Tweddell JS, Litwin SB, Mussatto K, Jaquiss RD, Baker JE: Cellular redistribution of inducible Hsp70 protein in the human and rabbit heart in response to the stress of chronic hypoxia: role of protein kinases. J Biol Chem 2003, 278:43636-43644
Kietzmann T, Samoylenko A, Immenschuh S: Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes. J Biol Chem 2003, 278:17927-17936
Bulger EM, Garcia I, Maier RV: Induction of heme-oxygenase 1 inhibits endothelial cell activation by endotoxin and oxidant stress. Surgery 2003, 134:146-152
Zhang X, Bedard EL, Potter R, Zhong R, Alam J, Choi AM, Lee PJ: Mitogen-activated protein kinases regulate HO-1 gene transcription after ischemia-reperfusion lung injury. Am J Physiol 2002, 283:L815-L829
Clavien PA, Selzner M, Rudiger HA, Graf R, Kadry Z, Rousson V, Jochum W: A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning. Ann Surg 2003, 238:843-852
Nuzzo G, Giuliante F, Vellone M, De Cosmo G, Ardito F, Murazio M, D??Acapito F, Giovannini I: Pedicle clamping with ischemic preconditioning in liver resection. Liver Transpl 2004, 10:S53-S57
Chouker A, Schachtner T, Schauer R, Dugas M, Lohe F, Martignoni A, Pollwein B, Niklas M, Rau HG, Jauch KW, Peter K, Thiel M: Effects of Pringle manoeuvre and ischaemic preconditioning on haemodynamic stability in patients undergoing elective hepatectomy: a randomized trial. Br J Anaesth 2004, 93:204-211
Serafin A, Rosello-Catafau J, Prats N, Gelpi E, Rodes J, Peralta C: Ischemic preconditioning affects interleukin release in fatty livers of rats undergoing ischemia/reperfusion. Hepatology 2004, 39:688-698
Koti RS, Yang W, Dashwood MR, Davidson BR, Seifalian AM: Effect of ischemic preconditioning on hepatic microcirculation and function in a rat model of ischemia reperfusion injury. Liver Transpl 2002, 8:1182-1191
Kobayashi H, Nonami T, Kurokawa T, Takeuchi Y, Harada A, Nakao A, Takagi H: Role of endogenous nitric oxide in ischemia-reperfusion injury in rat liver. J Surg Res 1995, 59:772-779
Kelly KJ, Baird NR, Greene AL: Induction of stress response proteins and experimental renal ischemia/reperfusion. Kidney Int 2001, 59:1798-1802
Shoskes DA: Effect of bioflavonoids Quercetin and Curcumin on ischemic renal injury: A new class of renoprotective agents. Transplantation 1998, 66:147-152
Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD, Zhao D, Zaky J, Melinek J, Lassman CR, Kolls JK, Alam J, Ritter T, Volk HD, Farmer DG, Ghobrial RM, Busuttil RW, Kupiec-Weglinski JW: Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 1999, 104:1631-1639
Ooie T, Takahashi N, Saikawa T, Nawata T, Arikawa M, Yamanaka K, Hara M, Shimada T, Sakata T: Single oral dose of Geranylgeranylacetone induces Heat-shock protein 72 and renders protection against ischemia/reperfusion injury in rat heart. Circulation 2001, 104:1837-1843
Kobayashi M, Takeyoshi I, Yoshinari D, Matsumoto K, Morishita Y: P38 mitogen-activated protein kinase inhibition attenuates ischemia-reperfusion injury of the rat liver. Surgery 2002, 131:344-349
Yoshinari D, Takeyoshi I, Kobayashi M, Koyama T, Iijima K, Ohwada S, Matsumoto K, Morishita Y: Effects of a p38 mitogen-activated protein kinase inhibitor as an additive to University of Wisconsin solution on reperfusion injury in liver transplantation. Transplantation 2001, 72:22-27
Zhao TC, Taher MM, Valerie KC, Kukreja RC: p38 triggers late preconditioning elicited by anisomycin in heart: involvement of NF-B and iNOS. Circ Res 2001, 89:915-922
Schwabe RF, Bradham CA, Uehara T, Hatano E, Bennett BL, Schoonhoven R, Brenner DA: c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration. Hepatology 2003, 37:824-832
Uchinami H, Yamamoto Y, Kume M, Yonezawa K, Ishikawa Y, Taura K, Nakajima A, Hata K, Yamaoka Y: Effect of heat shock preconditioning on NF-B/I-B pathway during I/R injury of the rat liver. Am J Physiol 2002, 282:G962-G971
Qu X, Seale JP, Donnelly R: Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker ratsCeffects of feeding. J Endocrinol 1999, 162:207-214
Peralta C, Closa D, Hotter G, Gelpi E, Prats N, Rosello-Catafau J: Liver ischemic preconditioning is mediated by the inhibitory action of nitric oxide on endothelin. Biochem Biophys Res Commun 1996, 229:264-270
Peralta C, Hotter G, Closa D, Prats N, Xaus C, Gelpi E, Rosello-Catafau J: The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by activation of adenosine A2 receptors. Hepatology 1999, 29:126-132
Anwar AA, Li FY, Leake DS, Ishii T, Mann GE, Siow RC: Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2. Free Radic Biol Med 2005, 39:227-236
Terry CM, Clikeman JA, Hoidal JR, Callahan KS: TNF-alpha and IL-1alpha induce heme oxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells. Am J Physiol 1999, 276:H1493-H1501
Beuers U, Throckmorton DC, Anderson MS, Isales CM, Thasler W, Kullak-Ublick GA, Sauter G, Koebe HG, Paumgartner G, Boyer JL: Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996, 110:1553-1563
Samuel VT, Liu ZX, Qu X, Elder BD, Bilz S, Befroy D, Romanelli AJ, Shulman GI: Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease. J Biol Chem 2004, 279:32345-32353
Ikeyama S, Kusumoto K, Miyake H, Rokutan K, Tashiro S: A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol. J Hepatol 2001, 35:53-61
Fudaba Y, Tashiro H, Ohdan H, Miyata Y, Shibata S, Shintaku S, Nishihara M, Ito H, Fukuda Y, Asahara T, Dohi K: Prevention of warm ischemic injury in rat liver transplantation by geranylgeranylacetone. Transplant Proc 2000, 32:1615-1616
Hirota K, Nakamura H, Arai T, Ishii H, Bai J, Itoh T, Fukuda K, Yodoi J: Geranylgeranylacetone enhances expression of thioredoxin and suppresses ethanol-induced cytotoxicity in cultured hepatocytes. Biochem Biophys Res Commun 2000, 275:825-830
Fudaba Y, Tashiro H, Ohdan H, Miyata Y, Shibata S, Shintaku S, Nishihara M, Asahara T, Ito H, Fukuda Y, Dohi K: Efficacy of HSP72 induction in rat liver by orally administered geranylgeranylacetone. Transpl Int 2000, 13:S278-S281
Fudaba Y, Ohdan H, Tashiro H, Ito H, Fukuda Y, Dohi K, Asahara T: Geranylgeranylacetone, a heat shock protein inducer, prevents primary graft nonfunction in rat liver transplantation. Transplantation 2001, 72:184-189
Yamagami K, Yamamoto Y, Ishikawa Y, Yonezawa K, Toyokuni S, Yamaoka Y: Effects of geranyl-geranyl-acetone administration before heat shock preconditioning for conferring tolerance against ischemia-reperfusion injury in rat livers. J Lab Clin Med 2000, 135:465-475
Newton AC: Protein kinase C: structure, function, and regulation. J Biol Chem 1995, 270:28495-28498
Kawamura S, Yoshida K, Miura T, Mizukami Y, Matsuzaki M: Ischemic preconditioning translocates PKC- and -, which mediate functional protection in isolated rat heart. Am J Physiol 1998, 275:H2266-H2271
Carini R, De Cesaris MG, Splendore R, Vay D, Domenicotti C, Nitti MP, Paola D, Pronzato MA, Albano E: Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes. Hepatology 2001, 33:131-139
Yamanaka K, Takahashi N, Ooie T, Kaneda K, Yoshimatsu H, Saikawa T: Role of protein kinase C in geranylgeranylacetone-induced expression of heat-shock protein 72 and cardioprotection in the rat heart. J Mol Cell Cardiol 2003, 35:785-794
Redaelli CA, Tian YH, Schaffner T, Ledermann M, Baer HU, Dufour JF: Extended preservation of rat liver graft by induction of heme oxygenase-1. Hepatology 2002, 35:1082-1092
Kato H, Amersi F, Buelow R, Melinek J, Coito AJ, Ke B, Busuttil RW, Kupiec-Weglinski JW: Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation. Am J Transplant 2001, 1:121-128
Cantoni L, Valaperta R, Ponsoda X, Castell JV, Barelli D, Rizzardini M, Mangolini A, Hauri L, Villa P: Induction of hepatic heme oxigenase-1 by diclofenac in rodents; role of oxidative stress and cytochrome P-450 activity. J Hepatol 2003, 38:776-783
