Feb. 15, 2011 -- Using inhaled steroids as a rescue medicine along with albuterol may help some children with mild persistent asthma avoid daily inhaled steroid therapy and one of its potential side effects, namely growth restriction, according to a new study.
The new findings, which appear in the Lancet, apply only to children with mild persistent asthma that is under control. This step-down treatment is not recommended for children with moderate to severe asthma or uncontrolled mild asthma.
Many children with asthma take one or two puffs of inhaled steroids such as beclomethasone each morning and evening to prevent an asthma attack. They also use a bronchodilator such as albuterol as a rescue medication to treat any breakthrough symptoms. Such symptom relief from albuterol doesn’t get at the underlying airway inflammation, which is why some people need daily inhaled steroids. Daily inhaled steroids are still considered the gold standard to prevent asthma attacks but are not risk-free. Risks of daily inhaled steroid therapy in children include possible restricted growth and problems with adherence.
“The strategy is to give rescue therapy with inhaled corticosteroids every time you need albuterol for relief of symptoms,” says study researcher Fernando D. Martinez, MD, the Swift-McNear Professor of Pediatrics and director of the Arizona Respiratory Center at the University of Arizonain Tucson. Forexample, “you can use two puffs on Monday and another two puffs on Friday during one week, none during another week, and six puffs every day on another week, depending on how many symptoms you have,” he says in an email.
The key is to know when you need help. “If the cold starts causing tightness and shortness of breath, the child will need more albuterol and thus will use more inhaled steroids,” he says. Colds can be an asthma trigger. “The number of inhaled steroid puffs is proportional to how many albuterol puffs are needed, and therefore, to how severe the symptoms are.”
In the study, 288 children aged 6 to 18 with persistent asthma were divided into four groups:
Children and adolescents in the daily group had fewer asthma exacerbations (28%) than those in the placebo group (49%).
Treatment failure occurred 23% in the placebo group, compared with 6% in the combined, 3% in the daily group, and 8.5% in the rescue group.
The researchers also looked at growth restriction and found that growth was on average 1.1 centimeter less over the course of the 44-week study period in the combined and daily groups than in the placebo group. There was no difference in growth in the rescue group compared to the placebo group.
More study is needed to confirm the new study findings, Martinez says. “Our results were very suggestive, but statistically borderline and with small numbers,” he says. “A larger definitive study is needed.”
Dec. 23, 2010 - The growing numbers of parents who turn toward complementary and alternative medicine (CAM) to treat their children’s illnesses may often assume that “natural” means safe and harmless.
But new research in the Archives of Childhood Diseases suggests that many complementary and alternative remedies can have significant -- even fatal -- side effects.
Complementary and alternative medicine includes vitamins, herbs, and special diets.
Alissa Lim, MD, a pediatrician at the Royal Children’s Hospital in Melbourne, Australia, and colleagues tracked and analyzed all CAM-related adverse events reported to the Australian Paediatric Surveillance Units from January 2001 through December 2003.
There were 39 reports of such events, including four deaths that occurred among children age from birth to 16 years. The greatest risks were seen among infants who were put on restrictive diets and children with chronic illnesses who were treated with complementary and alternative medicine (CAM) instead of conventional medicine. ?For example, a child with epilepsy died after being treated with alternative therapies instead of anticonvulsants, the study showed.
“Parents should be aware that, like any other treatments and medicines, adverse effects can be associated with CAM use,” she tells WebMD in an email. “They should talk with their doctor before changing prescribed medications or restricting their child's fluid or diet.”
According to Lim, “the take-home message for families is to be aware of potential side effects from the use of CAM [and] weigh up the benefits and risks of any treatment they use for their children."
Lawrence Rosen, MD, a pediatrician at the Whole Child Center in Oradell, N.J., and chairman of the American Academy of Pediatrics Section on Complementary and Integrative Medicine, says that the types of complementary and alternative medicine used vary from country to country, as to how and when such therapies are used.
Rosen does not think that the new findings apply to the U.S. “Most studies in the U.S. show that the use of these therapies is done in complement to conventional medications, not as an alternative,” he says. Restrictive diets in infants such as those cited in the new report are rarely used here, he says.
What’s more, many of the adverse effects seen in the new report occurred when these therapies were used in lieu of conventional, proven treatments.
“If you have a child with a chronic illness or a complex illness, do not stop conventional therapy to use alternative without discussing it with your physician,” he says.
“Talk to your doctor about everything you give your children,” he says. “Are there going to be adverse events? Yes. Do we need to do a better job monitoring them? Yes.”
Lim agrees that such surveillance is important to get a better handle on the risks associated with these therapies.
May 3, 2010 -- Many families are turning toward to special diets and/or psychotropic medications to help better manage autism spectrum disorder and its symptoms in their children, two new studies show.
The CDC estimates that about one in 110 children in the?U.S. have an autism spectrum disorder, the umbrella name given to a group of disorders that can range from the mild to the severe that often affect social and communication abilities.
One study shows that 21% of children with autism spectrum disorder are using complementary and alternative medical therapies. Of these, 17% were on special diets, most commonly a gluten-free or casein-fee diet.
Another study shows that more than one-quarter of children with autism spectrum disorder receive at least one psychotropic medication to treat some of their behavioral symptoms such as hyperactivity or irritability.
Both studies were presented at the Pediatric Academy Societies annual meeting in Vancouver, British Columbia, and were sponsored by the Autism Treatment Network, a network of 14 centers across the U.S. and Canada that is focused on developing standards of care for treating children with autism spectrum disorder.
"Complementary medicine is used for all sorts of things such as arthritis and attention deficit hyperactivity disorder (ADHD), so to see it being used for children on the spectrum is pretty much expected," says Daniel Coury, MD, chief of developmental behavioral pediatrics at Nationwide Children's Hospital in Columbus, Ohio, and the medical ?director of the Autism Treatment Network.
"Families may be looking at complementary treatment because traditional medical treatments may not be doing the job for their child," he says. There is some anecdotal evidence that these diets may improve symptoms among some children with autism.
Parents need to make sure that their child's doctors are aware of what they are taking as some alternative therapies may have side effects on their own or when used in combination with other therapies, he says.
The study shows that younger children with autism were less likely than older kids to receive psychotropic medications. Sixty percent of children aged 11 and older took one psychotropic drug, compared with 44% of children aged 6 to 10, 11% of children ages 3 to 5, and 4% of children under age 3. The most commonly used medications were stimulants to help treat ADHD symptoms and a drug called risperidone, which is prescribed to treat irritability. Older children were more likely to be taking more than one psychotropic drug, the study shows.
The study raises some questions about how, when, and even why these medications are being used in autism treatment, Coury says.
"It may be that parents and doctors are not treating these children when they are first diagnosed, which usually occurs at very young ages," Coury says. But "as the diagnosis is established, there is a higher likelihood of medications being prescribed. Or the use of these drugs may reflect those children who are more severely affected or don't have access to other nonmedical treatments such as intensive behavioral therapies," he says.
Children who are diagnosed with autism often see numerous specialists several times a week for various types of speech and behavioral therapy.
But "if these therapies are not available, parents may reach for plan B or plan C," he says.
The regulator of G-protein signaling (RGS2) contains a characteristic RGS domain flanked by short amino and carboxyl terminal sequences. The RGS domain mediates inhibition of Gq and Gi signaling, whereas the amino terminal domain (NTD) directs interaction with adenylyl cyclases, G-protein-coupled receptors, and other signaling partners. Here, we identify a set of novel RGS2 protein products that differ with respect to their amino terminal architecture and functional characteristics. An RGS2 expression reporter cassette revealed four distinct open reading frames (ORFs) that can be expressed from the RGS2 NTD. We hypothesized that alternative translation initiation from four AUG codons corresponding to amino acid positions 1, 5, 16, and 33 could produce the observed RGS2 expression profile. Selective disruption of each AUG confirmed that alternate sites of translation initiation accounted for each of the observed products. Proteins derived from ORFs 1 to 4 showed no difference in Gq inhibitory potential or recruitment from the nucleus in response to Gq signaling. By contrast, RGS2 products initiating from methionines at positions 16 (ORF3) and 33 (ORF4) were impaired as inhibitors of type V adenylyl cyclase (ACV) compared with full-length RGS2. We predicted that regulation of the RGS2 expression profile would allow cells to adapt to changing signaling conditions. Consistent with this model, activation of Gs/ACV but not Gq signaling increased the relative abundance of the full-length RGS2 protein, suggesting that alternative translation initiation of RGS2 is part of a novel negative feedback control pathway for adenylyl cyclase signaling.
Heterotrimeric G-protein-coupled receptors mediate cell responses to a variety of extracellular ligands (Ma and Zemmel, 2002). Coordination of G-protein signaling allows cells to adjust rapidly to dynamic physiological conditions. Mammalian regulators of G-protein signaling (RGS) proteins attenuate G-protein subunit activity via GTPase activating protein (GAP) domains (Berman et al., 1996; Watson et al., 1996) and thus are important for signal modulation and discrimination. A number of RGS proteins contain activities that extend beyond their GAP function. Proteins within the RGS7-like, RGS12-like, RhoGEF-containing, and G protein-coupled receptor kinase-like RGS protein subfamilies contain multiple modular protein-protein interaction domains that allow them to coordinate signaling between intracellular signaling networks (Zheng et al., 1999; Ross and Wilkie, 2000). By comparison, simply constructed RGS proteins in the RGSZ-like and RGS4-like (R4/B) subfamilies consist of little more than an RGS domain flanked by short (typically 10-70 residues) amino- and carboxyl-terminal extensions. It is evident that even such simple RGS proteins can be versatile integrators of G-protein signaling through their interaction with a diverse number of intracellular protein partners (Heximer and Blumer, 2007).
RGS2 belongs to the R4/B subfamily of simple RGS proteins. Despite its small size, RGS2 can interact with G-proteins and non-G-protein signaling partners. The GAP domain of RGS2 inhibits Gq- (Heximer et al., 1999) and Gi- (Ingi et al., 1998) signaling, whereas sequences within the RGS2 amino terminal domain (NTD) direct nuclear and plasma membrane targeting (Heximer et al., 2001). More recently, however, the NTD of RGS2 has also been shown to interact with additional signaling partners including G-protein-coupled receptor third intracellular loops and spinophilin (Bernstein et al., 2004; Hague et al., 2005; Wang et al., 2005), adenylyl cyclase (Sinnarajah et al., 2001; Salim et al., 2003; Roy et al., 2006a), TRPV6 (Schoeber et al., 2006), and tubulin (Heo et al., 2006). It is noteworthy that engagement of the versatile RGS2 NTD with various signaling partners is expected to direct the carboxyl terminal GAP domain into context-specific signaling compartments.
Several RGS genes produce more than one protein with unique functional properties using alternative mRNA splicing. Chidiac and coworkers recently showed that multiple RGS2 bands were evident in forskolin-stimulated mouse osteoblasts (Roy et al., 2006b). We examined the possible mechanisms that might result in the production of multiple RGS2 proteins. A search of the human-expressed sequence tag database (National Library of Medicine, National Center for Biotechnology Information) revealed no evidence of alternatively spliced RGS2 mRNAs. Furthermore, our own expression data suggested that multiple RGS2 proteins are expressed from the full RGS2 cDNA alone (Heximer et al., 1999). Together, these observations prompted us to study whether alternative translation of the human RGS2 mRNA was important for the regulation of its expression and function. Here, we report the discovery of a novel set of alternatively translated RGS2 proteins with distinct functional properties whose relative expression levels are coupled to changes in cell signaling status.
Materials. The pEGFP-C1 or pREV-TRE (Clontech, Mountain View, CA) plasmids were used to express RGS2 in this study. Constitutively active Gq (R183C) construct in pCIS was a kind gift from Dr. J. Hepler (Emory University, Atlanta, GA). Constitutively active Gs (GsQ227L) and the ACV clone were kindly provided by Drs. R. Feldman and P. Chidiac (University of Western Ontario, London, ON, Canada). Expression constructs for Fibrillarin-HcRed were kindly donated by Dr. K. Lukyanov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia). Polyclonal anti-green fluorescent protein (GFP) antibody was from Clontech, and horseradish peroxidase-coupled goat anti-rabbit IgG secondary and mouse 9E10 monoclonal anti-myc epitope antibodies were from Covance Research Products (Denver, PA). HEK293 cells stably expressing the M1 muscarinic receptor were kindly provided by P. Burgon and E. Peralta. Tet-ON HEK293 cells were from Clontech. All culture medium components were from Invitrogen (Burlington, ON, Canada). [myo-3H]Inositol for cell-labeling studies was from GE Healthcare (Baie D'Urfe, QC, Canada). FuGENE 6 transfection reagent was purchased from Roche Diagnostics (Laval, QC, Canada). The cAMP enzyme immunoassay kits were from Biomedical Technologies Inc. (Stoughton, MA) and Assay Designs, Inc. (Ann Arbor, MI). Unless otherwise stated, all other reagents and chemicals were from Sigma (Oakville, ON, Canada).
cDNA Constructs. cDNA expression constructs were amplified by high-fidelity polymerase chain reaction (Pfu; Stratagene, La Jolla, CA) and cloned into the NheI and AgeI cloning sites ahead of enhanced green fluorescent protein in pEGFP-C1. Where indicated, a Kozak consensus translation initiation sequence (GCCACCATGGCG) was introduced to increase the efficiency of translation of the different potential initiator codons. The following 5' polymerase chain reaction primers were used to generate the various RGS2 amino terminal constructs used in this study: wild-type full-length (no Kozak consensus), 5'-ACTAGTATGCAAAGTGCTATGTTC-3'; Kozak full-length (kzORF1), 5'-ACTAGTGGATCCGCCACCATGGCGCAAAGTGCTATGTTCTTG-3'; kzORF2, 5'-ACTAGTGGATCCGCCACCATGGCGTTCTTGGCTGTTCAACAC-3';kzORF3, 5'-ACTAGTGGATCCGCCACCATGGCGGACAAGAGCGCAGGCAGT-3'; and kzORF4, 5'-ACTAGTGGATCCGCCACCATGGCGAAACGGACCCTTTTAAAAGATTGG-3'; in combination with a common 3' primer, 5'-ACCGGTCGGTTCAAGTCTTCTTCTGA-3. To create the translation initiation reporter construct used to determine relative usage of initiator codon use in the RGS2 mRNA, cDNA sequences spanning the complete 5'-untranslated region (upstream primer, 5'-GCTAGCGCAAACAGCCGGGGCT-3') and coding sequence for amino acid residues 1 to 79 (downstream primer, 5'-ACCGGTCGCAGCTGTGCTTCCTCAGG-3') were cloned ahead of GFP as described above. RGS2 point mutations were made using the QuikChange mutagenesis kit (Stratagene). All constructs were purified using an Endo-Free Maxi large scale DNA purification kit (QIAGEN, Mississauga, ON, Canada) and verified by DNA sequencing of the entire protein-coding region.