Takahashi T, Morita K, Akagi R, Sassa S: Heme oxygenase-1: a novel therapeutic target in oxidative tissue injuries. Curr Med Chem 2004, 11:1545-1561
Kim YM, de Vera ME, Watkins SC, Billiar TR: Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor--induced apoptosis by inducing heat shock protein 70 expression. J Biol Chem 1997, 272:1402-1411
Motterlini R, Hidalgo A, Sammut I, Shah KA, Mohammed S, Srai K, Green CJ: A precursor of the nitric oxide donor SIN-1 modulates the stress protein heme oxygenase-1 in rat liver. Biochem Biophys Res Commun 1996, 225:167-172
Peralta C, Rull R, Rimola A, Deulofeu R, Rosello-Catafau J, Gelpi E, Rodes J: Endogenous nitric oxide and exogenous nitric oxide supplementation in hepatic ischemia-reperfusion injury in the rat. Transplantation 2001, 71:529-536
Lehmann TG, Wheeler MD, Froh M, Schwabe RF, Bunzendahl H, Samulski RJ, Lemasters JJ, Brenner DA, Thurman RG: Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat. Transplantation 2003, 76:28-37
Ravingerova T, Barancik M, Strniskova M: Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology. Mol Cell Biochem 2003, 247:127-138
Ping P, Zhang J, Huang S, Cao X, Tang XL, Li RC, Zheng YT, Qiu Y, Clerk A, Sugden P, Han J, Bolli R: PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits. Am J Physiol 1999, 277:H1771-H1785
Amersi F, Shen XD, Anselmo D, Melinek J, Iyer S, Southard DJ, Katori M, Volk HD, Busuttil RW, Buelow R, Kupiec-Weglinski JW: Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway. Hepatology 2002, 35:815-823
Miyazono M, Garat C, Morris KG, Jr, Carter EP: Decreased renal heme oxygenase-1 expression contributes to decreased renal function during cirrhosis. Am J Physiol 2002, 283:F1123-F1131
Rodgers PA, Seidman DS, Wei PL, Dennery PA, Stevenson DK: Duration of action and tissue distribution of zinc protoporphyrin in neonatal rats. Pediatr Res 1996, 39:1041-1049
Ward KW, Proksch JW, Azzarano LM, Mumaw JA, Roethke TJ, Stelman GJ, Walsch MJ, Zeigler KS, McSurdy-Freed JE, Kehler JR, Chokshi J, Levy MA, Smith BR: Preclinical pharmacokinetics of SB-203580, a potent inhibitor of p38 mitogen-activated protein kinase. Xenobiotica 2001, 31:783-797
Fujimoto H, Ohno M, Ayabe S, Kobayashi H, Ishizaka N, Kimura H, Yoshida K, Nagai R: Carbon monoxide protects against cardiac ischemia-reperfusion injury in vivo via MAPK and Akt-eNOS pathways. Arterioscler Thromb Vasc Biol 2004, 24:1848-1853
Joyeux M, Boumendjel A, Carroll R, Ribuot C, Godin-Ribuot D, Yellon DM: SB 203580, a mitogen-activated protein kinase inhibitor, abolishes resistance to myocardial infarction induced by heat stress. Cardiovasc Drugs Ther 2000, 14:337-343
Lai IR, Ma MC, Chen CF, Chang KJ: The protective role of heme oxygenase-1 on the liver after hypoxic preconditioning in rats. Transplantation 2004, 77:1004-1008
Huang TJ, McCoubrey WK, Jr, Maines MD: Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs. Antioxid Redox Signal 2001, 3:685-696
Davies SP, Reddy H, Caivano M, Cohen P: Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000, 351:95-105
Harada J, Sugimoto M: An inhibitor of p38 and JNK MAP kinases prevents activation of caspase and apoptosis of cultured cerebellar granule neurons. Jpn J Pharmacol 1999, 79:369-378
作者单位：From the Experimental Hepatology Unit,* Instituto de Investigaciones Biom?dicas de Barcelona-Consejo Superior de Investigaciones Cient?ficas, Institut d'Investigacions Biomdiques August Pi i Sunyer, Barcelona; and the Departament de Cincies Fisiol
【摘要】
Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson??s disease (PD), dementia with Lewy bodies, and multiple system atrophy are -synucleinopathies characterized by filamentous -synuclein (-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with -syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with -syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble -syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like -syn pathologies. Cell culture studies also revealed that -syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of -syn inclusions in PD and related -synucleinopathies.
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-Synuclein (-syn) is a constituent of filamentous Lewy bodies (LBs) and Lewy neurites (LNs) in Parkinson??s disease (PD).1-3 Three missense mutations in the -syn gene as well as gene duplications/triplications have been reported in familial PD with or without dementia.4-7 In addition, LBs and LNs are the defining lesions of dementia with LBs (DLB) and the LB variant of Alzheimer??s disease (LBVAD).8 Glial cytoplasmic inclusions (GCIs) consisting of filamentous -syn in oligodendrocytes are hallmarks of multiple system atrophy (MSA), a neurodegenerative movement disorder.9 Although -syn is ubiquitinated in -syn inclusions, the significance of this is not clear because native -syn fibrillizes in vitro and other abnormal modifications of -syn have been implicated in the formation of -syn inclusions.10-15 However, overexpression of mutant or wild-type -syn in transgenic (Tg) animals has yielded models of neurodegenerative -synucleinopathies with inclusions formed by aggregated -syn filaments.16-19
Accumulation of misfolded proteins due to a variety of stress situations leads to up-regulation of heat shock proteins (Hsps) to facilitate refolding and prevent aggregation of misfolded proteins.20,21 Hsps may help ubiquitinate and target nonrepairable proteins to the proteasome.22 Although proteasome impairments are linked to the formation of inclusions in neurodegenerative disorders, including Alzheimer??s disease (AD) and PD,23,24 Hsps can also be induced by proteasome inhibitors.25-27 Various Hsps have been found in cytoplasmic inclusions of neurons and glia in diverse neurodegenerative diseases.28,29 Hsp90 is an abundant molecular chaperone that prevents protein aggregation and increases Hsp expression.30 Although it has an ATPase domain, Hsp90 can act independently of ATP and can prevent protein aggregation alone or with other chaperones, such as Hsp70.30 Although several studies suggest Hsp70 plays a mechanistic role in -synucleinopathies,18,31-33 numerous other Hsps are expressed in brain, and it is not clear if Hsp70 alone versus multiple brain Hsps are involved in -synucleinopathies or if Hsps contribute to mechanisms of disease in other neurodegenerative disorders. We addressed these questions here by systematically screening the brains of patients with -synucleinopathies and other disorders as well as animal models of a PD-like -synucleinopathy for pathological alterations of multiple brain Hsps. Data from these studies plus other data from cell culture systems implicate Hsp90 as the predominant Hsp implicated in -syn pathologies in diverse -synucleinopathies but not in other neurodegenerative disorders.
【关键词】 convergence ubiquitin filamentous -synuclein inclusions -synucleinopathies
Materials and Methods
The panel of antibodies to Hsp, -syn, tau, and ubiquitin used in this study, including their respective dilutions and origins, are summarized in Table 1 .
Table 1. List of Antibodies Used in This Study
Human Brain Tissue and Patient Demographics
Brain tissue was obtained from the Center for Neurodegenerative Disease Research and the AD Center Core at the University of Pennsylvania School of Medicine. Established diagnostic criteria were used to assign brains to normal or disease groups as described.34-40 Demographic information for the patients analyzed is presented in Table 2 . Age, postmortem interval, or gender did not show a noticeable effect on the results of the following studies.
Table 2. Demographics of Patients Analyzed in This Study
-Syn Tg Mice
Mice from stable Tg mouse lines carrying the A53T human -syn mutation (line M83, 3 and 9 months old) and wild-type human -syn (line M7, 9 months old) as well as non-Tg mice (C57BL/C3H) (9 months old) were studied using immunohistochemical (IHC) methods previously reported in the characterization of these Tg mice.19
IHC Analyses
Preliminary studies were performed with antibodies against various Hsps and the carboxyl terminus of Hsp70 interacting protein (CHIP) to optimize IHC analyses, including examination of various fixatives (10% formalin or 70% ethanol) and antigen retrieval pretreatments (with or without microwaving, boiling, or 88% formic acid) on 6-µm-thick paraffin sections or fresh frozen brain and cryosectioning (10 µm thick) followed by 10% formalin fixation as described.41-43 As summarized in the Results, screening of multiple antibodies to six different Hsps showed that Hsp90 was most prominent in filamentous -syn inclusions, and paraffin-embedded sections cut from 70% ethanol-fixed tissues were used for IHC without antigen retrieval, except as noted. IHC was performed as described using the avidin-biotin complex (ABC) method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine (DAB) as chromogen.44,45 Double-labeling IHC with antibodies specific for Hsp90 and -syn (SLN4, syn303) was conducted using either the ABC method with DAB followed by the ß-galactosidase-based ABC method (Amersham Biosciences Corp., Buckinghamshire, UK) with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) visualization46 or two-color fluorescent IHC (FIHC) and secondary antibodies conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR) and Texas Red (Jackson ImmunoResearch Laboratory, West Grove, PA) or Alexa Fluor 594 (Molecular Probes) as described.45 Triple-labeling FIHC studies were performed by co-incubating sections with antibodies specific for Hsp90, -syn, and tau or ubiquitin, raised in different species, followed by visualizing bound antibodies with secondary antibodies conjugated with Alexa Fluor 488, Texas Red, or AMCA (Vector Laboratories) as described.45,47 Autofluorescence was blocked using published methods.48,49 These sections were coverslipped using Vector Shield mounting medium (Vector Laboratories) with (for double stain) or without (for triple stain) 4,6-diamino-2-phenylindole (DAPI) (1 µg/ml). Negative and positive controls for IHC and FIHC studies were performed as reported,44,47 and sections were analyzed with an Olympus BX51 microscope (Tokyo, Japan) equipped with bright-field and fluorescence light sources. Images were captured using a ProGres C14 digital camera (Jenoptik AG, Jena, Germany).
To estimate the relationship between Hsp90 and tau or -syn lesions, as well as -syn, Hsp90, and ubiquitin, semiquantitative analyses of double- and triple-labeled FIHC sections were conducted using a combination of different antibodies to these proteins applied to sections of the amygdala from PD, DLB, LBVAD, AD, and normal brains (n = 6), as well as the pons from MSA and normal brains (n = 6). Briefly, micrographs of five fields from the triple-label FIHC sections were captured (with x20 objective lens) within the anatomical area of interest. Images were opened with ImageProplus (Media Cybernetics, Silver Spring, MD), thresholds were established, manual editing was performed to eliminate artifacts, and positively stained profiles were counted. After importing images into Photoshop (Adobe System, San Jose, CA), sets of images labeled by Alexa Fluor488, Texas Red, and/or AMCA were overlaid, and double- as well as triple-labeled objects were counted and statistically analyzed. Positively labeled objects were included regardless of shape, and profiles smaller than the average diameter of axons (3 pixels in ImageProplus) were not included in this analysis.
Immunoelectron Microscopy
For immunoelectron microscopy, samples of midbrain were excised from the brains of three patients with PD (with a 3- to 4-hour postmortem interval), fixed in 4% paraformaldehyde and 0.1% glutaraldehyde overnight, and cut into 50-µm-thick sections using a Vibratome 3000 microtome (Vibratome, St. Louis, MO). Sections including LBs and LBs visualized by eosin staining were embedded in LR White. Ultrathin sections were used for single- or double-immunoelectron microscopy studies as described with minor modifications.50 Ultrathin sections (70 nm) were cut and double immunolabeled using mouse anti--syn monoclonal antibody (mAb) syn303 and rat anti-Hsp90 mAb 9D2 followed by anti-mouse IgG and anti-rat IgG conjugated with 10-nm protein A gold and 18-nm protein L gold (Rockland Immunochemical, Gilbertsville, PA), respectively. Chemicals for immunoelectron microscopy were purchased from Electron Microscopic Sciences (Fort Washington, PA).
Sequential Biochemical Fractionation and Western Blot Analyses
For sequential biochemical fractionation and Western blot studies, samples of frozen cingulate cortex from PD, DLB, LBVAD, and normal control as well as pons from MSA and normal control (n = 6) were examined using previously described methods.19 Brain tissue was homogenized in 3 ml/g of high-salt (HS) buffer and centrifuged at 100,000 x g for 30 minutes. The pellets were homogenized in SDS buffer (10 mmol/L Tris, pH 6.8, 1 mmol/L EDTA, 40 mmol/L dithiothreitol, 1% SDS, 10% sucrose) and centrifuged at 100,000 x g for 30 minutes. The pellet was sonicated in 1 ml/g 70% formic acid (FA). FA was removed by lyophilization. SDS-sample buffer (10 mmol/L Tris, pH 6.8, 1 mmol/L EDTA, 40 mmol/L dithiothreitol, 1% SDS, 10% sucrose) was added to HS, HS/T, RIPA, and FA fractions and samples were heated at 100??C for 5 minutes. The same amount of each fraction was loaded on separate lanes of 15% polyacrylamide gels and subjected to Western blot analysis as described previously.19,51
Cell Culture
Cell culture media was purchased from Invitrogen (Grand Island, NY). Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132) and lactacystin were purchased from Calbiochem (Bad Soden, Germany). Cells were maintained at 37??C, 10% CO2. To study the effects of proteasome inhibition on cultured cells, we used rodent OLN-93 cells, a permanent oligodendroglial cell line.52 These cells were stably transfected with cDNAs to express the longest human tau isoform (tau40)53 and -syn, respectively. OLN-tau40--syn cells were grown in Dulbecco??s modified Eagle??s medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Primary cultures of rat brain oligodendrocytes were examined. To do this, primary cultures of glial cells were prepared from the brains of 1- to 2-day-old Wistar rats, and oligodendrocytes were prepared from the flasks after 6 to 8 days as described.54 Precursor cells were replated on poly-L-lysine-coated culture dishes (2.7 x 106cells/10 cm dish) and kept for 5 to 7 days in serum-free Dulbecco??s modified Eagle??s medium to which insulin (5 µg/ml), transferrin (5 µg/ml), and sodium selenite (5 ng/ml) (Roche Diagnostics, Mannheim, Germany) were added. These cultures contain a highly enriched population of differentiated oligodendrocytes with a mature morphology.
For proteasome inhibition studies, cells were treated with MG-132 as indicated, and cellular monolayers of control and treated cells were washed with phosphate-buffered saline once, scraped into sample buffer, and heated for 10 minutes. Protein contents in the samples were determined as described.55 Total cellular extracts were separated by SDS-polyacrylamide gel electrophoresis followed by Western blot analysis as described above. The following antibodies were used for these studies: anti--tubulin (1:1000) (Sigma, Taufkirchen, Germany), anti-ubiquitin (P4G7-H11, 1:1000), and anti-Hsp90 (AC88; 1:1000) (StressGen, Victoria, BC, Canada) mouse mAb, and anti-myelin basic protein rabbit polyclonal antibody (1:1000) (gift from Dr. A. McMorris, Wistar Institute, Philadelphia, PA).
RNA Extraction and Reverse Transcription
RNA from oligodendrocytes (2.7 x 106 cells) was isolated with the RNeasy kit (Qiagen, Hilden, Germany) as described by the manufacturer for animal cells. One µg of RNA was used for reverse transcription in a final volume of 20 µl and polymerase chain reactions were performed in a Biometra Thermocycler (Göttingen, Germany) as previously described.56 Reaction products were analyzed on agarose gels and visualized by ethidium bromide staining. Primers were synthesized by Gibco-BRL Life Technologies (Karlsruhe, Germany). Primers were designed using PrimerSelect software (DNASTAR, Madison, WI).