Cell Lines and Tissue Culture. HEK293 and Tet-ON HEK293 cells were maintained in Dulbecco's modified Eagle's medium: Ham's F-12 medium (1:1) and -minimal essential medium, respectively, supplemented with 10% (v/v) heat-inactivated fetal calf serum (Atlanta Biologicals, Lawerenceville, GA), 2 mM glutamine, 10 µg/ml streptomycin, and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO2. For doxycycline-induction studies, transiently transfected Tet-ON HEK293 cells were treated for 48 h with the indicated doxycycline concentrations before harvesting for immunoblotting. All stably transfected HEK293 cell lines expressing epitope-tagged RGS2 were generated essentially as described previously (Heximer et al., 1999). In brief, a clonal population HEK293 cells (7 x 106 cells in 10-cm plates) was transfected with 5 µg of mammalian expression constructs that direct expression of translation start-site optimized and wild-type RGS2 constructs that had been tagged at their carboxyl termini with three tandem copies of the c-myc epitope. Cells were plated at limiting dilution, and stable RGS2-(myc)3-expressing clones were selected for in growth medium containing 0.5 mg/ml Geneticin. Cell lines expressing similar levels of RGS2 protein were identified by Western blotting, and clonality was verified by immunofluorescence staining using the mouse 9E10 monoclonal antibody. Clonal cell lines were immediately frozen in aliquots for storage at passage 3 to 4. The possibility of that loss of the appropriate signaling molecules occurred during clonal selection was minimized by examination of the relevant signaling readouts in 22 separate control (3 lines) and RGS (19 lines)-expressing cell lines. All vector control lines showed similar signaling efficiency. Two RGS-expressing clones showed greater inhibition than expected from their apparent low levels of expression and were not included. To determine the relative expression levels of RGS proteins in stably transfected cell lines, cells from trypsinized plates were counted, pelleted, and lysed (2 x 107 cells/ml) in Laemmli sample buffer and resolved by SDS-polyacrylamide gel electrophoresis. RGS2 protein expression patterns were determined by immunoblotting using antibodies directed against the indicated epitope tag in phosphate-buffered saline containing 0.1% Tween 20, 3% (v/v) skim milk powder, and 3% (v/v) bovine serum albumin (anti-GFP, 1:400; anti-myc, 1:1000) and enhanced chemiluminescence. Where indicated, densitometric quantitation of protein expression was performed using the gel analysis function of the ImageJ 1.32j software package.
Phosphoinositide Hydrolysis Assays. Inositol phosphate accumulation in stably and transiently transfected cell lines was measured essentially as described previously (Heximer, 2004).
Intracellular Calcium Imaging. HEK293 cells stably transfected with the M1 muscarinic receptor were seeded at 50% confluence on polylysine-coated #1 glass coverslips in 6-well plates before transfection with 1 µg of plasmid DNA in FuGENE 6 (Roche). After 24-h transfection, coverslips were washed and incubated in calcium imaging buffer (11 mM glucose, 130 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2, 17 mM HEPES, and 1 mM CaCl2, pH 7.3) containing 5 µM fura-2 AM and 0.05% Pluronic acid for 40 min at 37°C. Fura-2-loaded cells were washed again and incubated for at least 10 min in calcium imaging buffer to allow hydrolysis of the AM ester. Coverslips were mounted in a TC1-SL25 open-bath chamber (BioScience Tools, San Diego, CA) and imaged on an Olympus BX51WI upright microscope (Olympus, Tokyo, Japan) using a 10x water-dipping objective. Excitation light was provided by a DeltaRam V monochromator (PTI, Lawrenceville, NJ). Fluorescence imaging was performed with ImageMaster imaging software (PTI). Images were acquired with a Photometrics Cascade 512B cooled charge-coupled device camera (Roper Scientific, Tucson, AZ). GFP and RGS2-GFP expressing cells were identified using 488 ± 5 nm excitation and selected as regions of interest (ROIs). Relative GFP fluorescence (RGS expression) and fura-2 ratiometric (intracellular calcium) was determined for each ROI and was calculated as mean pixel fluorescence value after 200- and 100-ms exposure, respectively. For fura-2 imaging, alternating excitation wavelengths (355 ± 5/396 ± 5 nm) were provided at 1 excitation pair per second and paired images collected through a 510 ± 20-nm emission filter (Chroma Technology Corp., Brattleboro, VT). Fluorescent ratio (FR) values for the image pairs were determined for ROIs selected on the basis of their GFP expression. Baseline fluorescence ratios of nonstimulated cells were collected for 30 frames before the addition of 200 µM carbachol. The percentage of increase from baseline FR levels to the peak stimulated FR was determined specifically for low GFP or RGS2-GFP-expressing cells with relative GFP fluorescence between background levels (3300 relative fluorescent units, RFU) and an upper experimental limit of 10,000 RFU. Higher expression levels provide greater (even complete) attenuation of the intracellular calcium response; however, high intracellular GFP levels result interfere with the 396 nm channel during fura-2 excitation. For technical reasons, therefore, it is important to measure fura-2 ratios in GFP-expressing cells with a RFU of <10,000.
cAMP Level Determination. For stably transfected lines, cells (4 x 106 cells/well in six-well plates) were incubated overnight in starvation medium (1% serum). After 15-min preincubation with 1 mM 3-isobutyl-1-methylxanthine (IBMX), cells were stimulated with either vehicle or 100 µM isoproterenol for 15 min. Cells were washed with phosphate-buffered saline and lysed in hypotonic lysis buffer (50 mM Tris, pH 7.5, and 4 mM EDTA, plus protease inhibitors), immediately boiled, and spun at 14,000 rpm. After normalization with protein levels in separate controls plated at the same density, equal amounts of protein were used in a commercial cAMP radioimmunoassay kit to determine cAMP concentrations.
For cAMP measurement in transiently transfected cultures, subconfluent HEK293 cells were plated on six-well plates and transfected with constructs expressing either Gs(Q227L) (0.25 µg/well) along with RGS2 (0.5 µg/well) and type V AC (0.03 µg/well) using FuGENE6. The vector pcDNA3 (Invitrogen) was used to normalize all of the DNA concentrations to 1.28 µg/well. After 36 h of transfection, the cells were successively starved overnight in medium containing 1% fetal bovine serum and 2 h in medium devoid of serum. cAMP accumulation was measured after 15-min treatment with 1 mM IBMX as described previously (Salim et al., 2003).
Confocal Fluorescence Microscopy. Poly(lysine)-coated 25-mm circular #1 glass coverslips containing live transfected cells were mounted in a modified Leyden chamber containing HEPES-buffered saline solution, pH 7.4. Confocal microscopy was performed on live cells at 22°C using an Olympus FluoView 2.1 (single-wavelength) or FluoView 1000 (dual-wavelength colocalization) laser-scanning confocal microscope. Nucleolar localization was marked with Fibrillarin-HcRed (Fradkov et al., 2002), whereas mitochondrial staining was achieved by prelabeling cells in 25 nM tetramethylrhodamine (TMRM) for 15 min followed by incubation in 5 nM TMRM for the duration of the image collection. Images represent single equatorial planes obtained with a 60x oil objective. Confocal images were processed with Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).
Statistical Analysis. Unless otherwise stated, data were collected from triplicate wells for each experimental condition. Relative change from baseline data were collected from at least three independent experiments and presented as means ± S.E.M. In calcium signaling experiments, data were collected from n > 30 GFP or RGS2-GFP-expressing individual cells. Statistically significant differences were determined by unpaired Student's t test method, and a p value of <0.05 was deemed significant. Representative immunoblots shown reflect similar results obtained in at least three separate experiments.
Stable Expression of Human RGS2 mRNA Yielded Multiple Protein Products. HEK293 cell lines stably transfected with a wild-type RGS2-myc construct expressed multiple RGS2 proteins (30-35-kDa range) compared with empty vector controls (Fig. 1A; compare lanes wt-1 and control). Based on the migration of recombinant RGS2 on SDS-polyacrylamide gel electrophoresis, we predicted that the weak protein band at 34 kDa corresponded to full-length RGS2-myc; however, at least two more highly expressed bands were observed between 30 and 32 kDa. This multiband RGS2 expression pattern was very different from that from cell lines stably transfected with kzRGS2-myc, a construct that was modified to include an optimized translation start consensus [Kozak consensus (Kozak, 1986); kz]. kzRGS2 lines expressed a predominant protein species corresponding to the predicted size of the full-length protein (Fig. 1A; compare lanes kz-1 and kz-2 with wt-1). We examined whether the altered expression pattern correlated with altered signaling function in the wtRGS2 compared with kzRGS2 cell lines. Cell lines with relatively similar expression levels of total RGS2-myc protein (kz-1, 1.0; kz-2, 3.3; and wt-1, 3.7) were used to examine whether the RGS2 expression pattern had an impact on its ability to attenuate Gq or ACV signaling.
Fig. 1. Comparison of protein expression profile and function of wtRGS2 and kzRGS2. A, HEK293 cells stably transfected with wild-type RGS2 constructs express multiple protein species compared with cells transfected with Kozak-optimized RGS2 cDNAs. Western blot analysis of total cell lysates from control, wtRGS2-(myc)3 (wt-1) and 2 different kzRGS2-(myc)3-expressing HEK293 stable lines (kz-1 and kz-2). Proteins were separated on 12% Laemmli gels and characterized by immunoblotting. Bracket indicates the region of the gel in which the full-length and various alternative RGS2 products are found to migrate. Protein expression levels for each cell line were determined as total RGS2 expression within the bracketed region and are expressed relative to kz-1 line. *, the position of cross-reacting proteins observed in all HEK lysates. B, basal and carbachol-stimulated inositol phosphate production in control, wt-1, kz-1, and kz-2 lines described in A. Cells were labeled overnight with [myo-3H]inositol and treated with either water (control) or 200 µM carbachol (Carb) in the presence of 10 mM LiCl. IPx levels were measured 45 min after treatment. Top, IPx values expressed as the mean percentages (soluble IPx/total soluble inositol-containing material) of triplicate samples. Data are representative of three independent experiments. Bottom, IPx production data expressed relative to the IPx values for water-treated control in each experiment. Data are the mean of three independent experiments. S.E. values are indicated by error bars. C, basal and isoproterenol-stimulated cAMP accumulation in control, wt-1, and kz-2 lines. Cells were treated with 1 mM IBMX before treatment with water (Control) or 100 µM isoproterenol (Iso) for 15 min. Top, cAMP accumulation in the different lines expressed as the average amount of cAMP accumulated in duplicate samples. Data are representative of three independent experiments. Error bars represent the range of cAMP values for each cell line. Bottom, the average accumulation of isoproterenol-stimulated cAMP levels in the wt-1 and kz-2 relative to the control cell line. Data are from three independent experiments. S.E. values are indicated by error bars. *, p < 0.05.
RGS2-Mediated Inhibition of Gq Was Similar in kzRGS2- and wtRGS2-Expressing Lines. To determine whether changes in the RGS2 expression pattern correlated with changes in signaling function, we measured the ability of RGS2 to inhibit Gq and Gs/AC signaling in wtRGS2 and kzRGS2 cell lines. We have demonstrated that the Gq inhibitory function of stably expressed RGS proteins could be compared after stimulation of endogenous muscarinic receptors in HEK293 cells (Heximer et al., 1999; Heximer, 2004). Using a similar assay system, we here compared inositol phosphate accumulation in several RGS2-expressing lines. Basal and carbachol-stimulated inositol phosphate accumulation was lower in all of the RGS2-expressing compared with the control vector-containing cell lines (Fig. 1B) Thus, all of the RGS2 lines studied showed signaling characteristics consistent with the expression of functional RGS2 protein. The level of RGS2-dependent inhibition of inositol phosphate signaling seemed to be dependent on the levels of total RGS2 protein expression (summed expression of all products within 30-35-kDa range) under both basal and carbachol-stimulated conditions. In particular, compared with the control vector cell line, the two high RGS2-expressing lines, wt-1 and kz-2, inhibited the majority of carbachol-stimulated inositol phosphate accumulation, whereas the low RGS2-expressing line kz-1 showed the lowest inhibition of inositol phosphate accumulation (Fig. 1B). Together, these data suggested that RGS2-mediated Gq inhibition is more dependent on the total amount of RGS2 protein in the cell than on differences in its expression pattern.
RGS2-Mediated Inhibition of AC Was Higher in kzRGS2-Compared with wtRGS2-Expressing Lines. HEK293 cells express β-adrenergic receptors that can be stimulated with isoproterenol to increase AC activity and second-messenger intracellular cAMP levels. In Fig. 1C, this pathway was used to determine the relative AC inhibitory activity of RGS2 in wt-1 and kz-2, the two cell lines with the most similar total RGS2 protein levels (Fig. 1A). Isoproterenol stimulation of vector control and wt-1 lines resulted in similar stimulation of cAMP accumulation greater than baseline levels. Data from three independent experiments showed that the wt-1 cell line contained a similarly low level of ACV inhibitory activity as the empty vector control cell line (Fig. 1C). By contrast, the kz-2 cell line showed much less AC-dependent accumulation of cAMP, indicating a higher level of AC inhibitory activity in these cells. Thus, in contrast to the results for Gq inhibition, the inhibition of AC signaling seemed to be highly dependent on the expression of the largest protein species.
Alternative Translation Initiation Produced Four Distinct RGS2 Protein Products. Characterization of the product expression pattern from the endogenous RGS2 gene is difficult because of the lack of antibodies that can reliably detect low levels of protein. Therefore, we constructed an expression reporter construct by cloning the complete 32-base pair 5'-untranslated region and sequences encoding amino acids 1 to 79 of RGS2 in frame ahead of enhanced green fluorescent protein (EGFP) in the pEGFP-C1 vector. The resulting amino-terminal (NT) RGS2-GFP fusion reporter construct, NT-GFP, drove expression of the RGS2 reporter mRNA from the CMV promoter. Transfection of NT-GFP into HEK cells resulted in the expression of four distinct RGS2 protein products compared with nontransfected cells (Fig. 2A, arrows). In agreement with data from stable cell lines (Fig. 1), incorporation of an optimized translation initiation consensus sequence at the first in-frame methionine resulted in production of a single full-length NT-GFP protein (Fig. 2A, lane Kz).
Fig. 2. Comparison of wild-type RGS2 translation reporter protein profiles with predicted AUG initiation sites in the RGS2 mRNA. A, translation reporter expression pattern of the NT-GFP construct. HEK cells (0.5 x 106 cells in six-well plates) were transfected with either the wild-type (WT) or Kozak-optimized (KZ) RGS2 translation initiation reporter construct NT-GFP for 24 h before total cell lysates were examined for RGS2-GFP expression by immunoblotting. Arrows indicate the position four bands that are specifically labeled by a polyclonal rabbit anti-GFP antibody (1:400 dilution) in transfected compared with nontransfected cells (-). B, comparison of putative initiator codons with the optimal translation initiation (Kozak) sequence. Sequences flanking each of four potential initiator codons are aligned with the optimal translation initiation (Kozak) sequence (above). Similarity to the consensus is indicated by shading. The relative position of the initiator codon with respect to the first in-frame methionine (Met1) is indicated. C, alignment of RGS2 mRNA sequences for rat, mouse, and human genes. Putative AUG initiator codons are indicated (in uppercase letters), and those that are conserved in all sequences are underlined. D, analysis of putative AUG initiator codon use by site-directed mutagenesis of the NT-GFP construct. HEK cultures were transfected as above with the wild-type NT-GFP construct or constructs containing AUG to UUG mutations corresponding to amino acid changes at Met1 (M1L), Met5 (M5L), Met16 (M16L), and Met33 (M33L). Expression profiles were analyzed by immunoblotting as described above. E, proteasome inhibition does not alter the NT-GFP expression pattern. Cells expressing wild-type or mutant NT-GFP were treated with 10 µM MG132 for 5 h before harvesting for immunoblotting as above.