For the analysis of Hsp90 the following oligonucleotides were used: 5'-GAT TGA CAT CAT CCC CAA CC-3' and 5'-CTT CAT CAG ATC CCA CAT CC-3'. Control experiments were performed with the following primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-CCCACGGCAAGTTCAACGGCA-3' (nucleotides 220 to 240) and 5'-TGGCAGGTTTCTCCAGGCGGC-3' (nucleotides 805 to 825).57
OLN cells stably expressing tau and -syn were homogenized in 1 ml of RIPA buffer (150 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) with protease inhibitors. The homogenates were spun at 3000 rpm for 5 minutes to yield lysate. The lysate was precleared with protein A/G agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and subsequently used for immunoprecipitation reactions. Sufficient quantities of either anti--syn (syn310), anti-Hsp90 (AC88), anti-Hsp70 (C92 F3C4), anti-Hsc70 (IB5), or anti-Hsp40 (rabbit) antibody or no antibody was added to the precleared lysate in an individual test tube so as to completely immunoprecipitate the antigens in the lysate. The antibody-protein complex was precipitated with protein A/G agarose. The beads were washed several times with RIPA buffer, once with 50 mmol/L Tris, pH 6.8, and resuspended in 2x SDS loading buffer. Equivalent volumes of immunoprecipitates and supernatants from immunoprecipitates were loaded in each lane and subsequently analyzed by Western blotting as described.
Statistical Analyses
Data for the number of inclusions and double-labeled elements from the studies described above were expressed as mean ?? SEM. The ratio of Hsp90, tau, and ubiquitin immunoreactivity (IR) over -syn or tau IR values was assessed by analysis of variance and subsequently by unpaired, two-tailed Student??s t-test, taking into consideration disease type and protein type. Significance was set at P < 0.05.
IHC Screening of Hsps in -Synucleinopathies
The extent to which several major Hsps (B-crystallin, Hsp27, Hsp40, Hsp60, Hsp70, Hsc70, and Hsp90) co-localize in LBs and GCIs among various -syn inclusions in human disease brains was assessed by double-label FIHC screening with the combination of antibodies listed in Table 1 . Antibodies to Hsp27 (70%), Hsp40 (60%), Hsc70 (75%), and Hsp90 (95%) labeled large subsets of LBs, whereas B-crystallin, Hsp60, or Hsp70 were nearly undetectable in these inclusions (5%) (Figure 1, aCg) . GCIs were consistently detected by antibodies to B-crystallin (>90%) and Hsp90 (>90%) but rarely by Hsp27 (10%), Hsp40 (10%), or Hsp70 (<5%) (Figure 1, hCn) . We used confocal microscopy to confirm co-localization of these proteins in studies preliminary to the more detailed analyses reported in our manuscript. In view of this, and because epifluorescence microscopy is much more practical and feasible for large scale quantitative analysis as conducted here, we do not describe the data from these preliminary confocal analyses in the present study.
Figure 1. Screening of Hsps using double-label FIHC with different anti-Hsp and anti--syn antibodies visualized by Alexa Fluor 594 or Texas Red and Alexa Fluor 488, respectively, shows co-localization of a subset of Hsps examined in LBs (aCg) and GCIs (hCn). Yellow color represents co-localization of Hsp and -syn. Hsp27 (b), Hsp40 (c), Hsc70 (e), and Hsp90 (g) are located in LBs, whereas B-crystallin (h) and Hsp90 (n) are located in GCIs and threads. Specificity of Hsp90 antibodies (o), 9D2 (lanes 1 and 2), and AC88 (lanes 3 and 4) is shown in HS fraction of human (lanes 1 and 3) and mouse (lanes 2 and 4) brain homogenates. Rat 9D2 does not recognize mouse Hsp90 (lane 2). Photomicrographs in pCx show modest Hsp90 IR in neurons of normal brain (p), which also is seen in -synucleinopathy brains, in addition to more intense Hsp90 IR in LBs (arrowhead) of the PD SN (q), DLB amygdala (r), and GCIs (arrow) in the MSA pons (s). Little or no iHsp90 IR is seen in neurofibrillary tangles of AD (t, double arrowheads) and DLB (r). t: IHC profile with an antibody against Hsp90 (9D2) or tau (AT8) (inset) from adjacent sections in the CA1 region of AD hippocampus are presented for comparison. It is apparent that Hsp90 IR is reduced in neurofibrillary tangles. u: Modest Hsp90 IR is noted in ubiquitin inclusions in the hippocampus of the FTD-MND brain. Inset shows ubiquitin IR in inclusions on the adjacent section. v and w show double-label IHC with horseradish peroxidase-DAB and ß-galactosidase-X-gal to illustrate co-localization of iHsp90 IR (brown) with -syn IR (blue) in LBs (arrowhead) in the midbrain of PD (v) as well as GCIs (arrow) in the pons of MSA (w). x: No CHIP IR was found on GCI in the pons of MSA, whereas the adjacent section indicates descent -syn IR inclusions (inset). Scale bars, 10 µm.
Hsp90 Immunoreactivity in Human Brain
Specificity of the Hsp90 mAbs used was confirmed as shown in Figure 1 , where rat 9D2 recognized a discrete band at 84 kd in HS extracts of human, but not mouse, brain extracts (Figure 1o) . Mouse AC88 labeled a distinct 84-kd band in both mouse and human brain HS homogenates (Figure 1o) .
In normal human brain, both anti-Hsp90 antibodies stained neuronal perikarya and proximal dendrites throughout all brain regions examined and, to a lesser extent, glial cells (Figure 1p) . Brain sections from patients with PD, DLB, LBVAD, MSA, and frontotemporal dementia with motor neuron disease type (FTD-MND) showed ubiquitin inclusions with a modest basal staining pattern similar to normal control brains (Figure 1u) . However, in contrast to controls, intense Hsp90 immunoreactivity (iHsp90) IR was observed in LBs, LNs, spheroids of the PD, DLB, and LBVAD brain sections (Figure 1, q and r) , and in the GCIs of MSA (Figure 1s) . In sharp contrast, neurofibrillary tangles in AD and DLB brain sections showed little or no Hsp90 IR (Figure 1, r and t) compared with prominent tau-positive staining of neurofibrillary tangles by AT8 in the identical area in the adjacent section of CA1 region of AD brain (Figure 1t , inset). Occasionally increased Hsp90 IR was noted in nontangle-bearing neurons (data not shown). The composition of the diagnostic inclusions in FTD-MND is not well characterized, but they are identified by ubiquitin IHC and showed modest Hsp90 IR (Figure 1u and inset therein).
In double-label IHC studies using horseradish peroxidase-DAB (for Hsp90) and ß-galactosidase-X-Gal (for -syn), Hsp90 IR clearly co-localized with -syn in LBs and LNs of PD, DLB, and LBVAD brains (Figure 1v) and in GCIs and threads in MSA brains (Figure 1w) . This result was further confirmed using double-label FIHC methods, which demonstrated iHsp90 IR in -syn IR LBs and LNs (Figure 2, aCc) as well as in GCIs (Figure 2, dCf) . Because variable amounts of tau pathologies were noted in the brains of patients with different neurodegenerative diseases, including tauopathies and -synucleinopathies, as well as in normal control, and tau may co-exist in filamentous -syn lesions,51,58 triple-label FIHC was performed on sections of control and disease brains using antibodies raised in different species, ie, rabbit anti-tau (17024), mouse anti--syn, and rat anti-Hsp90 (9D2), or rabbit anti-tau (N-tau), mouse anti--syn, and rat anti-Hsp90 (9D2). Figure 2 illustrates representative data from these studies, including the presence of tau-positive neurites, some of which were iHsp90-positive. However, there was greater co-localization of iHsp90 IR and -syn than iHsp90 IR and tau in neuronal or glial inclusions containing either tau or -syn (Figure 2, gCj) . Localization of CHIP in -syn inclusion was also examined, but no CHIP-positive staining was found on LBs, LNs, or GCI (Figure 1x) .
Figure 2. Double-label FIHC confirms Hsp90 IR (Texas Red) co-localizes with -syn IR (Alexa Fluor 488) in LBs of PD amygdala (arrowhead in aCc) and GCIs in the MSA pons (dCf). gCj: Representative images from triple-label FIHC of DLB amygdala shows differential co-localization of Hsp90 with -syn (arrow) rather than tau-positive inclusions (arrowhead) in these preparations. Even when tau IR is located in iHsp90 IR cells, the subcellular localization of these proteins is normally discordant (asterisk). k: Semiquantitative analysis reveals the number of iHsp90 IR with -syn- or tau IR lesions including intracytoplasmic inclusions, dystrophic neurites, and spheroids. l: iHsp90 co-localizes with -syn to a significantly greater extent (P < 0.01) than tau in disease lesions. Asterisks indicate statistically significant difference. P < 0.05. Scale bars, 10 µm (aCj).
Semiquantitative analyses were conducted on sections of amygdala, which commonly harbors inclusions in diverse neurodegenerative diseases. These studies confirmed that 1) the occurrence of both tau and -syn pathologies in amygdala of AD and -synucleinopathies; 2) the abundance of tau-positive thread pathology in -synucleinopathies; and 3) co-localization of iHsp90 IR with -syn IR in the inclusions of -synucleinopathies, including LBs, LNs, and GCIs, significantly more often than with tau IR (Figure 2, k and l) .
Hsp90 and Ubiquitin in -Syn Pathologies
Because ubiquitinated -syn in -syn inclusions suggests a functional relationship between Hsp90 and ubiquitin, we analyzed the distribution of Hsp90 and ubiquitin in -syn inclusions, including cell body and thread pathologies, in the amygdala of PD, DLB, LBVAD brains, and the pons from MSA brains using triple-label FIHC. These studies showed that not every -syn IR element was Hsp90 IR or ubiquitin IR and that Hsp90 IR was more common in ubiquitin-positive, as compared to ubiquitin-negative, -syn lesions in all -synucleinopathy brains examined (Figure 3, aCh) . These observations were confirmed using semiquantitative analysis (Figure 3, i and j) . In follow-up semiquantitative analyses using double-label FIHC and DAPI staining, the ratio of co-localization of iHsp90 IR or ubiquitin IR with -syn IR was 90% in DLB and MSA (Figure 3, i and j) . Thus, both Hsp90 and ubiquitin may be sequestered into -syn inclusions by linked mechanisms because they co-occur in the same -syn lesions more often than separately.
Figure 3. Triple-label FIHC micrographs of iHsp90 (Texas Red), ubiquitin (Alexa Fluor 488), and -syn (AMCA) of DLB amygdala (aCd) and MSA pons (eCh) indicate that iHsp90 co-localizes preferentially with ubiquitinated -syn lesions (arrow), whereas a subset of -syn-positive profiles are ubiquitin-negative (arrowhead). i and j: Data from semiquantitative analyses of triple-label FIHC studies of amygdala from DLB brain (n = 6 in i) and pons of MSA brains (n = 6 in j) showing that a similar percentage (30 to 45%) of the total number -syn lesions (neurites plus perikaryal inclusions) are iHsp90C (-syn + Hsp90) and ubiquitin IR (-syn + Ubiq), whereas >90% of perikaryal -syn inclusion lesions are iHsp90C (-syn + Hsp90) and ubiquitin IR (-syn + Ubiq). There were no significant differences between the number of Hsp90-positive and ubiquitin-positive -syn inclusions. Scale bars, 10 µm (aCh).
Western Blot Analysis of Human -Synucleinopathy Brains
-Syn, Hsp90, Hsp70, Hsc70, Hsp40, Hsp27, B-crystallin, and ubiquitin protein levels were assessed and compared biochemically using sequential fractionation and Western blot analysis. In HS fractions of cingulate cortex from PD, DLB brains, and pons of MSA brains, similar amounts of monomeric -syn were noted, whereas high-molecular weight species of -syn were visible in the FA-insoluble fractions from PD, DLB, and MSA cases in association with moderately elevated levels of Hsp90 protein in these same fractions in a consistent manner (Figure 4) . Also there was a slight increase in Hsc70 in FA fractions of PD, DLB, and MSA, whereas Hsp70 levels did not differ in disease versus normal brains throughout the fractions, and, in addition, Hsp70 was not detectable in FA fraction (Figure 4) . Moderate accumulation of Hsp40 and Hsp27 relative to normal brains also was noted in FA fractions of PD and DLB brains, and slight accumulation of B-crystallin was observed in the FA fraction of MSA brains (Figure 4) . Polyubiquitinated protein species were detectable only in FA fractions, and the levels of these proteins were greater in -synucleinopathy than normal brains (Figure 4) . These finding are consistent with the distribution of these proteins in -syn inclusions of different disease brains as described above.
Figure 4. Representative images from Western blot analyses (WB) of HS, RIPA, and FA samples of cingulate cortex from normal (norm), PD (PD), DLB brains (DLB), and pons of MSA brain (MSA). Equal amounts of sample were analyzed by SDS-polyacrylamide gel electrophoresis. These results show that the FA fraction from PD, DLB, and MSA brains harbors accumulations of oligomeric -syn species, while variably increased amounts of Hsp90 and Hsc70, but not Hsp70, are seen in FA fractions of all -synucleinopathy disease brains. Additionally, some pathological accumulations of Hsp40 as well as Hsp27 and B-crystallin were noted in PD/DLB and MSA brains. Polyubiquitinated protein species in the FA fraction were more apparent in the disease brains than normal brain.
Up-Regulation of Hsp90 and Ubiquitin in Oligodendroglial and Neuronal Cell Cultures
Because impairments in proteasome activity have been implicated in -synucleinopathies,59 we examined if proteasomal inhibition might be causally related to the sequestration of Hsp90 in LBs and GCIs. To address this question, rat primary oligodendrocytes were prepared and treated with MG-132 or lactacystin. MG-132 induced the accumulation of ubiquitinated proteins and Hsp90, which was maximal after 18 hours of treatment (Figure 5Aa) , but there was no change in levels of tubulin or myelin basic protein (Figure 5Aa) . This effect was observable after treatment with MG-132 at concentrations as low as 0.1 µmol/L (Figure 5Ab) . Similarly, treatment with lactacystin (10 µmol/L, 18 hours), a more specific proteasome inhibitor, caused the induction of ubiquitinated proteins and Hsp90 (Figure 5Ab) . MG132 or lactacystin did not cause apparent alterations of -syn protein levels or noticeable oligomerization of the molecule under the specific conditions used in these experiments. Notably, these data are in agreement with previous studies using oligodendrocytes or OLN cells without -syn transfection, indicating that proteasome inhibition causes up-regulation of Hsps independent of the overexpression of -syn.27,60
Figure 5. A: Effects of MG-132 and lactacystin (LC) on cultured cells. In oligodendrocyte primary culture (a and b), exposure to MG-132 (1 µmol/L) for 3, 5, 7, and 18 hours leads to the appearance of polyubiquitinated proteins after 18 hours, with slight up-regulation of Hsp90 levels (a). By 18 hours of incubation of different doses of MG-132 (1, 5, 10 µmol/L) or LC at one (10 µmol/L) dose there is a sharp induction of ubiquitinated proteins, accompanied by up-regulation of Hsp90 protein and mRNA level (b, bottom). Similar results are seen in OLN (c) cells transfected with tau40 plus -syn and N2A cells (d) transfected with -syn although the levels of tubulin and -syn remain unchanged. Bar graphs indicate quantitation of variable Hsp90 protein levels due to MG132 treatment in respective cell types, confirming up-regulation of the protein level in all cases. B: Co-immunoprecipitation analysis using OLN-tau40--syn cells revealed noticeable protein-protein interactions between -syn and Hsp90 as well as Hsc70 (bound), but not between -syn and Hsp70 or Hsp40 (unbound). The protein complex of interest was isolated from the OLN cell homogenate using either no antibody (no Ab), -syn Ab, Hsp90, Hsc70, Hsp70, or Hsp40 (IP Ab) and detected by SNL1 (-syn) or AC88 (Hsp90) by Western blot (WB Ab). Input represents original material.