Insertion of an optimized translation initiation consensus sequence at the beginning of the RGS2 open reading frame (ORF) might affect the protein expression pattern by one of two different mechanisms. First, if alternative translation start site use is responsible for the observed multiband profile, then optimization of initiation from the most upstream initiator codon might be expected to reduce the translation start from downstream initiator codons. Second, if the band pattern is due primarily to proteolytic breakdown, a pathway mediated by the position 2 glutamine in RGS2 (Yang et al., 2005; Bodenstein et al., 2007), then mutation of this residue to a stabilizing alanine (required for codon optimization) might stabilize the full-length RGS2 protein and prevent accumulation of smaller breakdown products.
The following observations led us to focus our attention on alternative translation start site use as an explanation for this unique expression profile. Cross-species comparison of human, mouse, and rat RGS2 mRNAs revealed the presence of four conserved in-frame AUG initiator codons that mark the beginning four putative RGS2 ORFs (ORFs 1-4) corresponding to proteins initiated from amino acid positions Met1, Met5, Met16, and Met33 (Fig. 2C). It is noteworthy that the relative migration rates of the four NT-GFP-derived proteins (Fig. 2A) were consistent with translation from four such initiator codons. Alignment of the translation initiation consensus sequence with sequences flanking each putative initiation codons indicated that the third in-frame methionine (Met16) showed the highest degree of sequence similarity to the established translation initiation consensus sequence (Fig. 2B). Moreover, the third-slowest migrating NT-GFP protein was expressed more much strongly than the others (Fig. 2A), consistent with the possibility of strong relative translation initiation from Met16.
To determine whether the expression profile observed in Fig. 2A was produced by alternative translation initiation, AUG codons at positions Met1, Met5, Met16, and Met33 in NT-GFP were mutated to UUG (leucine) codons, and the resulting protein expression profiles were compared on immunoblots. Ablation of the first two AUG codons, corresponding to Met1 and Met5, completely eliminated expression of full-length and second-most slowly migrating RGS2 bands (Fig. 2D, M1L and M5L). Likewise, ablation of AUG codons at Met16(M16L) and Met33(M33L) selectively abolished expression of the third- and fourth-most slowly migrating protein bands, respectively. Treatment of cells expressing NT-GFP with the proteasome inhibitor MG132 increased expression of all four products but did not dramatically reduce the amount of smaller products, suggesting that the faster-migrating species were not stable byproducts of proteasome-dependent degradation (Fig. 2E). To rule out the unlikely possibility that point mutations in the NT-GFP reporter construct altered the transcription rate or stability of the RGS2 mRNA, reverse transcription-polymerase chain reaction was performed on total RNA samples from cells transfected with the various constructs. Steady-state levels of reporter mRNAs were not different in NT-GFP and NT(M33L)-GFP-transfected cells (data not shown). Therefore, data from stable cell lines and mutant NT-GFP translation reporter constructs suggest that the existence of the four protein bands can be explained by alternative translation initiation from four different initiator AUGs corresponding to amino acid positions Met1, Met5, Met16, and Met33 in the RGS2 protein.
Fig. 3. Schematic representation of cDNA expression constructs showing predicted protein products and domain structures. Predicted RGS2 protein products expressed from cDNA constructs for myc epitope-tagged proteins used in stable cell lines (A), wild-type and mutant NT-GFP translation initiation reporter (B), and kzORFs (C). All of the predicted protein translation initiation sites are indicated by arrows and amino acid numbers above their corresponding AUG codons shaded in dark gray. Amino acid positions and ORF numbers are indicated relative to the full-length protein sequence (accession number NP_002914[GenBank]). In cases in which initiation codons have been optimized with a Kozak consensus sequence, the shaded AUG codon is highlighted with black. The position of functional domains and protein sequence tags relative to the predicted translation initiation sites are shown below. These are denoted as follows: AC inhibitory domain (AC); plasma membrane-targeting sequence (PM); and GTPase activating protein or RGS core domain (GAP). The 5'-untranslated region of the endogenous RGS2 mRNA has been incorporated into the NT-GFP reporter construct series and is shown above as 5'-UTR. Type and locations of the epitope tags are indicated above the appropriate construct sets and are indicated as follows: GFP and triple myc epitope tag (3xmyc).
Differential Translation Start Site Model Was Consistent with Stable Cell Line Signaling Data. Signaling data in Fig. 1 suggest that the RGS2 cDNA can produce a set of proteins that differ in their ability to inhibit AC but not Gq signaling. The diagram in Fig. 3 summarizes the location of the four putative initiator codons relative to known functional domains in RGS2 (GAP and adenylyl cyclase inhibition, AC, shown below). Figure 3A compares the predicted architecture of proteins expressed in the different stable cell lines from Fig. 1, in which initiation sites are shown as gray shaded bars with forward-facing arrows and are labeled by their amino acid position number relative to Met1. Black shaded bars indicate optimization of a translation initiation consensus sequence. Figure 3B compares the wild-type and mutant NT-GFP translation reporter constructs compared in Fig. 2D. We asked whether a single unifying model could explain the relationship between the RGS2 expression pattern and its biological function. Because the kz-1 and kz-2 cell lines apparently express mainly Met1-derived protein compared with Met16-derived protein in the wt-1 line, we inferred that loss of specific sequences between Met1 and Met16 explained the observed lower adenylyl cyclase inhibition by RGS2 in wt-1 cells (Fig. 1). Indeed, the Met16-derived product lacks the AC inhibitory domain (AC) and would be expected to show weaker inhibition of β2-adrenergic signaling than Met1-derived protein (Fig. 3A). Thus, the evidence suggests that alternative translation of RGS2 can produce several RGS2 proteins with different abilities to inhibit adenylyl cyclase activity. Because one test of this model is the functional characterization of each putative RGS2 ORF in isolation, Fig. 3C shows the design of kzORF1 to 4, the expression constructs used for this purpose.
AC Inhibition Domain Was Not a Key Modulator of RGS2 Plasma Membrane Association. We showed previously that the RGS2 NTD is required for its association with the plasma membrane and that amphipathic helical sequences between residues 39 and 52 were necessary and sufficient for this function (Heximer et al., 2001). RGS2 can also be found in plasma membrane signaling complexes containing a seven-transmembrane receptor (β2-adrenergic receptor), Gs, and type IV or VI adenylyl cyclase (Roy et al., 2006a). Thus, it seems that there are multiple discrete domains within the RGS2 NTD that are capable of cooperatively regulating its localization and signaling function. The relative contribution of the AC-inhibition domain to membrane localization is currently unknown and may have important functional implications in cells with different RGS2 expression profiles. Because proteins driven from Met16 and Met33 lack the AC-inhibition domain, the NT-GFP and the AUG-UUG mutant constructs provided a unique opportunity to study the contribution of this domain to membrane association (Fig. 4A). The four NT-GFP products were strongly localized to the plasma membrane with very little GFP fluorescence in the cytoplasm, consistent with the pattern of localization reported previously for the complete amino terminal domain (Heximer et al., 2001). The combined mutation of the first two in-frame AUGs in NT (M1L, M5L)-GFP did not alter tonic plasma membrane targeting efficiency, consistent with the notion that the primary determinants for plasma membrane are located downstream of Met16 in the RGS2 amino terminus (Fig. 4A). Because the AC inhibitory domain is located within amino acids Val9 to His11, it seems unlikely that this domain contributes to basal association of RGS2 with the plasma membrane but rather that it is required for specific recruitment or coordination of activated Gs-coupled receptor signaling complexes after exposure to a physiological stimulus.
Fig. 4. Subcellular localization of RGS2 amino terminal domains produced from wild-type and mutant translation initiation reporter constructs. A, localization of wild-type and indicated NT-GFP mutation constructs were analyzed in transfected living HEK cells using confocal microscopy. Images show cells with low/medium relative fluorescence and are representative of at least 50 cells transfected with the same construct. Confocal images were taken of HEK cells transfected with NT-GFP. Shown are GFP images collected from the basal region of the cell as determined by a z-axis series. B, colocalization of NT-GFP constructs with nucleolar markers in live cells. HEK cells were cotransfected with wild-type NT-GFP and the nucleolar marker protein fibrillarin (Fibrillarin-HcRed). Using different lasers for excitation (488 nm, GFP; 543 nm, HcRed) and emission spectrum discrimination capabilities of the Olympus FV1000 confocal microscope, green- and red-channel images were collected from the same confocal plane to determine the subcellular localization of NT-GFP and Fibrillarin-HcRed, respectively. Merged images were created to demonstrate the extent of colocalization (yellow) of these constructs. C, colocalization of NT-GFP constructs with mitochondrial dyes in live cells. HEK cells transfected with the wild-type or M33L NT-GFP constructs were incubated in the mitochondrial targeted fluorescent dye TMRM. Colocalization was determined as in B.
Mitochondrial but Not Nuclear/Nucleolar Localization RGS2 Was Dependent on Translation Start Site Use. It is becoming more widely appreciated that NTD of RGS2 can interact with an increasing number of cellular partners to coordinate localized signaling events (Heximer and Blumer, 2007). Compared with the GFP protein, which is evenly distributed throughout the cytosol and nucleus of HEK293 cells (Heximer et al., 2001), the RGS2 NTD directs nucleoplasmic and possibly nucleolar localization (Fig. 4A). It may be that the cell sequesters RGS2 in the nucleus to prevent its potent inhibition of signaling pathways or that there is a specific purpose for RGS2 inside the nuclear compartment. We therefore examined its localization to identify new potential sites of RGS2 function. The NT-GFP proteins showed strong colocalization (arrows) with the nucleolar marker Fibrillarin-HcRed (Fig. 4B), indicating a possible role for RGS2 in nucleoli. NT-GFP-derived proteins also associated with punctate organellar structures in the cytosol predicted previously to be mitochondria (Heximer et al., 2001). These features were shown to precisely colocalize (arrow-heads) with the mitochondrial-specific dye TMRM (Fig. 4C). It is the only Met33-initiated protein that targets mitochondria, because TMRM colocalization was abolished for the NT(M33L)-GFP construct. It remains to be determined whether RGS2, and more specifically its Met33-derived ORF, plays a role in the regulation of mitochondrial function. kzORF1 to 4 are recruited from the nucleus by Gq but not Gs/ACV signaling. Although the RGS2 NTD mediates localization and AC inhibition, the RGS2 GAP domain mediates its function as a Gq inhibitor. Our group and others have shown that these protein domains cooperate to mediate recruitment of RGS2 from the nucleus in response to a Gq stimulus (Heximer et al., 2001; Roy et al., 2003). We asked whether long-term Gs/ACV signaling can also recruit these four RGS2 proteins (kzORFs) out of the nucleus (Fig. 5). We predicted that RGS2 proteins containing the AC-inhibition domain (kzORF1 and kzORF2) would be more efficiently recruited to the plasma membrane. Expression of each RGS2 product was achieved by polymerase chain reaction cloning and inclusion of an optimized translation initiation sequence at the upstream AUG codon (Fig. 3C) The resulting clones were named kzORF1 through kzORF4. Each kzORF construct expressed a predominant protein band on anti-GFP immunoblots (Fig. 5A). Confocal microscopy was used to examine the subcellular localization of kzORF1 to -4 in control cells and in cells coexpressing constitutively active Gq or Gs/ACV (Fig. 5, B and C). All four kzORF clones showed efficient recruitment from the nucleus to the plasma membrane/cytosol compartment in response to Gq activation (Fig. 5B). By contrast, none of the different kzORF constructs tested was efficiently recruited from the nucleus to the plasma membrane/cytosol in Gs/ACV-stimulated cells. Relative pixel intensity values indicated that Gq activation resulted in recruitment of kzORF1 from the nucleus, whereas Gs/ACV activation had no effect on its relative distribution, despite the presence of an intact AC inhibition domain (Fig. 5C).
Fig. 5. Effect of GAP domain function and G-protein-signaling status on RGS2 localization determinants. A, Western blot of total cell lysate from cells transfected with the specified construct shows that the presence of a Kozak consensus sequence results in the production of only one protein species. B, localization of the indicated RGS2 kzORF-GFP fusion constructs with and without either constitutively active Gq(Gq*) or Gs and ACV[(Gs*)/ACV] was examined in transiently transfected cells as described above. C, the ratio of GFP signal between the nucleus and plasma membrane was measured using ImageJ. Shown are means ± S.E.M.
Taken together, the subcellular localization data for the NT-GFP and kzORF constructs do not support a role for alternative translation initiation in the differential control of RGS2 targeting to the plasma membrane or recruitment from the nucleus. Therefore, functional differences between the RGS2 ORFs are most likely to be the result of their intrinsic inability to inhibit Gq or ACV.
Fig. 6. Analysis of Gq inhibitory potential of the different RGS2 kzORF-derived products. A, HEK cells were cotransfected with constitutive Gq (Gq*) and control plasmid DNA with and without the indicated RGS2 kzORF expression plasmids. Total DNA in each transfection was 6 µg. Triplicate wells containing cells (1 x 106) were incubated in the presence of [myo-3H]inositol and 10 mM LiCl. Inositol phosphate levels were assayed as described under Materials and Methods. B, M1-HEK cells on coverslips were transiently transfected with the indicated construct and loaded with fura-2 AM. Transfected cells identified as low fluorescence intensity (<10,000 relative fluorescence units) were selected for analysis of their intracellular calcium responsiveness to carbachol. Changes in intracellular calcium levels were recorded as changes in fluorescence ratio [FR = (emission at 510 nm upon excitation at 355 nm)/(emission at 510 nm upon excitation at 396 nm)]. Shown are mean FR trace values (n > 50 kinetic cells) expressing yellow fluorescent protein control, RGS2, and RGS5 in a typical experiment showing baseline and relative FR change after the addition of 100 µM carbachol (arrow). Peak relative increases in intracellular calcium levels for each cell were calculated as the percentage of FR increase above baseline = [(peak stimulated FR/unstimulated baseline FR) - 1] x 100%. Experiments show mean percentage of FR increase above baseline ± S.E.M. for n > 30 cells.
KzORFs 1 to 4 Showed Similar Levels of Gq Inhibitory Function. Data from our translation reporter system and RGS2 stable lines suggest that all four RGS2 proteins produced by alternative translation were functionally competent with respect to their Gq inhibition activity. However, it was not possible to determine the relative function of the individual products because these proteins were expressed simultaneously from the wild-type RGS2 mRNA construct. First, we examined Gq inhibition by each individual RGS2 ORF under long- and short-term signaling conditions. We measured the ability of kzORF1 to -4 to inhibit inositol phosphate accumulation in cells cotransfected with constitutively active Gq(R183C). Transfection of Gq(R183C) alone resulted in a 50-fold increase in accumulated inositol phosphate (IPx) levels relative to nontransfected HEK293 cells. It is noteworthy that Gq(R183C)-dependent phosphoinositide hydrolysis was attenuated to a similar extent (>80% reduction of maximum signal) by each of the different RGS2 proteins (Figs. 6A). In a separate series of experiments in which 3-fold less RGS2 plasmid was used, kzORF1 and kzORF3 both attenuated signaling to a similar extent (40% reduction of maximal signal; data not shown). In short-term assays, HEK293 cells stably expressing the M1 muscarinic receptor (M1-HEK) were used to study the function of kzORFs 1 to 4 as inhibitors of agonist-mediated increases in intracellular calcium. In particular, M1-HEK cells were transiently transfected with pEGFP control plasmid or the indicated RGS2 kzORF-GFP fusion construct before fura-2 loading and stimulation with carbachol. The Gq inhibitory function of ORFs 1 to 4 was determined by measuring intracellular calcium responses in single cells that had been preselected on the basis of their kzORF-GFP expression. When RGS2 activity was compared between cells expressing similar levels, the four kzORFs all showed similar inhibition of intracellular calcium elevation (40%) in response to a 200 µM carbachol bolus (Fig. 6B). Taken together, these data indicated that the four RGS2 ORFs produced by alternative translation initiation were not functionally different at the level of their Gq inhibition.