To test if Hsp90 induction by proteasomal inhibition is caused by the accumulation of nondegradable proteins or of increased transcription, total RNA was extracted from oligodendrocytes after various times of treatment with MG-132 (1 µmol/L). After reverse transcription, the resulting cDNA was subjected to polymerase chain reaction (reverse transcriptase-polymerase chain reaction). Reaction conditions were chosen so that amplification was in a linear range, and amplification of GAPDH was performed to control for equal loading of cDNA samples. Figure 5Ab (bottom) demonstrates that MG-132 caused an increase in mRNA encoding Hsp90.
Because mature primary oligodendrocytes contain very low levels of -syn,61 we performed another set of experiments using OLN-tau40--syn, representing a cell line with oligodendroglial characteristics,27,62 and the N2A cell line after stable transfection to express human -syn. Cells were treated with MG-132, and cell extracts were analyzed by Western blotting. Similar to primary rat brain oligodendrocytes, proteasomal inhibition by MG-132 led to a concentration-dependent increase in Hsp90 and ubiquitin in OLN-tau40--syn cells and N2A cells overexpressing -syn; no effect on -syn protein level was observed (Figure 5A, c and d) . Hence, proteasome inhibition specifically leads to the recruitment of Hsp90 and ubiquitin (Figure 5A, c and d) . Finally, co-immunoprecipitation analysis confirmed interactions between -syn and Hsp90 as well as Hsc70 in OLN cells (Figure 5B) , but these immunoprecipitation methods did not demonstrate an association between -syn and Hsp70 or Hsp40.
Hsp90 and -Syn Pathology in -Syn Tg Mice
Since previously established -syn Tg mice develop LB-like -syn inclusions that are ubiquitinated similar to human -synucleinopathies,19,63 this model was studied to determine whether these -syn inclusions also sequestered Hsp90 like their human counterparts. IHC revealed that 3-month-old non-Tg mice (Figure 6, aCc) , wild-type -syn M7 Tg mice (not shown), and A53T mutant -syn M83 Tg mice (asymptomatic) (Figure 6, dCf) had ubiquitous and modest expression of Hsp90 in neurons similar to normal human control brains (see Figure 1q ). Among these lines of mice, only M83 Tg mice form -syn lesions in an age-dependent manner. At 9 months of age, double-label FIHC revealed numerous -syn IR inclusions in symptomatic M83 Tg mouse brain and co-localization of iHsp90 IR in a subgroup of these inclusions (Figure 6, gCi) , while, in addition, a subset of these also were ubiquitinated (Figure 6, jCl) . In contrast, the various control mouse lines showed unappreciable changes in -syn, Hsp90, and ubiquitin IHC profiles between 3 and 9 month of age (data not shown). Further, Western blot analysis revealed the accumulation of Hsp90 and Hsc70 in the detergent insoluble FA soluble fraction selectively in symptomatic M83 Tg mouse brain, where aggregated -syn and ubiquitinated protein species were detected (Figure 6, mCo) . Other Hsps, including Hsp70, Hsp40, and B-crystallin failed to show detectable levels in the same FA fractions.
Figure 6. In the brainstem of a 3-month-old control non-Tg mouse (aCc), M7 Tg mouse (data not shown), and asymptomatic -syn (M83) Tg mouse (dCf), there are no -syn lesions and modest Hsp90 IR. However, in older symptomatic M83 Tg mouse (9 months old), numerous -syn inclusions are formed, which are frequently associated with iHsp90 IR (gCi, arrow) and ubiquitin IR (jCl, arrow). Western blots of non-Tg (lanes 1 to 3 in mCo) and M83Tg mouse (lanes 4 to 6 in mCo) show accumulation of -syn (m), Hsp90, and Hsc70, but not Hsp70, Hsp40, or B-crystallin (n) and polyubiquitinated (o) proteins (likely including -syn) exclusively in the FA fraction (lane 6) of 9-month-old symptomatic M83 Tg mouse brain, but not in the 3-month-old non-Tg mouse brain (mCo) or 9-month-old non-Tg mouse (data not shown). Scale bars, 10 µm (aCl).
Immunoelectron Microscopy of Hsp90 and -Syn in Substantia Nigra LBs of PD
Postembedding double-immunoelectron microscopy studies were performed using anti-Hsp90 and anti--syn antibodies applied to midbrain sections, including substantia nigra (SN) dopaminergic neurons with LBs, of PD brains and visualized by 18-nm (Hsp90) and 10-nm (-syn) protein gold. These studies confirmed co-localization of both proteins to the same bundles of filaments in LBs (Figure 7, a, c, and e) , whereas neurons without -syn inclusions revealed sporadic distribution of Hsp90-positive gold staining (Figure 7, b, d, and f) . These data suggest that -syn and Hsp90 are closely associated in -syn protein inclusions.
Figure 7. Double-immunoelectron microscopy reveals a close association of Hsp90 and -syn in filamentous LBs in dopaminergic neurons of the SN in PD brain (a, c, e). -Syn-positive (10 nm gold, arrow) filaments in the LB are also Hsp90-positive (18 nm gold, arrowhead), whereas unaffected dopaminergic neurons in the same SN section (f) reveal infrequent Hsp90 labeling (arrowhead). Image e is a high-power view of the inset in c, from the center of a LB. PG, neuronal pigment; N, nucleus.
Filamentous intracytoplasmic inclusions are common pathological features of diverse neurodegenerative disorders, and these lesions are often associated with Hsps and ubiquitin,64 as exemplified by -synucleinopathies wherein -syn fibrillizes to form ubiquitinated LBs, GCIs, and LNs.63,65 Previous studies detected several different Hsps in LBs and other types of -syn inclusions in diverse -synucleinopathies, but Hsp70 has been the major focus of investigation.18,31-33,66 However, we show that here among a large group of brain Hsps examined, Hsp90 most prominently and consistently co-localized in -syn inclusions such as LBs, GCIs, LNs, many of which were ubiquitinated, as previously reported,63 while Hsp90 also was most prominently associated with -syn biochemically and with -syn filaments ultrastructurally. Notably, Tg mouse models of -synucleinopathies recapitulated this association between Hsp90 and -syn pathologies, whereas cell culture studies suggested that impaired proteasome function may be causally linked to the accumulation of Hsp90, which then may interact with pathologically altered -syn in the disease state. However, it is important to emphasize that although we show that here Hsp90 is the predominant Hsp linked to pathological -syn in diverse -synucleinopathies, other Hsps also are present in -syn inclusions and pathologically altered thereby implicated them in these disorders as well. Significantly, by systematically assessing the differential involvement of multiple brain Hsps in -syn pathologies, we provide a roadmap for focused next steps toward elucidating how Hsps, and especially Hsp90, contribute to the onset and/or progression of -synucleinopathies.
Using a panel of different antibodies against a variety of Hsps, our data indicate that Hsp90 is the predominant Hsp sequestered in LBs, LNs, and GCIs throughout disease brains, but only rarely is Hsp90 found in tau inclusions. Furthermore, biochemical characterization of PD, DLB, and MSA brains and Tg mouse brains indicated that -syn and Hsp90 shift into detergent insoluble fractions compared to control brains, whereas other Hsps (ie, Hsp70, Hsp40) did not. Thus, the predominant engagement of Hsp90 with -syn in disease inclusions of -synucleinopathies appears to be a selective process rather than the result of passive or nonspecific trapping of this Hsp. Although several studies implicate Hsp70 in -syn pathogenesis,18,31-33,66,67 other Hsps have not been as extensively examined in -synucleinopathies and other neurodegenerative diseases. Thus, the predominant involvement of Hsp90 in mechanisms of -synucleinopathies that we report here is substantiated by investigation of multiple Hsps in diverse -synucleinopathies compared to other neurodegenerative disorders, as well as with a Tg mouse model of PD-like -synucleinopathies. However, although Hsp90 is most abundant, this does not mean it is the most significant Hsp in these inclusions. The percentage of Hsp40- (60%) and Hsp27-positive (70%) LBs reported by McLean and colleagues32 are similar to those we found here, whereas we found a much higher percentage of Hsp90 (95%) and lower percentage of Hsp70 (5%) in LBs than McLean??s report.32 However, our data on Hsp70 are identical to those of Auluck and colleagues?? report.18 There are several factors that could explain the differences between our study and McLean and colleagues.32 For example, we examined amygdala and McLean and colleagues32 focused on substantia nigra, but other technical or methodological differences (eg, antibodies fixation, antigen retrieval) could also account for these differences.
Hsp90 is a highly conserved protein with chaperone activity.30 It interacts with many proteins, including co-chaperones as well as substrates involved in signal transduction and cell cycle control. Hsp90 binds to these substrates, termed client proteins, and assists in final folding and stabilization. On stress, Hsp90 maintains nonnative client proteins in a folding-competent structure and prevents irreversible denaturation, a process that requires ATP binding. When ATP is bound, Hsp90 may promote ubiquitination and degradation by directing client proteins to the proteasome.68 Hence, Hsp90 is possibly involved in the balance between protein stabilization and degradation of its client proteins. Indeed, Hsps constitute a major defense against protein misfolding, and accumulation of misfolded proteins activates the ubiquitin/proteasome pathway,69 whereas proteasome inhibitors up-regulate Hsps and ubiquitin.25,27 Defects in the ubiquitin/proteasome pathway have been linked to PD and decreased proteasomal activity was reported in SN of sporadic PD.70 Because Hsp90 may promote ubiquitination as well as target client proteins to the proteasome, it is plausible that Hsp90 is causally related to the ubiquitination of -syn in fibrillary aggregates such as LBs and GCIs.
Understanding the molecular interactions among -syn, Hsp90, and ubiquitin in -syn pathogenesis is a question relevant to both biological and pathological processes, and our data suggest a variable co-localization among -syn, Hsp90, and ubiquitin. The explanation for this is not entirely clear, but it is plausible that when -syn initially becomes denatured/aggregated by stress caused by genetic or environmental factors, Hsp90 may engage -syn and successfully rescue -syn from further denaturing processes at this step. However, if stress persists chronically, -syn may eventually attain a firmly aggregated stage, wherein Hsp90 fails to rescue -syn from misfolding. Subsequently Hsp90 may redirect the protein to the proteasome system by facilitating its ubiquitination for degradation. Alternatively, Hsp90 may engage -syn preferably when it becomes denatured and/or aggregated, to promote its degradation by facilitating ubiquitination. These speculations notwithstanding, future studies would clarify the nature of this pathological cascade. However, the presence of Hsps and ubiquitin in inclusions points to an unsuccessful attempt to remove aggregated proteins by the proteasomal machinery. As we show in cell culture systems here, proteasomal inhibition by MG-132 or lactacystin causes up-regulation of Hsp90, and leads to the accumulation of ubiquitinated proteins. Thus, a chronic impairment of the proteasomal system, which might occur insidiously during the onset or progression of neurodegenerative diseases, may not be counteracted by the induction of Hsps, and the increase and association of Hsp90 with -syn aggregates might contribute to cell death.
In conclusion, our demonstration here of the predominant association of Hsp90 with ubiquitinated yn inclusions in human -synucleinopathies and animal models thereof, as well as a functional linkage between Hsp90 and proteasomal inhibition in cell culture models opens new avenues of investigation for elucidating the role of Hsps in mechanisms of neurodegenerative -synu-cleinopathies.
We thank Charles Graves for technical assistance, the colleagues in the Center for Neurodegenerative Disease Research, the staff of the biomedical imaging core of the University of Pennsylvania for technical support and advice, and the families of patients whose generosity made this research possible.
【参考文献】
Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M: Alpha-synuclein in Lewy bodies. Nature 1997, 388:839-840
Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, Trojanowski JQ, Iwatsubo T: Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson??s disease and dementia with Lewy bodies. Am J Pathol 1998, 152:879-884
Giasson BI, Duda JE, Murray IV, Chen Q, Souza JM, Hurtig HI, Ischiropoulos H, Trojanowski JQ, Lee VM: Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions. Science 2000, 290:985-989
Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL: Mutation in the alpha-synuclein gene identified in families with Parkinson??s disease. Science 1997, 276:2045-2047
Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols L, Riess O: Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson??s disease. Nat Genet 1998, 18:106-108
Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J, Gwinn-Hardy K: Alpha-synuclein locus triplication causes Parkinson??s disease. Science 2003, 302:841
Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, Ampuero I, Vidal L, Hoenicka J, Rodriguez O, Atares B, Llorens V, Gomez TE, del Ser T, Munoz DG, de Yebenes JG: The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 2004, 55:164-173
Goedert M, Spillantini MG, Serpell LC, Berriman J, Smith MJ, Jakes R, Crowther RA: From genetics to pathology: tau and alpha-synuclein assemblies in neurodegenerative diseases. Philos Trans R Soc Lond B Biol Sci 2001, 356:213-227
Goedert M, Spillantini MG: Lewy body diseases and multiple system atrophy as alpha-synucleinopathies. Mol Psychiatry 1998, 3:462-465
Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT: Chronic systemic pesticide exposure reproduces features of Parkinson??s disease. Nat Neurosci 2000, 3:1301-1306
Paxinou E, Chen Q, Weisse M, Giasson BI, Norris EH, Rueter SM, Trojanowski JQ, Lee VM, Ischiropoulos H: Induction of alpha-synuclein aggregation by intracellular nitrative insult. J Neurosci 2001, 21:8053-8061
Przedborski S, Chen Q, Vila M, Giasson BI, Djaldatti R, Vukosavic S, Souza JM, Jackson-Lewis V, Lee VM, Ischiropoulos H: Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson??s disease. J Neurochem 2001, 76:637-640
Uversky VN, Li J, Fink AL: Pesticides directly accelerate the rate of alpha-synuclein fibril formation: a possible factor in Parkinson??s disease. FEBS Lett 2001, 500:105-108
Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL, Di Monte DA: The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in mice: paraquat and alpha-synuclein. J Biol Chem 2002, 277:1641-1644
Norris EH, Giasson BI, Ischiropoulos H, Lee VM: Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications. J Biol Chem 2003, 278:27230-27240
Feany MB, Bender WW: A Drosophila model of Parkinson??s disease. Nature 2000, 404:394-398
Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L: Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science 2000, 287:1265-1269
Auluck PK, Chan HY, Trojanowski JQ, Lee VM, Bonini NM: Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson??s disease. Science 2002, 295:865-868
Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM: Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron 2002, 34:521-533
Fink AL: Chaperone-mediated protein folding. Physiol Rev 1999, 79:425-449
Sherman MY, Goldberg AL: Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron 2001, 29:15-32
Schwartz AL, Ciechanover A: The ubiquitin-proteasome pathway and pathogenesis of human diseases. Annu Rev Med 1999, 50:57-74
Keller JN, Gee J, Ding Q: The proteasome in brain aging. Ageing Res Rev 2002, 1:279-293
McNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW: Altered proteasomal function in sporadic Parkinson??s disease. Exp Neurol 2003, 179:38-46
Lee DH, Goldberg AL: Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. Mol Cell Biol 1998, 18:30-38
Ito H, Kamei K, Iwamoto I, Inaguma Y, Garcia-Mata R, Sztul E, Kato K: Inhibition of proteasomes induces accumulation, phosphorylation, and recruitment of HSP27 and alphaB-crystallin to aggresomes. J Biochem (Tokyo) 2002, 131:593-603
Goldbaum O, Oppermann M, Handschuh M, Dabir D, Zhang B, Forman MS, Trojanowski JQ, Lee VM, Richter-Landsberg C: Proteasome inhibition stabilizes tau inclusions in oligodendroglial cells that occur after treatment with okadaic acid. J Neurosci 2003, 23:8872-8880
Slavotinek AM, Biesecker LG: Unfolding the role of chaperones and chaperonins in human disease. Trends Genet 2001, 17:528-535