Alternative RGS2 Translation Start Sites Produced Functionally Distinct Inhibitors of AC. Data from Fig. 1C suggest that the different RGS2 proteins produced by alternative translation of the RGS2 mRNA may behave differently in their abilities to attenuate G-protein-coupled receptor-mediated cAMP accumulation in HEK cells. We predicted that these differences were attributed to the specific loss of the AC inhibitory domain in Met16 (ORF3)- and Met33 (ORF4)-derived proteins. To determine the relative AC inhibitory potential of the various alternatively translated proteins, we used a cotransfection assay that was developed to study the function of the RGS2 as a direct inhibitor of ACV function (Salim et al., 2003). The various kzORFs were transiently cotransfected with constitutively active Gs(Q227L) and ACV in HEK293 cells, after which cAMP accumulation was measured (Fig. 7). In the presence of active Gs(Q227L) and ACV, steady-state intracellular cAMP levels were increased by 20-fold compared with unstimulated controls. The coexpression of the full-length RGS2 (kzORF1) and kzORF2 proteins each resulted in a >50% decrease in cAMP levels. As predicted from the expected downstream initiator codon positions relative to the AC inhibitory domain, kzORF3 and kzORF4 were completely deficient at inhibiting cAMP accumulation by Gs(Q227L) and ACV.
Fig. 7. Inhibition of cAMP accumulation by the different RGS2 kzORF-derived proteins. Production of cAMP was measured in HEK293 cells transiently transfected as indicated with ACV (ACV), Gs*, and the kzORF derived proteins (kzORF1-4). Data are expressed as mean ± S.E. from a single experiment and are representative of three experiments each performed in duplicate. *, a significant decrease in Gs*/ACV-stimulated cAMP levels (p < 0.05) for the indicated kzORF constructs compared with control lane (with no RGS construct).
Activation of Gs but Not Gq Signaling Pathways Altered the Expression Profile of RGS2 Translation Products. Because the above data show that the biological activity of RGS2 depends on the relative expression levels of different proteins produced from different translation initiation sites, we next determined whether the relative abundance of the alternative translation products was regulated by different long-term G-protein signaling conditions (Fig. 8). Therefore, the translation reporter vector NT-GFP was expressed alone or together with either Gq(R183C) or Gs(Q227L) and ACV. The resulting protein expression profiles were compared on immunoblots. Although no changes in the RGS2 profile were observed in response to Gq stimulation, coexpression of Gs and ACV resulted in an increase in the expression of the Met1-derived protein (Fig. 8A). Also evident was a concurrent decrease in the expression of the Met16-derived protein such that the ratio of Met1 to Met16-derived protein was greatly increased in response to Gs and ACV (Fig. 8B). Because the transcriptional activity of both the RGS2 and CMV promoters is increased in a cAMP-dependent manner, we asked whether the observed increase in the Met1-derived protein was caused by transcriptional up-regulation after cotransfection with Gs and ACV. The NT-GFP cassette was cloned into a tetracycline-inducible vector (pREV-TRE), and the RGS2 expression profile was examined at different rates of transcription that were controlled by the amount of doxycycline added to the culture medium. Increased transcription from this reporter construct was evident from stepwise increases in RGS2 protein expression; however, this was not associated with an increased level of the Met1-derived compared with the Met16-derived protein (Supplemental Data). Moreover, the coexpression of Gs and ACV resulted in increased relative expression of the Met1-derived protein irrespective of the doxycycline concentration used. Together, these data suggest that the Met1-derived protein is up-regulated independently from cAMP-dependent changes in transcription rate.
Fig. 8. Characterization of the RGS2 expression profile under different G-protein signaling conditions. A, profile of NT-GFP expression in cells with long-term Gs or Gq signaling. Cells were cotransfected with NT-GFP, and either activated Gq (Gq*) or activated Gs (Gs*) and ACV (ACV). Lysates were analyzed by immunoblotting with the GFP antibody as described in the legend to Fig. 2. B, densitometric analysis of the relative expression of the Met1-derived and Met16-derived effect of initiation codon mutations on NT-GFP reporter plasmid expression profile in the presence and absence of constitutive Gs/ACV signaling. The indicated mutants were cotransfected with Gs* and ACV, and the expression profiles were compared as in A. *, p < 0.001.
RGS Protein Genes Expressed Multiple Gene Products with Different Functional Properties. As completion of the human and mouse genome sequencing projects draw near, the search for novel RGS protein products with different biological functions is an emerging area of interest. Alternative mRNA splicing is a common mechanism by which several RGS proteins are produced from a single gene. Genes such as RGS3 and RGS12 produce alternatively spliced mRNAs that furnish their respective GAP domains with varying complements of PDZ domain or PDZ domain binding sequences (Snow et al., 1998; Kehrl et al., 2002). Likewise, RGS6, RGS8, RGS9, RGS10, and RGS11 yield splice variants of their GAP domain sequences with more than one complement of regulatory domains (Granneman et al., 1998; Giudice et al., 2001; Haller et al., 2002; Saitoh et al., 2002; Chatterjee et al., 2003). It is intriguing that nearly all of the RGS proteins derived from alternatively spliced mRNAs contain the RGS GAP domain sequences. Thus, it seems that cells modulate their G-protein signaling profiles via alternative splicing of appropriate regulatory domains onto RGS domain sequences. It is of interest, therefore, to characterize the mechanisms for alternative RGS protein production as a step toward understanding cellular modulation of G-protein signaling.
Multiple RGS2 Proteins Were Expressed from a Single mRNA. Our analysis showed that RGS2, like many of the RGS protein-encoding genes, is capable of producing more than one protein product. By contrast, however, RGS2 did not seem to use differential splicing to generate these species. Some RGS proteins (RGS2, RGS4, RGS5, and RGS16) are targeted for proteasome-mediated degradation through the N-end rule pathway, a mechanism that is dependent on cleavage of the first methionine and the protein stabilizing/destabilizing nature of the second amino acid (Davydov and Varshavsky, 2000; Hu et al., 2005; Lee et al., 2005; Bodenstein et al., 2007). We asked whether such a mechanism could produce the observed expression profile. Our current data do not seem to support this notion. First, according to the eukaryotic N-end rule (Varshavsky, A., 1996), the glutamine, phenylalanine, aspartic acid, and lysine residues at position 2 (Table 1) are all destabilizing residues and therefore should not promote selective accumulation of any of the four species. Second, the addition of the proteasome inhibitor MG132 to cells expressing NT-GFP did not selectively stabilize the full-length RGS2 band at the expense of the smaller proteins. Although we cannot rule out the possibility that some of the ORF1- to -4-derived proteins have a higher intrinsic stability than the others, the specific loss of single protein bands after AUG mutagenesis clearly points to alternative translation initiation as the primary explanation for the four species.
TABLE 1 Amino terminal sequence of ORFs 1 to 4 derived from the human RGS2 gene
The first 14 amino acids of each predicted RGS2 ORF are shown. Amino acid positions correspond to the full-length protein sequence (accession number NP_002914). Second-position residues glutamine, leucine, aspartic acid, and lysine of each ORF are highlighted in boldface type.
Alternative Translation Initiation Yields RGS2 Proteins with Varying Biological Functions. To the best of our knowledge, this is the first example of alternative translation initiation leading to the expression of RGS protein products capable of conferring different biological activities. Indeed, the abilities of the different RGS2 products to inhibit AC were dramatically different. Consistent with their lack of an AC inhibitory domain, kzORF3 and kzORF4 were deficient of AC inhibitory function. These data provide supporting evidence for the notion that alternative translation start site initiation is another potential mechanism for the regulation of RGS protein function in mammalian cells. It is of interest to determine whether these alternatively translated proteins are capable of differentially regulating other recently identified RGS2 effectors such as TRPV6, for which the interaction domain in RGS2 amino terminus is not known.
Leaky Ribosome Scanning Promotes Alternative Translation Initiation of RGS2. Our data support the use of four different initiation codons in the RGS2 mRNA and predict that a number of ribosomes are able to bypass the upstream initiator codon(s) in the wild-type RGS2 mRNA. Three mechanisms have been proposed to explain how translation from multiple ORFs in a single mRNA is achieved: 1) internal ribosome entry; 2) ribosome shunting; and 3) leaky ribosome scanning (Kozak, 1991). Internal ribosome entry has been described for a number of genes, including c-myc and the p58 and p110 PITSLRE protein kinases (Nanbru et al., 1997; Cornelis et al., 2000). Inclusion of a strong translation initiation signal at the first in-frame AUG codon resulted in loss of expression of the other smaller RGS2 products. Although this mutant also incorporates a stabilizing alanine residue at the second amino acid position that could increase its relative stability compared with the other ORFs, this mechanism cannot explain the loss of expression of the other products. Moreover, the loss of specific protein bands in the M>L mutagenesis experiments suggests that translation start site use is the primary factor controlling the observed change in expression pattern.
What is the mechanism controlling differential translation start site use in the RGS2 cDNA? Ablation of downstream ORF initiation in the RGS2-myc cell lines and the cells expressing the NT-GFP reporter argues strongly against the possibility that the RGS2 mRNA contains one or more strong internal ribosome entry site elements. At present we cannot rule ribosome shunting on the RGS2 mRNA, a mechanism in which ribosomes are repositioned across strong RNA hairpins. However, this mechanism requires termination of translation of the upstream ORF and reinitiation of the shunted ribosomes (Hemmings-Mieszczak et al., 2000). The multiple RGS2 ORFs in question are long overlapping sequences that would make a termination/reinitiation event via ribosome shunting highly improbable.
In the ribosome scanning model of translation, the 43S ribosomal complex scans the 5'-UTR in a 5' to 3' direction until it reaches an AUG within the context of a good consensus initiation sequence, where translation begins. Leaky ribosome scanning can produce multiple protein products if the translation machinery does not efficiently recognize the upstream initiator codons. To date, only a small subset of cellular mRNAs has been reported to express protein products from more than one start codon. Among these are CCAAT/enhancer-binding proteins and β that each give rise to multiple products because of weak translation initiation consensus sequences at their upstream AUG codons (Calkhoven et al., 2000). One predicted consequence of the leaky scanning model is that initiation from downstream AUG codons should increase if upstream alternative start sites are disrupted. Indeed, our studies with the NT-GFP translational reporter show that this occurs in the RGS2 mRNA because disruption of the initiator sequence for the strongly recognized AUG codon for Met16 results in increased expression of the Met33-derived protein. Thus, it seems likely that RGS2 can be added to this small set of genes whose protein expression profile is mediated by leaky ribosome scanning.
G-Protein Signaling Status Regulates the RGS2 Protein Expression Profile. RGS2 gene expression is highly tuned to the signaling status of the cell. RGS2 is an immediate-early gene whose mRNA levels are increased in a number of cell types in response to stimuli that increase intracellular calcium and cAMP (Kehrl and Sinnarajah, 2002). Thus, it has been proposed that RGS2 mRNA levels may be increased as part of a negative feedback mechanism to reciprocally modulate Gq- and AC-dependent signaling (Roy et al., 2006b). The current study suggests that the regulation of post-transcriptional events may be equally important for integrating signaling feedback loops. In particular, long-term Gs signaling increased expression of the Met1-derived compared with the Met16-derived protein, an observation consistent with signaling-dependent modulation of translation efficiency at Met1. It is noteworthy that in forskolin-treated osteoblasts, the largest RGS2 protein is also apparently expressed at much higher levels than the other proteins, suggesting that a similar regulatory mechanism may be present in other cell types and tissues (Roy et al., 2006b). The precise mechanism for this unique type of regulation, however, remains to be determined. Nonetheless, this unique adaptation of the RGS2 expression profile to a change of cell signaling status represents a new type of signaling feedback mechanism that implicates regulated alternative translation start site use in the regulation of G-protein-coupled signaling.
We gratefully acknowledge the technical support of Janet He and Ashley Misquitta.
ABBREVIATIONS: RGS, regulator of G-protein signaling; GAP, GTPase activating protein; AC, adenylyl cyclase; ACV, type V adenylyl cyclase; NTD, amino terminal domain; NT, amino terminus; ORF, open reading frame; GFP, green fluorescent protein; ROI, region of interest; IBMX, 3-isobutyl-1-methylxanthine; TMRM, tetramethyl rhodamine methyl ester; RFU, relative fluorescent unit; AM, acetoxymethyl ester; EGFP, enhanced green fluorescent protein; wt, wild type; HEK, human embryonic kidney; FR, fluorescence ratio; IPx, inositol phosphate; kz, Kozak; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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作者单位：Department of Physiology, Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada (S.G., A.A., S.P.H.); Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas (S.
July 30, 2009 -- Americans spend almost a third as much money out-of pocket on herbal supplements and other alternative medicines as they do on prescription drugs, a new government report shows.
Out-of-pocket spending on herbal supplements, chiropractic visits, meditation, and other forms of complementary and alternative medicines (CAM) was estimated at $34 billion in a single year.
The estimate was based on responses to a national health survey conducted in 2007 by the CDC’s National Center for Health Statistics (NCHS).
“The bottom line is that Americans spend a lot of money on CAM products, classes, materials and practitioner visits,” National Center for Complementary and Alternative Medicine (NCCAM) Director Josephine P. Briggs, MD, said in a media briefing today. “We estimate that this (represents) approximately 11% of the total out-of-pocket spending on health care.”
Overall out-of-pocket expenditures for complementary and alternative medicines accounted for 1.5% of the $2.2 trillion spent on health care during the year prior to the survey.
Other highlights from the report, released today by NCHS and NCAAM, include:
Previously reported figures based on the same national survey showed that 38% of adults and 12% of children under the age of 18 used some type of alternative medicine in 2007.
Clinical and experimental data indicate that anti-neutrophil cytoplasmic autoantibodies (ANCAs) cause glomerulonephritis and vasculitis. Here we report the first evidence that complement is an important mediator of ANCA disease. Transfer of anti-myeloperoxidase (MPO) IgG into wild-type mice or anti-MPO splenocytes into immune-deficient mice caused crescentic glomerulonephritis that could be completely blocked by complement depletion. The role of specific complement activation pathways was investigated using mice with knockout of the common pathway component C5, classic and lectin binding pathway component C4, and alternative pathway component factor B. After injection of anti-MPO IgG, C4C/C mice developed disease comparable with wild-type disease; however, C5C/C and factor BC/C mice developed no disease. To substantiate a role for complement in human ANCA disease, IgG was isolated from patients with myeloperoxidase ANCA (MPO-ANCA) or proteinase 3 ANCA (PR3-ANCA) and from controls. Incubation of MPO-ANCA or PR3-ANCA IgG with human neutrophils caused release of factors that activated complement. IgG from healthy controls did not produce this effect. The findings suggest that stimulation of neutrophils by ANCA causes release of factors that activate complement via the alternative pathway, thus initiating an inflammatory amplification loop that mediates the severe necrotizing inflammation of ANCA disease.
Anti-neutrophil cytoplasmic autoantibodies (ANCA) are specific for proteins in the cytoplasm of neutrophils and monocytes. The major target antigens in patients with vasculitis and glomerulonephritis are myeloperoxidase (MPO) and proteinase 3 (PR3). ANCAs occur in greater than 80% of patients with active untreated Wegener??s granulomatosis, microscopic polyangiitis, and pauci-immune crescentic glomerulonephritis.1 There is compelling clinical and experimental evidence that ANCA IgG causes ANCA-associated vasculitis and glomerulonephritis. The strongest clinical evidence for causation is the observation that a newborn child developed glomerulonephritis and pulmonary hemorrhage shortly after delivery from a mother with MPO-ANCA-associated microscopic polyangiitis, apparently caused by transplacental transfer of ANCA IgG.2,3 Two compelling animal models of ANCA vasculitis and glomerulonephritis have been described by two different research groups.4,5 Xiao and colleagues4 induced glomerulonephritis and systemic vasculitis by intravenous injection of either anti-MPO IgG or anti-MPO splenocytes derived from MPO knockout mice immunized with murine MPO. Induction of glomerulonephritis by anti-MPO IgG in this model is enhanced by cytokines6 and requires neutrophils.7 Little and colleagues5 immunized rats with human MPO, resulting in the production of antibodies that cross reacted with rat MPO and caused vasculitis and glomerulonephritis. The pathogenic effects of these anti-MPO antibodies were augmented by cytokines.
Numerous in vitro studies indicate that ANCA IgG can activate neutrophils and cause them to release proinflammatory factors. The expression of ANCA antigens (MPO and PR3) at the surface of neutrophils where they are accessible to interact with ANCA IgG is enhanced by proinflammatory cytokines, such as tumor necrosis factor (TNF)-.8,9 Incubation of TNF--primed neutrophils with ANCA IgG induces full activation with release of lytic and toxic granule enzymes and reactive oxygen species.8,9 Interaction of ANCA IgG with ANCA antigens in the microenvironment of neutrophils causes activation through both Fc receptor engagement and Fab'2 binding.10-13 Activation of neutrophil by ANCA IgG in the presence of cultured endothelial cells results in neutrophil adherence,14 neutrophil transmigration,15 and endothelial cell death.16,17 Little and colleagues5 have documented this process in vivo using their rat model to show by intravital microscopy that leukocytes activated with MPO-ANCA IgG adhere to and injure the microvasculature.
Before the observations reported in this article, a role for complement in the pathogenesis of ANCA-induced inflammation has not been suspected. This is in part because there is less complement deposition in vessel walls in ANCA vasculitis and glomerulonephritis as compared with the substantial complement deposition that is observed with immune complex disease and anti-glomerular basement membrane disease.18,19 However, an important mediator of vascular inflammation does not have to be present in vessel walls at high concentrations. For example, there is a paucity of IgG in the vascular lesions of ANCA vasculitis and glomerulonephritis, yet, as reviewed earlier, there is compelling evidence that ANCA IgG is the primary pathogenic factor causing these inflammatory lesions. ANCA disease is not associated with hypocomplementemia; however, this is not a sensitive indicator of complement involvement because certain forms of glomerulonephritis and vasculitis that have substantial vascular deposits of complement do not have hypocomplementemia, such as Henoch-Schönlein purpura and anti-GBM disease. In addition, complement activation has been identified as a major mediator of injury and inflammation in a variety of diseases in which there is little or no complement localization at the site of injury, for example, complement activation, probably through the alternative pathway, is an important mediator in ischemia reperfusion injury.20 The complement system can be activated through three different pathways: classic, lectin, and alternative.21-23 Among the many factors that can activate complement are mediators released by activated neutrophils.24-27 Based on the observations reported herein, we hypothesize that ANCA-induced activation of neutrophils results in the release of factors that activate the alternative complement pathway amplification loop, which in turn augments recruitment and activation of more neutrophils, resulting in the severe necrotizing leukocytoclastic inflammation that is so characteristic of acute ANCA disease.
【关键词】 alternative complement pathogenesis mediated anti-neutrophil cytoplasmic autoantibodies
Materials and Methods
MPO knockout (MpoC/C) mice initially generated by Aratani and colleagues28 were maintained by the University of North Carolina Division of Laboratory Animal Medicine. MpoC/C mice (8 to 10 weeks old) were used for immunization and as donors of anti-MPO antibodies and splenocytes. Rag2 mice were purchased from Taconic Farms (Germantown, NY). Factor BC/C mice that were initially generated by Matsumoto and colleagues29 were kindly provided by Dr. Zhi Liu, Department of Dermatology, University of North Carolina, Chapel Hill, NC. Age- and sex-matched wild-type (WT) B6 controls, C4C/C mice, and C5C/C mice were purchased from Jackson Laboratory (Bar Harbor, ME). All complement-deficient mice had a C57BL/6 (B6) background. Rag2C/C mice, 10 to 12 weeks old, were used as recipients for adoptive splenocyte transfer experiments. Factor BC/C, C4C/C, C5C/C, and WT B6 mice, 9 to 10 weeks old, were used for IgG transfer experiments. All animal experiments were approved by the Animal Studies Committee of the University of North Carolina at Chapel Hill, School of Medicine, and were used in accordance with National Institutes of Health guidelines.
Kinetics of C3 Depletion by Cobra Venom Factor (CVF)
To determine the kinetics of C3 depletion by CVF, WT B6 mice (n = 6) were injected intraperitoneally with a single dose of 30 µg of CVF (Sigma-Aldrich, St. Louis, MO) in 0.3 ml of phosphate-buffered saline (PBS). Control mice (n = 6) received 0.3 ml of PBS. Serum C3 levels was measured on days 0, 1, 3, 5, 7, 9, 11, and 13 after injection of CVF by C3 antibody capture enzyme-linked immunosorbent assay (ELISA) using F(ab')2 fragments of goat anti-mouse C3 as capture antibody and peroxidase-conjugated goat anti-mouse C3 as the second antibody (Cappel, Organon Teknika Corporate, West Chester, PA), according to the manufacturer??s protocol. C3 levels monitored by ELISA were markedly reduced with the lowest level within 24 hours after injection and remained at this low level for up to 7 days. Thereafter, C3 returned toward normal and reached the normal level at approximately day 11. Control mice injected with the same volume of PBS exhibited normal levels of circulating C3 (Figure 1) .
Figure 1. Profile of depletion of circulating complements by CVF. WT mice were given a single intraperitoneal injected dose of CVF (30 mg/0.3 ml PBS) or PBS (0.3 ml) and the kinetics of complement depletion were monitored by ELISA analysis of circulating C3 at indicated days after injection. Open squares represent data from CVF-treated mice (n = 6). Filled squares represent control mice that received PBS (n = 6). Bars indicate the SD.
Preparation of Nephritogenic Mouse Anti-Murine MPO Splenocytes and Antibodies
Purification of mouse MPO and the immunization of MpoC/C mice were performed as previously described.4 In brief, mouse MPO was purified from WEHI-3 cells by Dounce homogenization, Concanavalin A affinity chromatography, ion exchange, and gel filtration chromatography. MpoC/C mice were immunized intraperitoneally with 10 µg of purified murine MPO or bovine serum albumin in complete Freund??s adjuvant and subsequently boosted with antigen in incomplete Freund??s adjuvant. Development of antibodies was monitored by ELISA and anti-MPO antibody reactivity with neutrophils was confirmed by indirect immunofluorescence microscopy on murine neutrophils. The IgG fraction was isolated from serum by 50% ammonium sulfate precipitation and protein G affinity chromatography. Splenocytes were isolated from immunized and nonimmunized MpoC/C mice by disrupting the spleens into cold RPMI 1640 medium and then washing twice with RPMI 1640. Red blood cells were removed with lysis buffer (Sigma-Aldrich) followed by washing with RPMI 1640 and final suspension in sterile PBS.
Induction of Glomerulonephritis with Anti-MPO IgG and Anti-MPO Splenocytes
Induction of experimental necrotizing and crescentic glomerulonephritis (NCGN) with anti-MPO IgG in WT mice or anti-MPO splenocytes in Rag2C/C immune-deficient mice was performed as previously described.4 Briefly, in antibody transfer experiments, mice were injected intravenously with 50 µg/g body weight (BW) of anti-MPO or control IgG in PBS and sacrificed on day 6. In splenocyte transfer experiments, Rag2C/C mice were injected intravenously with 5 x 107 anti-MPO or control splenocytes in 0.5 ml of PBS and sacrificed on day 13. At the point of sacrifice, serum and urine samples were collected to evaluate kidney disease, and serum samples also were collected for serological assays of antibody and complement activity.
In IgG transfer CVF complement depletion experiments, WT and control mice received intravenously 50 µg/g BW of anti-MPO IgG 4 hours after injection of one dose of CVF or PBS, respectively, and were sacrificed on day 6. The effect of CVF on circulating complement was determined by C3 antibody capture ELISA. The urine, serum, and kidney tissue samples were collected for further evaluation as below.
In splenocyte transfer CVF complement depletion experiments, Rag2C/C mice were injected intraperitoneally with 30 µg of CVF in 0.3 ml of PBS on days 0 and 6, and control mice received 0.3 ml of PBS on days 0 and 6. Both experimental and control mice received 5 x 107 anti-MPO splenocytes by intravenous injection 4 hours after receiving the first dose of CVF or PBS. The mice were sacrificed on day 13.
In IgG transfer experiments in complement knockout mice, C5C/C, C4C/C, fBC/C, or WT mice were injected intravenously with 50 µg/g BW of anti-MPO IgG and sacrificed on day 6. The urine, serum, and kidney tissues were collected for evaluation.
Functional Evaluation of Renal Injury
Mice were placed in metabolic cages 1 day before sacrifice and urine was collected for 12 hours. Urine was tested by dipstick (Roche Diagnostics Corp., Indianapolis, IN) for hematuria and leukocyturia. The extent of hematuria and leukocyturia was expressed as the mean on a scale of 0 (none) to 4 (severe). Albuminuria was determined by a mouse albumin ELISA quantitation kit (Bethyl Laboratories Inc., Montgomery, TX). Using the mean ?? 2 SDs for the reference range established in 305 healthy B6 mice, abnormal hematuria was set at >0.9 and leukocyturia >0.2.7 Serum samples were collected at the time of sacrifice. Serum creatinine and blood urea nitrogen were measured using a Johnson & Johnson Ortho-Clinical Diagnostics VITROS 250 (Raritan, NJ).
Pathological Evaluation of Renal Injury
Samples of kidney tissue were collected at the time of sacrifice and fixed in 10% formalin, embedded in paraffin using routine protocols, sectioned at 4-µm, stained with hematoxylin and eosin and periodic acid Schiff, and evaluated by light microscopy. The extent of glomerular crescents and necrosis were expressed as the mean percentage of glomeruli with crescents and necrosis in each animal.
For immunofluorescence microscopy to detect glomerular localization of immune determinants, 4-µm frozen sections were stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Molecular Probes Invitrogen, Carlsbad, CA), IgM, IgA, and MPO (ICN/Cappel, Aurora, OH), respectively. Deposition of complement C3 was visualized with fluorescein isothiocyanate-conjugated goat anti-mouse C3 (ICN/Cappel). Immunofluorescence microscopy staining of glomeruli for IgG, IgM, IgA, C3, and MPO was expressed as the mean intensity of staining on a scale of 0 (none) to 4 (severe).
For immunohistochemistry to detect renal leukocytes infiltration, 4-µm frozen sections were stained with rat anti-mouse neutrophil Gr-1 (clone RB6-8C5; BD Pharmingen, Franklin Lakes, NJ) and rat anti-mouse monocytes/macrophages CD68 antibodies (clone FA11; Serotec, Raleigh, NC). Rat antibody binding was detected using peroxidase-conjugated secondary rabbit anti-rat IgG and tertiary goat anti-rabbit IgG antibodies (DAKO, Carpinteria, CA). Staining was visualized with 3-amino-9-ethylcarbazole and hydrogen peroxide. Sections were counterstained with hematoxylin. Leukocyte localization was expressed as the mean number of leukocytes per cross-section of glomeruli based on evaluating an average of 75 glomeruli per specimen (range, 60 to 90 glomeruli).
Measurement of Complement Activation by Factors Released by ANCA-Stimulated Human Neutrophils
IgG from healthy controls (n = 8), MPO-ANCA patients (n = 6), or PR3-ANCA patients (n = 8) was prepared from plasma by ammonium sulfate precipitation and protein G affinity column chromatography. IgG preparation were passed over AffinityPak endotoxin removal columns (Pierce, Rockford, IL) before use. The plasma was obtained during plasma exchange on ANCA-positive patients with active, biopsy-proven necrotizing and crescentic glomerulonephritis. The patients also were receiving immunosuppressive treatment. Neutrophils were isolated from healthy volunteers using Ficoll sedimentation. Neutrophils from a single donor were used for each experimental run that compared the effect of control IgG to various experimental preparations. Autologous serum was obtained from the same donors as the neutrophils. In preparation to determination of the ability of IgG to stimulate neutrophils to release complement-activating factors, neutrophils were primed for 15 minutes at 37??C with 2 ng/ml TNF-. The primed neutrophils then were incubated with 200 µg/ml of either control IgG, MPO-ANCA IgG, or PR3-ANCA IgG for 15 minutes at 37??C. Additional controls included reaction mixtures that contained control IgG, MPO-ANCA IgG, or PR3-ANCA IgG but no neutrophils (to determine whether the IgG alone could activate complement) and reaction mixtures that contained TNF-primed neutrophils but no immunoglobulin (to determine whether primed neutrophils alone could activate complement). The reaction mixtures were centrifuged at 300 g for 5 minutes at 4??C. A 100-µl aliquot of supernatant was added to precooled tubes containing 100 µl of autologous serum and incubated for 1 hour at 37??C. The reaction was stopped by adding ethylenediaminetetraacetic acid to a final concentration of 10 mmol/L. The supernatants were stored frozen at C70??C until assayed for C3a. C3a, which is a sensitive marker of complement activation, was assayed in the supernatant using the Quidel C3a EIA kit (Quidel Corp., San Diego, CA) by the manufacturer??s instructions. All determinations were made in duplicate. Two or more control IgG samples were included with each assay, and results for individual control and experimental samples were expressed as a percentage of the result of the control IgG mean.
Complement Is Required for Induction of Glomerulonephritis by Anti-MPO IgG
Pauci-immune NCGN that closely mimics human ANCA glomerulonephritis is induced in mice by a single injection of anti-MPO IgG derived from MpoC/C mice that have been immunized with mouse MPO.4,6,7 By day 6, all mice that receive anti-MPO IgG develop segmental fibrinoid necrosis and crescents in 5 to 15% of glomeruli. Immunohistology demonstrates only mild staining for immunoglobulins and complement. The effect of complement depletion on the induction of NCGN in this model was evaluated. WT B6 mice were pretreated with (n = 7) or without (n = 7) a single intraperitoneal injection of CVF 4 hours before injection of anti-MPO IgG. Six days after injecting anti-MPO IgG, all mice that did not have complement depletion developed albuminuria, leukocyturia, and hematuria whereas the mice that received CVF stayed within the reference range (Figure 2, A and B) . At the time of sacrifice, mice that received CVF had the same level of circulating anti-MPO IgG (Figure 2C) as the mice that did not receive CVF; however, as expected, they had decreased serum C3 levels (Figure 2D) . All of the mice that received anti-MPO IgG without CVF developed NCGN with neutrophil and macrophage infiltration and low-level glomerular IgG and C3 deposition (Figure 3, ACC ; and Tables 1 and 2 ). In contrast, none of the complement-depleted mice injected with the same dose of anti-MPO IgG developed glomerular necrosis or crescents (Figure 3, DCF ; and Tables 1 and 2 ). The mice that did not receive CVF had a paucity of staining for C3 (0.5+) and mice that received CVF had no staining for C3 (Table 2) .