Richter-Landsberg C, Goldbaum O: Stress proteins in neural cells: functional roles in health and disease. Cell Mol Life Sci 2003, 60:337-349
Picard D: Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life Sci 2002, 59:1640-1648
Auluck PK, Meulener MC, Bonini NM: Mechanisms of suppression of alpha-synuclein neurotoxicity by geldanamycin in Drosophila. J Biol Chem 2005, 280:2873-2878
McLean PJ, Kawamata H, Shariff S, Hewett J, Sharma N, Ueda K, Breakefield XO, Hyman BT: TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation. J Neurochem 2002, 83:846-854
McLean PJ, Klucken J, Shin Y, Hyman BT: Geldanamycin induces Hsp70 and prevents alpha-synuclein aggregation and toxicity in vitro. Biochem Biophys Res Commun 2004, 321:665-669
McKeith IG, Galasko D, Kosaka K, Perry EK, Dickson DW, Hansen LA, Salmon DP, Lowe J, Mirra SS, Byrne EJ, Lennox G, Quinn NP, Edwardson JA, Ince PG, Bergeron C, Burns A, Miller BL, Lovestone S, Collerton D, Jansen EN, Ballard C, de Vos RA, Wilcock GK, Jellinger KA, Perry RH: Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB International Workshop. Neurology 1996, 47:1113-1124
: The National Institute on Aging and Reagan Institute Working Group: Consensus recommendations for the postmortem diagnosis of Alzheimer??s disease. The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer??s Disease. Neurobiol Aging 1997, 18:S1-S2
Harding AJ, Halliday GM: Simplified neuropathological diagnosis of dementia with Lewy bodies. Neuropathol Appl Neurobiol 1998, 24:195-201
Hurtig HI, Trojanowski JQ, Galvin J, Ewbank D, Schmidt ML, Lee VM, Clark CM, Glosser G, Stern MB, Gollomp SM, Arnold SE: Alpha-synuclein cortical Lewy bodies correlate with dementia in Parkinson??s disease. Neurology 2000, 54:1916-1921
Harding AJ, Stimson E, Henderson JM, Halliday GM: Clinical correlates of selective pathology in the amygdala of patients with Parkinson??s disease. Brain 2002, 125:2431-2445
Trojanowski JQ, Dickson D: Update on the neuropathological diagnosis of frontotemporal dementias. J Neuropathol Exp Neurol 2001, 60:1123-1126
Munoz DG, Dickson DW, Bergeron C, Mackenzie IR, Delacourte A, Zhukareva V: The neuropathology and biochemistry of frontotemporal dementia. Ann Neurol 2003, 54(Suppl 5):S24-S28
Trojanowski JQ, Schuck T, Schmidt ML, Lee VM: Distribution of tau proteins in the normal human central and peripheral nervous system. J Histochem Cytochem 1989, 37:209-215
Horiguchi T, Uryu K, Giasson BI, Ischiropoulos H, LightFoot R, Bellmann C, Richter-Landsberg C, Lee VM, Trojanowski JQ: Nitration of tau protein is linked to neurodegeneration in tauopathies. Am J Pathol 2003, 163:1021-1031
Pawlyk AC, Giasson BI, Sampathu DM, Perez FA, Lim KL, Dawson VL, Dawson TM, Palmiter RD, Trojanowski JQ, Lee VM: Novel monoclonal antibodies demonstrate biochemical variation of brain parkin with age. J Biol Chem 2003, 278:48120-48128
Uryu K, Laurer H, McIntosh T, Pratico D, Martinez D, Leight S, Lee VM, Trojanowski JQ: Repetitive mild brain trauma accelerates Abeta deposition, lipid peroxidation, and cognitive impairment in a transgenic mouse model of Alzheimer amyloidosis. J Neurosci 2002, 22:446-454
Uryu K, Giasson BI, Longhi L, Martinez D, Murray I, Conte V, Nakamura M, Saatman K, Talbot K, Horiguchi T, McIntosh T, Lee VM, Trojanowski JQ: Age-dependent synuclein pathology following traumatic brain injury in mice. Exp Neurol 2003, 184:214-224
Sanes JR, Rubenstein JL, Nicolas JF: Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J 1986, 5:3133-3142
Higuchi M, Ishihara T, Zhang B, Hong M, Andreadis A, Trojanowski J, Lee VM: Transgenic mouse model of tauopathies with glial pathology and nervous system degeneration. Neuron 2002, 35:433-446
Romijn HJ, van Uum JFM, Breedijk I, Emmering J, Radu I, Pool CW: Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy. J Histochem Cytochem 1999, 47:229-236
Schnell SA, Staines WA, Wessendorf MW: Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 1999, 47:719-730
Smith RM, Jarret L: Electron microscopic immunocytochemical approaches to the localization of ligands, receptors, transducers, and transporters. F de Pablo eds. Handbook of Endocrine Research Techniques. 1993:pp 227-246 Academic press, San Diego
Giasson BI, Forman MS, Higuchi M, Golbe LI, Graves CL, Kotzbauer PT, Trojanowski JQ, Lee VM: Initiation and synergistic fibrillization of tau and alpha-synuclein. Science 2003, 300:636-640
Richter-Landsberg C, Heinrich M: OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures. J Neurosci Res 1996, 45:161-173
Brand A, Leibfritz D, Richter-Landsberg C: Oxidative stress-induced metabolic alterations in rat brain astrocytes studied by multinuclear NMR spectroscopy. J Neurosci Res 1999, 58:576-585
Goldbaum O, Richter-Landsberg C: Activation of PP2A-like phosphatase and modulation of tau phosphorylation accompany stress-induced apoptosis in cultured oligodendrocytes. Glia 2002, 40:271-282
Neuhoff V, Philipp K, Zimmer HG, Mesecke S: A simple, versatile, sensitive and volume-independent method for quantitative protein determination which is independent of other external influences. Hoppe Seylers Z Physiol Chem 1979, 360:1657-1670
Richter-Landsberg C, Gorath M: Developmental regulation of alternatively spliced isoforms of mRNA encoding MAP2 and tau in rat brain oligodendrocytes during culture maturation. J Neurosci Res 1999, 56:259-270
Fort P, Marty L, Piechaczyk M, el Sabrouty S, Dani C, Jeanteur P, Blanchard JM: Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res 1985, 13:1431-1442
Galloway PG, Bergeron C, Perry G: The presence of tau distinguishes Lewy bodies of diffuse Lewy body disease from those of idiopathic Parkinson disease. Neurosci Lett 1989, 100:6-10
McNaught KS, Perl DP, Brownell AL, Olanow CW: Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson??s disease. Ann Neurol 2004, 56:149-162
Goldbaum O, Richter-Landsberg C: Proteolytic stress causes heat shock protein induction, tau ubiquitination, and the recruitment of ubiquitin to tau-positive aggregates in oligodendrocytes in culture. J Neurosci 2004, 24:5748-5757
Richter-Landsberg C, Gorath M, Trojanowski JQ, Lee VM: Alpha-synuclein is developmentally expressed in cultured rat brain oligodendrocytes. J Neurosci Res 2000, 62:9-14
Richter-Landsberg C, Heinrich M: OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures. J Neurosci Res 1996, 45:161-173
Sampathu DM, Giasson BI, Pawlyk AC, Trojanowski JQ, Lee VM: Ubiquitination of alpha-synuclein is not required for formation of pathological inclusions in alpha-synucleinopathies. Am J Pathol 2003, 163:91-100
Chin SS, Goldman JE: Glial inclusions in CNS degenerative diseases. J Neuropathol Exp Neurol 1996, 55:499-508
Giasson BI, Lee V-Y, Trojanowski JQ: Parkinson??s disease, dementia with Lewy bodies, multiple system atrophy and the spectrum of diseases with alpha-synuclein inclusions. Esiri M Lee V-Y Trojanowski JQ eds. The Neuropathology of Dementia. 2004:pp 353-375 Cambridge University Press, Cambridge
Dedmon MM, Christodoulou J, Wilson MR, Dobson CM: Heat shock protein 70 inhibits alpha-synuclein fibril formation via preferential binding to prefibrillar species. J Biol Chem 2005, 280:14733-14740
Lindersson E, Beedholm R, Hojrup P, Moos T, Gai W, Hendil KB, Jensen PH: Proteasomal inhibition by alpha-synuclein filaments and oligomers. J Biol Chem 2004, 279:12924-12934
Neckers L: Heat shock protein 90 inhibition by 17-allylamino-17-demethoxygeldanamycin: a novel therapeutic approach for treating hormone-refractory prostate cancer. Clin Cancer Res 2002, 8:962-966
Glickman MH, Ciechanover A: The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 2002, 82:373-428
McNaught KS, Mytilineou C, Jnobaptiste R, Yabut J, Shashidharan P, Jennert P, Olanow CW: Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures. J Neurochem 2002, 81:301-306
Braak H, Braak E: Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 1991, 82:239-259
作者单位：From The Center for Neurodegenerative Disease Research,* the Institute of Aging,|| the Department of Pharmacology, and the Department of Biology and Howard Hughes Medical Institute,¶ University of Pennsylvania, Philadelphia, Pennsylvania; the Department of Medicine and Physiology, University of