Figure 2. Mice depleted of complement are resistant to anti-MPO IgG-induced experimental glomerulonephritis. WT mice were pretreated with a single dose of CVF or PBS (n = 7 for each group). Four hours later, the mice received anti-MPO IgG (50 µg/g body weight). A and B: Urine analysis showed that untreated mice and complement-depleted mice that received anti-MPO IgG had no urine abnormalities, whereas noncomplement-depleted mice that received anti-MPO IgG had albuminuria, hematuria, and leukocyturia. C and D: ELISA analysis demonstrated the same circulating anti-MPO IgG titers in noncomplement-depleted and complement-depleted mice (C); and low circulating C3 in complement-depleted mice (D). Extent of hematuria and leukocyturia is expressed as the mean on a scale of 0 (none) to 4 (severe). *P < 0.05 and #P < 0.001 versus untreated WT. **P < 0.004 compared with noncomplement-depleted mice. Bars represent the mean ?? SD.
Figure 3. Renal tissue was examined 6 days after mice received anti-MPO IgG. WT mice that received pathogenic IgG without complement depletion developed focal glomerular necrosis (long arrow) and crescents (short arrows) (A) and low-level glomerular IgG (B) and C3 (C) deposition. In contrast, the mice depleted of complement had no glomerular lesions after the same dose of anti-MPO IgG (D) and had no IgG (E) or C3 (F) in glomeruli. A and D are stained with PAS.
Table 1. Pathology Finding in ANCA Mouse Model with and without CVF
Table 2. Immunofluorescence Microscopy Observations in ANCA Mouse Models with and without CVF Complement Depletion
Complement Is Required for Induction of NCGN by Anti-MPO Splenocytes
Intravenous administration of anti-MPO splenocytes causes more severe disease than injection of anti-MPO IgG, although it is complicated by a background of immune complex disease.4 In our previously reported experiments,4 all mice that received 1 x 108 or 5 x 107 anti-MPO splenocytes developed NCGN with crescents in 80% of glomeruli. None of the mice that received 1 x 107 anti-MPO splenocytes developed NCGN. Immunohistology demonstrates a moderate accumulation of immunoglobulins and complement in glomeruli of mice that receive either anti-MPO splenocytes or control splenocytes. The effect of complement depletion with CVF was evaluated in this more aggressive form of NCGN induced by anti-MPO splenocytes. Rag2C/C mice were treated intraperitoneally with two doses of 30 µg of CVF in 0.3 ml of PBS (n = 8) or PBS alone (n = 8) at day 0 and day 6. Four hours after injection of the first dose of either CVF (n = 8) or PBS (n = 8), mice received an intravenous injection of 5 x 107 splenocytes from MpoC/C mice that had been immunized with mouse MPO. Control Rag2C/C mice received no CVF and no splenocytes. On day 13, all mice injected with anti-MPO splenocytes without complement depletion developed albuminuria, hematuria, leukocyturia, elevated serum creatinine, and elevated blood urea nitrogen (Figure 4, ACD) . In contrast, mice with complement depletion injected with anti-MPO splenocytes had unremarkable urine findings, creatinine, and blood urea nitrogen that did not differ significantly frommice that received neither CVF nor anti-MPO splenocytes (Figure 4, ACD) . Pathological evaluation of kidneys revealed that all noncomplement-depleted mice that received anti-MPO splenocytes developed severe NCGN with glomerular neutrophil and macrophage infiltration (Figure 5, ACD ; and Table 1 ). In contrast, none of the complement-depleted mice that received anti-MPO splenocytes developed glomerular necrosis or crescents or increased infiltration of inflammatory cells (Figure 5, FCH ; and Table 1 ). There was a marked decrease in circulating C3 in mice given CVF that occurred within 2 days and persisted throughout the experiment (Figure 6A) . There was no significant decrease in C3 levels in the mice that received splenocytes but no CVF. Both noncomplement-depleted and complement-depleted mice developed circulating anti-MPO antibodies within 2 days, and the titers steadily increased at similar levels until sacrifice at 13 days after receiving anti-MPO splenocytes (Figure 6B) . Previous experiments have demonstrated that immune-deficient Rag2C/C mice that receive splenocytes from immune-competent mice develop an immune complex mediated mesangioproliferative glomerulonephritis without crescents.4 Severe crescent formation is induced only when the splenocytes are derived from MpoC/C mice that have been immunized with MPO.4 Thus, the anti-MPO immune response and not the immune complex glomerulonephritis is the cause for the NCGN in this model. In the current experiments, the interpretation of the blockade of NCGN by CVF after anti-MPO splenocyte transfer is complicated by the marked reduction of immune complexes and complement deposition caused by CVF complement depletion (Table 2) . The complement depletion could be blocking crescent formation by inhibiting a specific anti-MPO-mediated mechanism or by reducing the synergistic stimulation of concurrent immune complex disease or both.
Figure 4. Complement depletion by CVF abolishes the development of urine abnormalities, and elevation of serum blood urea nitrogen and creatinine in Rag2C/C mice 13 days after they received 5 x 107 anti-MPO splenocytes (ACD). Bars represent the SD. *P < 0.05, **P < 0.01, #P < 0.001 compared with untreated Rag2C/C mice (ie, with no anti-MPO splenocytes and no CVF).
Figure 5. Complement depletion prevents anti-MPO splenocyte-induced glomerular necrosis and crescents. Rag2C/C mice were treated with CVF or PBS (n = 8 per group), and then, 4 hours later, were injected with 5 x 107 anti-MPO splenocytes. Pathological examinations were performed at day 13 of receiving anti-MPO splenocytes. In noncomplement-depleted Rag2C/C mice, transfer of anti-MPO splenocytes induced glomerular necrosis (long arrow) and crescent formation (short arrow) (A) (H&E), moderate glomerular IgG deposition (B) (fluorescein isothiocyanate anti-IgG), and neutrophil and macrophage infiltration (C, D). Complement-depleted Rag2C/C mice that received anti-MPO splenocytes had no lesions by light microscopy (E) (H&E), low-level IgG deposition (F), and no significant increase in glomerular neutrophils or macrophages (G, H).
Figure 6. Circulating C3 and anti-MPO IgG levels in Rag2C/C mice during induction of NCGN. Rag2C/C mice that received pretreatment of CVF or PBS (n = 8 for each group) were injected with 5 x 107 anti-MPO splenocytes. Circulating C3 and anti-MPO IgG were monitored by ELISA at different time points up to 13 days after receiving splenocytes. A: Circulating C3 levels in CVF-treated Rag2C/C mice (open squares) and control Rag2C/C mice (filled squares). B: Circulating anti-MPO IgG levels in CVF-treated Rag2C/C mice (open squares) and control Rag2C/C mice (filled squares). Bars represent the SD.
Mice Deficient in C5 Are Resistant to Anti-MPO ANCA-Induced NCGN
To confirm the observation that complement plays a role in anti-MPO-induced NCGN, C5-deficient mice (n = 7) were injected with anti-MPO IgG and compared with WT mice (n = 8) injected with the same dose of anti-MPO IgG. By day 6 after receiving anti-MPO antibodies, all WT mice developed albuminuria, leukocyturia, and hematuria, whereas none of the C5C/C mice that received anti-MPO developed significant albuminuria, leukocyturia, and hematuria (Figure 7, A and B) . All eight WT mice sacrificed 6 days after injecting anti-MPO IgG developed glomerulonephritis with crescents (mean 13% of glomeruli with crescents) and necrosis (mean 5% of glomeruli with necrosis) (Figures 7C and 8A) . In contrast, none of the C5C/C mice injected with the same dose of anti-MPO IgG had glomerular necrosis or crescents (Figures 7C and 8B) . At day 6, immunofluorescence microscopy of glomeruli showed a paucity of glomerular IgG and C3 in WT mice and no IgG or C3 staining in C5C/C mice; and ELISA showed similarly high levels of circulating anti-MPO IgG in both WT and C5-deficient mice (data not shown).
Figure 7. Lack of C5 blocked induction of NCGN by anti-MPO IgG. WT (n = 8) and C5C/C (n = 7) mice were injected intravenously with anti-MPO IgG. Six days after injection, urine and kidney samples were taken and examined. A and B: Urine albumin was determined by ELISA (A), and leukocyturia and hematuria were analyzed by dipsticks and expressed as the mean on a scale of 0 (none) to 4 (severe) (B). C: The extent of glomerular crescents and necrosis were expressed as the mean percentage of glomeruli with necrosis and crescents. Bars represent the SD. *P < 0.004.
The Alternative Pathway but Not the Classic or Lectin-Binding Pathway Is Required for Induction of NCGN by Anti-MPO IgG
To gain insight into the relative roles of the three different pathways of complement activation in the mediation of anti-MPO induced NCGN, we evaluated the induction of NCGN by anti-MPO IgG in mice deficient in C4 or factor B (fB). C4 is required for activation through both the classic pathway and the lectin-binding pathway; whereas fB is required for activation through the alternative pathway. WT (n = 8), C4C/C (n = 6), and fBC/C (n = 8) were injected intravenously with the same nephritogenic dose of anti-MPO IgG. Six days after injection, all WT mice and C4C/C mice developed albuminuria, leukocyturia, and hematuria; however, none of the fBC/C mice developed urine abnormalities outside the reference range (Figure 9, A and B) . Histological examination of glomeruli on day 6 after injection of anti-MPO IgG revealed that all WT and C4C/C mice had glomerular necrosis and crescents of similar severity (Figures 9C and 10) . Crescents involved an average of 16.7% of glomeruli in WT mice and 17.6% in C4C/C mice. Necrosis involved an average of 7.3% of glomeruli in WT mice and 8.7% in C4C/C mice. In striking contrast, none of the fBC/C mice that received the same dose of anti-MPO IgG developed renal lesions (Figures 9C and 10) . Anti-MPO IgG serum levels in WT, C4C/C, and fBC/C mice determined by ELISA were the same in all three groups of mice on day 6 (Figure 9D) .
Figure 9. Mice deficient in fB but not C4 were resistant to anti-MPO IgG-induced NCGN. WT (n = 8), fBC/C (n = 8), and C4C/C (n = 6) mice were injected intravenously with anti-MPO IgG and examined 6 days later. A and B: WT and C4C/C mice that received anti-MPO IgG showed albuminuria, leukocyturia, and hematuria, but fBC/C mice that received anti-MPO antibody had no urine abnormalities. Crescents and necrosis were observed in glomeruli of all WT and C4C/C mice that received anti-MPO IgG. No glomerular injury was seen in fBC/C mice injected with same amount of anti-MPO IgG. C: The extent of glomerular damage was expressed as the mean percentage of glomeruli with crescents and necrosis. D: Circulating anti-MPO antibody titers determined by ELISA were similar in WT, fBC/C, and C4C/C mice. Bars represent the SD. *P < 0.005.
Stimulation of Human Neutrophils by ANCA IgG Causes Complement Activation
We hypothesized that activation of neutrophils by ANCA IgG results in the release of factors that cause complement activation, which in turn amplifies the recruitment and activation of more neutrophils. To test this hypothesis, we incubated TNF--primed normal human neutrophils with IgG isolated from patients with MPO-ANCA (anti-MPO) (n = 6) or PR3-ANCA (anti-PR3) (n = 8), or IgG from ANCA-negative healthy controls (n = 8). After 15 minutes, the suspensions were centrifuged and the supernatants from the reaction mixtures were added to normal human serum from the donor of the neutrophils. Complement activation was determined by ELISA measurement of C3a generation. Figure 11 demonstrates that anti-MPO IgG and anti-PR3 IgG but not control IgG induced neutrophils to release factors that caused complement activation with generation of C3a. An analysis of variance comparing the ranked values of the three groups gave a P value of 0.0016. Using a Duncan??s multiple range test both the MPO-ANCA and PR3-ANCA groups had statistically greater complement activation than the control group; however, the MPO-ANCA group was not different from the PR3-ANCA group. A nonparametric version of the analysis of variance (Kruskal-Wallis test) gave a P value of 0.0058, which further confirmed the differences between the groups. Anti-MPO IgG, anti-PR3 IgG, and control IgG in the absence of neutrophils did not cause C3a generation (Figure 11) . TNF--primed neutrophils incubated under the same conditions in the absence of any IgG did not release factors that activated complement. Thus, ANCA-induced activation of human neutrophils results in the release of factors that cause complement activation.
Figure 11. Incubation of TNF--primed normal human neutrophils with human anti-MPO IgG or anti-PR3 IgG caused release of factors that caused complement activation in normal serum as detected by generation of C3a. Normal TNF--primed neutrophils were first incubated with IgG and then the supernatant was reacted with normal serum and the activation of complement measured by C3a ELISA. C3a generation is expressed as a percentage of the mean of the results for control IgG. Anti-MPO IgG and anti-PR3 IgG caused C3a generation at 173.3 and 146.4%, respectively, compared with control IgG. The normal control replicate assays averaged 98.2%. The C3a generation by both anti-MPO and anti-PR3 ANCA was statistically significant compared with control (P = 0.0016). No C3a generation was caused by anti-MPO IgG, anti-PR3 IgG, or normal control IgG alone; or by TNF--primed neutrophils in the absence of IgG.
Microscopic polyangiitis, Wegener??s granulomatosis, Churg-Strauss syndrome, and renal limited NCGN are manifestations of ANCA-associated small vessel vasculitis and glomerulonephritis.30 The acute pathological lesions in each of these expressions of ANCA disease are characterized by extensive neutrophil infiltration, leukocytoclasia, and necrosis.18,19,30 As demonstrated in the current and previous studies,4,6,7 vascular and glomerular inflammation and necrosis that is virtually identical to human ANCA disease is produced by administration of anti-MPO IgG to mice. Neutrophils are required for the induction of this injury.7 In this study, we have demonstrated that induction of glomerulonephritis with anti-MPO IgG or anti-MPO splenocytes requires the alternative complement pathway but not the classic pathway or the lectin pathway.
The complement system complements the mediation of inflammation through three pathways that are initiated by diverse stimuli and augment inflammation initiated both by the innate and adaptive immune responses.21 The classic pathway amplifies injury initiated by antibody binding to antigen, and thus interfaces with adaptive immune responses. The classic pathway also can be activated by innate immune responses through interaction with C-reactive protein.22 The lectin pathway is activated by pattern recognition of carbohydrate ligands, such as mannose, that are expressed by infectious pathogens. The classic and lectin pathways are unidirectional and thus are not self-sustaining if the stimulus is eliminated. In contrast, the alternative pathway is self-sustaining and thus acts as an amplification loop for inflammation that continues until it is down-regulated by specific control proteins, such as factor H and factor I.23 The alternative pathway begins with amplification of constitutive low-level C3 autoactivation, called "tickover," which occurs normally and is held in check by control proteins unless an added impetus fully activates the amplification loop.
Activation of the classic, lectin, and alternative complement pathways all result in the conversion of C3 to C3b and C3a.21 The classic and lectin pathways generate the same C3 convertase (C4bC2b), whereas the alternative pathway generates a different C3 convertase (C3bBbP). Likewise, the classic and lectin pathways generate the same C5 convertase (C3bC4bC2b), whereas the alternative pathway generates a different C5 convertase (C3b2BbP). Thus, all three pathways converge at the activation of C5 to form the potent chemoattractant C5a and the membrane attack complex C5b-9. Thus C3 and C5 are required for activation of complement by each of the three pathways.
In both the anti-MPO IgG and anti-MPO splenocyte transfer mouse models of ANCA disease, pretreatment with Cobra venom factor, which causes extensive depletion of C3,31 completely blocked the induction of NCGN (Table 1) . Likewise C5C/C mice were completely protected from the induction of NCGN by anti-MPO IgG (Figure 7) . The absence of complement activation not only prevented the induction of glomerular necrosis and crescents, but also abrogated the marked influx of neutrophils and monocytes that is typical of this model (Table 1) , probably because of blockade of generation of C5a, which is a potent chemoattractant and activator of neutrophils. Rather than total absence of complement-activating capacity, there may be a critical level of complement depletion below which lesions do not develop. Future studies will be needed to determine how much depression of complement activation is required to completely block disease induction.
Both the classic and lectin pathways require activation of C4 to produce C3 convertase (C4bC2b) and C5 convertase (C3bC4bC2b). C4C/C mice are unable to generate these convertases and thus cannot activate C3 or C5. Nevertheless, there was no protection of C4C/C mice from the induction of NCGN by anti-MPO IgG (Figure 9) . This observation indicates that neither the classic nor lectin pathways of complement activation are required for the induction of this model of ANCA NCGN. This observation also substantiates the contention that ANCA disease is not a form of typical immune complex mediated inflammation, which involves activation of complement via the classic or alternative pathways.21,32 In marked contrast, the absence of the alternative pathway component factor B completely protected fBC/C mice from induction of NCGN by anti-MPO IgG (Figure 9) . This requirement for factor B in the absence of a requirement for C4 indicates that the mediation of the inflammation involves the alternative pathway but not the classic or lectin pathways.
Based on the evidence reviewed in the introduction that ANCA IgG activates cytokine-primed neutrophils8-17 and reports in the literature that activated neutrophils release factors that can activate the alternative complement pathway,24-27 we hypothesized that activation of neutrophils by ANCA IgG causes the release of factors that activate complement. This hypothesis was substantiated by demonstrating that activation of human neutrophils by human MPO-ANCA or PR3-ANCA IgG releases factors that activate complement with the generation of C3a (Figure 11) . These studies do not identify what factor or factors are responsible for the complement activation, but a number of candidates are suggested by the literature, including reactive oxygen radicals,24 MPO,25 proteases,26 and properdin.27 Properdin is synthesized by neutrophils and stored in secondary granules that are released on activation.33 C5a stimulates properdin release from neutrophils.32 Thus, ANCA activation of neutrophils could set in motion a cycle of properdin release with C5a generation that in turn causes more neutrophil recruitment, activation, and properdin release to perpetuate the cycle.
Once the alternative pathway is activated, it is self-sustaining and thus acts as an amplification loop for inflammation that continues until it is down-regulated by specific control proteins, such as factor H and factor I.23 In a microenvironment containing immune complexes, eg, ANCA antibodies bound to ANCA antigens, C3b can associate with the Fc region of immunoglobulins and be protected from regulatory proteins,34 which could further enhance the amplification of inflammatory injury at sites of ANCA disease.
In conclusion, supported by the observations reported in this article, we propose that stimulation of neutrophils by ANCA IgG caused the release of factors that activate complement via the alternative pathway, which initiates an inflammatory amplification loop that mediates the severe necrotizing inflammation of ANCA disease (Figure 12) .
Figure 12. Diagram depicting a putative pathogenic mechanism for ANCA glomerulonephritis and vasculitis. Beginning in the top left, neutrophils are primed by cytokines to express more ANCA antigens (MPO and PR3) at the surface where they can interact with ANCA antibodies. This results in neutrophil activation both by Fc receptor engagement and Fab'2 binding. ANCA-activated neutrophils release factors (eg, properdin, proteases, oxygen radicals, and MPO) that activate the alternative complement pathway with the generation of the powerful neutrophil chemoattractant C5a and the membrane attack complex C5b-9. This complement activation amplifies neutrophil influx, neutrophil activation, and vessel damage, resulting in the aggressive necrotizing inflammation of ANCA disease.
Figure 8. Six days after injection of anti-Mpo IgG, histological examination revealed glomerular necrosis and crescents (arrows) in all WT mice (A) but no glomerular lesion in C5C/C mice that received the same dose of anti-MPO IgG (B). H&E stain.
Figure 10. A and B: Crescents and necrosis were observed in glomeruli of all WT mice (A) and all C4C/C mice (B) that received anti-MPO IgG (arrows). C: No glomerular injury was seen in fBC/C mice injected with same amount of anti-MPO IgG. H&E stain.
We thank Dr. Susan Hogan for statistical analysis, Dr. Jiajin Yang for ANCA IgG, and Dr. Bei Zhang and Ms. Jue Yao for expert technical assistance.
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作者单位：From the Department of Pathology and Laboratory Medicine,* University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and the Department of Pathology and Laboratory Medicine, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, The Netherla
Although cancer cells are not generally controlled by normal regulatory mechanisms, tumor growth is highly dependent on the supply of oxygen, nutrients, and host-derived regulators. It is now established that tumor vasculature is not necessarily derived from endothelial cell sprouting; instead, cancer tissue can acquire its vasculature by co-option of pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis, or vasculogenic mimicry. The best-known molecular pathway driving tumor vascularization is the hypoxia-adaptation mechanism. However, a broad and diverse spectrum of genetic aberrations is associated with the development of the "angiogenic phenotype." Based on this knowledge, novel forms of antivascular modalities have been developed in the past decade. When applying these targeted therapies, the stage of tumor progression, the type of vascularization of the given cancer tissue, and the molecular machinery behind the vascularization process all need to be considered. A further challenge is finding the most appropriate combinations of antivascular therapies and standard radio- and chemotherapies. This review intends to integrate our recent knowledge in this field into a rational strategy that could be the basis for developing effective clinical modalities using antivascular therapy for cancer.
Until recently, vascularization of malignant tumors was considered the exclusive result of directed capillary ingrowth (endothelial sprouting). However, recent advances have been made in identifying the processes involved in angiogenesis and vascular remodeling. Consequently, the simplistic model of an invading capillary sprout has been deemed insufficient to describe the entire spectrum of morphogenic and molecular events required to form a neovascular network. Before discussing the different ways a tumor is vascularized, we should emphasize that these mechanisms are not mutually exclusive; in fact, in most cases they are interlinked, participating concurrently in physiological as well as in pathological angiogenesis. Although the various types of cancer vascularization share some molecular features and may be controlled in part by similar sets of regulatory factors, a considerable variety of differences also exists. Although the molecular regulation of endothelial sprouting has been extensively studied and reviewed in the literature, the morphogenic and molecular events associated with alternative cancer vascularization mechanisms are less understood. Therefore, this review focuses on the pathogenesis of the different forms of "nonsprouting angiogenesis" and, more specifically, on the possibilities and the potential use of novel antiangiogenic and vascular targeting strategies against alternative tumor vascularization mechanisms.
Vascularization Mechanisms in Cancer
【关键词】 alternative vascularization mechanisms
The best-known mechanism by which tumors promote their own vascularization is inducing new capillary buds from pre-existing host tissue capillaries. The first description of this process dates back to the 1970s, when Ausprunk and Folkman1 suggested the following sequence for tumor-induced capillary sprouting (Figure 1 , Alt. 1). 1) The basement membrane is locally degraded on the side of the dilated peritumoral postcapillary venule situated closest to the angiogenic stimulus, interendothelial contacts are weakened, and endothelial cells (ECs) emigrate into the connective tissue, toward the angiogenic stimuli. 2) There is formation of a solid cord by ECs succeeding one another in a bipolar fashion. 3) Lumen formation occurs by cell-body curving of a single EC or by participation of more ECs in parallel with the synthesis of the new basement membrane and the recruitment of pericytes/mural cells. The main disadvantages of this model are its inability to identify the nature and origin of the stimulus necessary for lumen formation and the assumption that dedifferentiation and redifferentiation take place during the same process, manifest in the loss and regaining of luminal-basal EC polarity. Furthermore, although it has been well established that the stimulus necessary for lumen formation comes from the developing basement membrane, according to this model, basement membrane deposition occurs after lumen formation. In the early 1990s, a different sprouting model was described2 (Figure 1 , Alt. 2). This model suggests a three-stage sequence to explain ultrastructural changes during tumor-induced endothelial sprouting. 1) There is structural alteration of the basement membrane characterized by the loss of electron density (gel-sol transition) over the entire circumference of the dilated "mother vessel" (although basement membrane components such as laminin and collagen IV can still be detected by immunohistochemistry). Partial and regulated degradation of the altered basement membrane occur only at places where EC processes (connected by intercellular junctions) are projecting into the connecting tissue. 2) Further migration of ECs, which are arranged in parallel, maintaining their basal-luminal polarity and forming a slit-like lumen, takes place continuously with the lumen of the mother vessel and sealed by intact interendothelial junctions. Basement membrane of low electron density is deposited continuously by the polarized ECs while only the very tip of the growing capillary bud is free of basement membrane material. 3) Proliferating pericytes of the mother vessel migrate along the basement membrane of the capillary bud, resulting in complete pericyte coverage of the new vessel. In parallel, the appearance of electron-dense basement membrane around the maturing capillary buds (sol-gel transition) can be observed. According to the above model, no stimulus is necessary for the induction of lumen formation, because ECs do not lose their polarity during the process.
Figure 1. Endothelial sprouting. Schematic representation of the EC sprouting models suggested by Ausprunk and Folkman (Alt. 11 ) and by Paku and Paweletz (Alt. 22 ). Red cells represent endothelial cells; brown cells are pericytes. Yellow cells are mural cells of other origin (fibroblasts or bone marrow-derived cells). See Vascularization Mechanisms in Cancer for details.
The molecular background of capillary sprouting has been extensively studied and reviewed in the literature.3 During the process, vessels initially dilate and become leaky in response to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF).4 This is mediated by the up-regulation of nitric oxide, the development of fenestrations and vesiculo-vacuolar organelles, and by the redistribution of CD31/PECAM-1 and vascular endothelial (VE)-cadherin. The so-called gel-sol transition of the basement membrane, probably mediated by matrix metalloproteases (MMPs), gelatinases, and the urokinase plasminogen activator system, could be partly responsible for the initiation of EC proliferation and migration. Ang-2 (Angiopoetin-2, a mediator of Tie-2 signaling) is involved in the detachment of pericytes and loosening of the matrix. A vast number of molecules stimulate endothelial proliferation and migration, including transforming growth factor (TGF)-ß1, tumor necrosis factor (TNF)-, members of the chemokine system and the VEGF, fibroblast growth factor, and platelet-derived growth factor (PDGF) families.3 It could be argued that integrins represent the most important adhesion receptors in migrating ECs.5 A wide variety of integrins have been shown to be expressed during sprouting, including 1ß1, 2ß1, 3ß1, 5ß1, vß5, and vß3. Perhaps the most important among them is vß3, which mediates the migration of ECs in the fibrin-containing cancer stroma and maintains the sol state of the basement membrane because of its ability to bind to MMP-2. During maturation of nascent vessels, PDGF-BB recruits pericytes and smooth muscle cells, whereas TGF-ß1 and Ang-1/Tie-2 stabilize the interaction between endothelial and mural cells.3 All in all, sprouting is controlled by a tightly regulated balance of proangiogenic factors and inhibitors: an angiogenic cytokine promotes EC proliferation, migration, or lumen formation, whereas an inhibitor interferes with these steps and modulates the proliferation or migration activity of ECs. However, individual tumor types use various combinations of proangiogenic and inhibitory cytokines.3
When tumors arise in or metastasize to a pre-existing, usually well-vascularized, tissue, their growth not only depends on expansion, like a balloon, more typical of slow-growing benign tumors, but also on the invasion of host tissue, allowing the cancer cells close contact with the surface of blood vessels. Therefore, malignant cells may initially associate with and grow preferentially along pre-existing microvessels. Until recently, however, no studies have focused on the role played by the host vasculature in the process of tumor vascularization. Although in 1987 Thompson6 had already proposed that tumors acquire their vasculature by incorporation of host tissue capillaries, the first study suggesting the existence of vessel co-option was not published until 1999 by Holash et al.7 In their model, Holash and colleagues found that co-option is limited to the initial phases of tumorigenesis.7 However, additional morphological evidence in human malignancies suggests that co-option of pre-existing blood vessels might persist during the entire period of primary or metastatic tumor growth. In cutaneous melanoma, we found that during tumor growth, there are no signs of directed vessel ingrowth; instead, these tumors appear to grow by co-opting the massive vascular plexus present in the peritumoral connective tissue.8 In non-small cell lung cancer, a putatively nonangiogenic growth pattern was observed.9 In this "alveolar type" of growth, cancer cells filled the alveoli, entrapping but not destroying the co-opted alveolar capillaries. In liver metastases of human colorectal carcinomas, different growth patterns (replacement, pushing, and desmoplastic) were observed, depending on the degree of differentiation. In replacement growth type, the architecture of the liver was preserved, and the ECs of sinusoids showed low mitotic activity. However, pushing and desmoplastic tumor types destroyed the liver architecture.10 According to our previous results in experimental hepatic metastases, during growth of sinusoidal-type metastases, invading cancer cells advance between the basement membrane and the endothelial lining of the sinusoids and evoke proliferation of ECs. This process resulted in the development of large tortuous vessels without basement membrane inside the tumor nodules. Conversely, sprouting-type angiogenesis was observed in portal-type metastases. The replacement growth pattern corresponded to sinusoidal-type metastases of undifferentiated tumors, whereas desmoplastic tumors showed similarities to portal-type metastases.11 In the pushing-type growth pattern, we recently described a mechanism for the development of blood supply and supportive connective tissue12 (Figure 2) . This process includes the proliferation of smooth muscle actin-positive stellate, but not endothelial, cells on the surface of the tumor spheroid accompanied by capillarization of the sinusoids in this region. Because of the pressure of the tumor and the proliferating stellate cells, the hepatocytes disappear from the closest vicinity of the tumor, leading to the fusion of the sinusoids and the appearance of vascular lakes at the surface of the tumor. Together with the collagen-producing cells, these vascular lakes are incorporated into the tumor, resulting in the development of vessel-containing connective tissue columns that traverse the tumor. These columns represent the main structural and functional unit, providing blood supply for the inner part of the growing metastasis. Thus, the presence of the above mechanisms further supports earlier observations that vascularization of metastases in the liver is a heterogeneous process, depending on the degree of tumor differentiation or localization of the metastases within the liver.13
Figure 2. Examples for vessel co-option. ACD: Pushing-type angiogenesis in liver metastases of colorectal cancer. A: Cross-section of a compressed invagination. SMA-expressing cells (blue fluorescence) facing the tumor tissue, hepatocytes are crowded in the middle of the invagination (pan-cytokeratin, green fluorescence). Continuous CD31 staining (red fluorescence), representing fused sinusoids (arrows), is visible in contact with the SMA-positive cells. Note the paucity of sinusoids between the hepatocytes. B: Laminin (blue fluorescence) co-localizes with 6 integrin within the columns. The column tightly packed with SMA-positive cells (red fluorescence). C: 6 integrin (green fluorescence) is present at the periphery of the column and around the central vessel. D: Schematic representation of the development of vasculature in pushing-type liver metastases. For better visibility of the vessels, hepatocytes are depicted only in the upper part of the drawings. At the early stage of the tumor development, the tumor faces normal liver architecture. As the compression of the tumor grows, the hepatocytes "step back," and fusion of the sinusoids takes place. The fused vessel, together with the newly synthesized connective tissue, is incorporated into the tumor. The pressure of the tumor results in the separation of the vessel from the liver parenchyma. The vessel in the direction of the axis of the column remains connected to the sinusoidal system of the liver. Column formation is finished by the back-to-back fusion of the basement membranes of the tumor bulges. Green, tumor; brown, hepatocytes; red, sinusoids and central vessel.
Although sprouting capillaries are more vulnerable to apoptosis than their quiescent counterparts,14 maintenance of incorporated mature microvessels depends on the survival of ECs as well. The continued survival of co-opted ECs is intimately tied to their local microenvironment and, in particular, to the presence of pericytes, survival-promoting cytokines, and extracellular matrix proteins. Thus, the molecular repertoire that ECs may use to survive during vessel co-option is diverse and may vary for a given tumor type or host environment. The major players that control this process are angiopoetins and VEGF.7 Based on the model of vessel co-option described by Holash et al7 and in other recent studies,15 Ang-1 activates Tie-2 and induces subsequent signal transduction pathways favoring EC survival, endothelial quiescence, and tumor-vessel maintenance. Conversely, Ang-2 is thought to act as a nonsignaling Tie-2 ligand that binds to endothelial Tie-2 and thereby negatively interferes with agonistic Ang-1/Tie-2 signals. In co-opted blood vessels, the up-regulation of Ang-2 disrupts the interaction between Tie-2 and Ang-1, which in turn causes the destabilization of capillary walls (ie, the detachment of pericytes from the endothelial tube).16 Once ECs are separated from pericytes, they become particularly vulnerable. In the presence of VEGF, EC survival and new vessel growth are promoted; however, the lack of stimulatory factors results in the regression of destabilized vessels.17
VEGF was first described as a survival factor for retinal ECs and has now been shown to promote survival in different EC models. This antiapoptotic and survival function of VEGF seems to depend on an interaction between vascular endothelial growth factor receptor (VEGFR)-2, ß-catenin, and VE-cadherin.18 However, targeting of VEGF has been shown to result in apoptosis only in newly formed tumor vessels and in the developing vasculature of the neonatal mouse but not that of adult mice or of quiescent tumor vascular networks.17 In summary, although cytokines responsible for EC survival could be the key molecules, their precise role in initiation and maintenance of vessel co-option still requires investigation.
Intussusceptive Microvascular Growth (IMG)
IMG refers to vessel network formation by insertion of connective tissue columns, called tissue pillars, into the vessel lumen and to subsequent growth of these pillars, resulting in partitioning of the vessel lumen (Figure 3) . This type of angiogenesis, which has been observed in a wide variety of normal and malignant tissues, is faster and more economical than sprouting, occurs within hours or even minutes and does not primarily depend on EC proliferation, basement membrane degradation, and invasion of the connective tissue.19 However, in contrast to sprouting, IMG can work only on existing vessel networks. The most important feature of IMG, therefore, seems to be its ability to increase the complexity and density of the tumor microvessel network already built by sprouting, independent of EC proliferation. In addition, IMG can provide more surface area for further sprouting. Its molecular regulation, however, is poorly understood since IMG was first described only a few years ago. Nevertheless, the role of some players is gradually becoming clearer. We know that local stimuli, such as intravascular shear stress, might induce a cascade of physiological or pathological reactions in ECs, and new capillary development by tissue pillar formation could be one of them.20 Furthermore, intussusception is certainly synchronized by several cytokines. Major candidates are those capable of mediating information between ECs or from ECs to mural cells, such as PDGF-BB, angiopoietins, and their Tie receptors, TGF-ß, monocyte chemotactic protein-1, and ephrins and Eph-B receptors.19
Figure 3. Intussusceptive microvascular growth. Schematic representation of intussusceptive microvessel growth. The first step of the process is the development of the transluminal endothelial bridge. This is followed by the reorganization of the endothelial lining, a process that is largely unknown. The division of the vessel is completed by the development of a connective tissue pillar through the vessel lumen. Red cells are endothelial cells; brown cells are pericytes. Gray, basement membrane.
After the initial stage of immature capillary network formation by sprouting, additional vascular growth and development of complex vascular beds, including their continuous remodeling and adaptation, may occur by intussusception in cancers. The absence of intense EC proliferation in IMG implies that neovascularization by this mechanism would be resistant to angiosuppressive treatment in itself.
Glomeruloid bodies (GBs) are best known in high-grade glial malignancies, where they are one of the diagnostic histopathological features of glioblastoma multiforme. However, these complex vascular aggregates have also been described in a wide variety of other malignancies.21 They are composed of several closely associated microvessels surrounded by a variably thickened basement membrane within which a limited number of pericytes are embedded. In recent studies, the presence of GBs was associated with markers of aggressive tumor behavior and significantly reduced survival in cancer patients.22 In the first animal model,23 GBs developed in mother vessels from recruitment and proliferation of ECs and pericytes (in the absence of tumor cells), and VEGF was essential for their induction and maintenance. In contrast to this model and based on our previous results in the first experimental tumor model of glomeruloid angiogenesis,24 we believe that GB formation starts immediately after tumor cell extravasation, much earlier than necrosis appears within the metastases. We found that the proliferating and migrating tumor cells are able to pull the capillaries and the adjacent capillary branching points into the tumor cell nests. This process leads to the appearance of simple coiled vascular structures that later develop into GBs with multiple narrowed afferent and efferent capillaries (Figure 4) . Despite the absence of sprouting angiogenesis, necrosis was scarce in these lesions, suggesting that the blood supply from the pre-existent vascular bed is sufficient to provide the tumor cells with oxygen and nutrients. This type of GB formation cannot be termed as true angiogenesis; it rather represents a remodeling of the existing vasculature of the host tissue. Whether GBs represent an accelerated form of angiogenesis or a dysfunctional, possibly abortive, form remains an open question. However, it cannot be excluded that "active" and "passive" types of glomeruloid angiogenesis can operate concurrently in various cancer types.
Figure 4. Glomeruloid angiogenesis. A: Experimental brain metastases stained for laminin (green fluorescence) and CD31 (blue fluorescence), 28 days following intracarotid inoculation of the A2058 human melanoma cell line. Glomeruloid bodies are connected to each other by a capillary that is very small in diameter (arrows). The outlines of the metastases are clearly visible because of the strong laminin positivity of the tumor cells (arrowheads). B: Schematic representation of glomeruloid body formation. Following extravasation, the tumor cells (green) adhere firmly to the abluminal surface of the capillary basement membrane (gray). In the first step, because of the contractile force of the tumor cell a loop develops on the capillary. Proliferating tumor cells pull the capillary inward, resulting in the development of further loops and reduction of the diameter of the capillary segment lying outside the glomeruloid body. The last drawing shows the cross-section of a fully developed glomeruloid body built by ECs (red), pericytes (brown), and tumor cells (green). Extreme large cytoplasmic projections of the tumor cells adhere to different segments of the capillary.
Postnatal Vasculogenesis: The Role of Endothelial Progenitor Cells
Vasculogenesis (defined as the in situ differentiation of vascular ECs from primitive precursor cells) has long been thought to occur only in the early phases of vascular development. Recent studies, however, have demonstrated that circulating bone marrow-derived endothelial progenitor cells (EPCs) home to sites of physiological and pathological neovascularization and differentiate into ECs (Figure 5) . EPCs may be mobilized by tumor tissue-derived cytokines from the bone marrow by a mechanism recently described by Asahara et al.25 Best characterized among these cytokines is VEGF. During tumor progression, the level of circulating VEGF has been shown to rise, and this level was found to correlate with the number of EPCs in the circulation. Furthermore, PDGF-CC promoted vascularization in part by stimulating outgrowth of EPCs. In contrast, Ang-1 was shown to reduce EPC mobilization from bone marrow (reviewed in Ref. 26 ).
Figure 5. Endothelial progenitor cells. Schematic representation of postnatal vasculogenesis. The term "EPC" encompasses a group of cells existing in a variety of stages ranging from common hemangioblasts to fully differentiated ECs. Although their putative precursors and the exact differentiation lineage of EPCs remain to be determined, to date it is widely accepted that early EPCs (localized in the bone marrow or immediately after migration into the circulation) are AC133+/CD34+/VEGFR-2+ cells, whereas circulating EPCs are positive for CD34 and VEGFR-2, lose AC133, and begin to express cell surface markers typical of mature ECs such as CD31, VE-cadherin, and von Willebrand Factor (vWF).
After homing, ie, after adhesion and insertion of EPCs into the monolayer of surrounding mature vascular ECs, additional local stimuli may promote the activation of local endothelium to express adhesion molecules to recruit EPCs. This process may be completed by mechanisms not yet elucidated. In addition to the physical contribution of EPCs to newly formed microvessels, the angiogenic cytokine release of EPCs may be a supportive mechanism to improve neovascularization as well.27 It is also important to note that Lyden et al recently identified VEGFR-1+ hematopoietic progenitor cells that multiply in the bone marrow, mobilize to the peripheral blood along with VEGFR-2+ EPCs, and incorporate into pericapillary connective tissue, thus stabilizing tumor vasculature.28 More interestingly, these cells seem to home in before the tumor cells arrive, promoting metastatic growth by forming niches where cancer cells can locate and proliferate.29
Although EPCs obviously participate in the vascularization process of malignant tumors, it is still unclear whether they are essential for these processes or what the relative contribution of EPCs is compared with that of in situ proliferating ECs. Moreover, it has yet to be determined whether EPCs can be targeted to treat certain types of malignancies, or alternatively??as they are endowed with the capacity to home to the tumor vasculature??can be used to deliver toxins or vascular-targeting agents.
"Vasculogenic mimicry" is defined by the unique ability of aggressive melanoma cells to express an EC phenotype and to form vessel-like networks in three-dimensional culture, "mimicking" the pattern of embryonic vascular networks and recapitulating the patterned networks seen in patients?? aggressive tumors correlating with a poor prognosis.30 Comparative global gene analyses of aggressive and poorly aggressive human cutaneous and uveal melanoma cell lines unexpectedly revealed the ability of aggressive tumor cells to express genes (and proteins) associated with multiple cellular phenotypes and their respective precursor stem cells, including endothelial, epithelial, pericyte, fibroblast, and several other cell types.31-33 These new and intriguing findings support the premise that aggressive melanoma cells acquire a multipotent, plastic phenotypea concept that challenges our current thinking on how to target tumor cells that can possibly masquerade as other cell types, particularly with embryonic stem cell-like properties. The etiology of the melanoma vasculogenic phenotype remains unclear; however, it seems to involve dysregulation of the lineage-specific phenotype and the concomitant transdifferentiation of aggressive cancer cells into other cell types??such as endothelial-like cells. Vasculogenic mimicry has been confirmed in breast, prostate, ovarian, chorio-, and lung carcinomas; synovial-, rhabdomyo-, and Ewing sarcomas; and phaeochromocytoma.34 Expression profiling studies revealed that the most significantly up-regulated genes by aggressive melanoma cells include those that are involved in angiogenesis and vasculogenesis, such as the genes encoding VE-cadherin, erythropoietin-producing hepatocellular carcinoma-A2 (EphA2), MMPs, and laminin 52 chain (LAMC2). These molecules, with their binding partners, are a few of the factors required for the formation and maintenance of blood vessels and also for vasculogenic mimicry in melanomas. Perhaps equally significant is the down-regulation of the gene MART-1 (melanoma antigen recognized by T cell 1, also called Melan-A), a classic marker for melanocytes and melanoma, by aggressive melanoma cells. The concept of vasculogenic mimicry was developed further to include the existence of a fluid-conducting, laminin-containing extracellular matrix meshwork, providing a site for nutritional exchange for aggressive tumors, and therefore possibly preventing necrosis (Figure 6) .34,35 Functional studies revealed the close association of tumor-cell-lined networks with angiogenic mouse vessels at the human-mouse interface and the cooperation between the two systems.36,37 The molecular dissection of the physiological mechanisms critical to the function of the fluid-conducting meshwork revealed the biological relevance of the up-regulated expression of tissue factor pathway-associated genes??essential for the anticoagulation properties of the intratumoral, extracellular matrix-rich extravascular fluid-conducting pathway. Gene profiling, protein detection, and immunohistochemistry validation demonstrated up-regulation of tissue factor (TF), TF pathway inhibitor 1 (TFPI-1), and TFPI-2??critical genes that initiate and regulate the coagulation pathways??in aggressive, as opposed to poorly aggressive, melanoma. It was found that TFPI-2 contributes to vasculogenic mimicry and endothelial transdifferentiation by melanoma cells, whereas TFPI-1 has anticoagulant functions for perfusion of fluid-conduction meshworks formed by TF-expressing melanoma cells. Additional studies have focused on the signal transduction pathways that regulate blood vessel formation and stabilization during vasculogenesis and angiogenesis, addressing critical signaling events that regulate melanoma vasculogenic mimicry and their endothelia-like phenotype.38-40 It was demonstrated that VE-cadherin and EphA2 were co-localized in cell-cell junctions and VE-cadherin can regulate the expression of EphA2 at the cell membrane by mediating its ability to become phosphorylated through interactions with its membrane-bound ligand, ephrin-A1. These studies illuminate a novel signaling pathway that could be potentially exploited for therapeutic intervention. Additional investigation uncovered the role of phosphoinositide 3-kinase (PI3K) as a critical regulator of vasculogenic mimicry, specifically affecting membrane type-1 MMP (MT1-MMP) and MMP-2 activity. Both MMPs are essential for the process of vasculogenic network formation by aggressive melanoma tumor cells, and the downstream effect on the cleavage of laminin 52 chain into the 2' and 2x promigratory fragments.38,39 Furthermore, these results showed that blocking PI3K resulted in abrogation of vasculogenic mimicry. Most recent studies have identified focal adhesion kinase (FAK)-mediated signal transduction pathways to promote not only the aggressive phenotype but also vasculogenic mimicry of melanoma cells as well.40 In addition, expression of a negative regulator of FAK signaling, the FAK-related non-kinase in aggressive melanoma cells, resulted in an inhibition of melanoma vasculogenic mimicry concomitant with a decrease in melanoma cell invasion and migration. This biological effect was mediated in part through an extracellular signal-regulated kinase 1/2 signaling pathway that resulted in a down-regulation of urokinase and MMP-2/MT1-MMP activity.40 These results suggest that FAK may serve as a new target for therapeutic intervention in treating aggressive melanomas with capabilities for vasculogenic mimicry.
Figure 6. Vasculogenic mimicry. This diagram represents the current interpretation of data generated from several studies involving the use of tracers and perfusion analyses of mice containing aggressive melanoma cells (green) during tumor development. The endothelial-lined vasculature is closely apposed to the tumor cell-formed fluid conducting meshwork, and hypothetically, it is presumed that as the tumor remodels, the vasculature becomes leaky, resulting in the extravascular conduction of plasma. There is also evidence of a physiological connection between the endothelial-lined vasculature and the extravascular melanoma meshwork.
Antivascular Therapy of Cancer
It has been over 30 years since Judah Folkman hypothesized that tumor growth is angiogenesis dependent.41 Subsequent research has led to the identification of several regulators of angiogenesis, some of which represent therapeutic targets. However, although antivascular agents are often highly active in preclinical studies, recent clinical trials including these agents have been both encouraging and disappointing. Because of the predominant role of capillary sprouting and its main molecular mediator VEGF in tumor vascularization, inhibition of VEGF seems to be necessary but is probably insufficient to halt tumor progression permanently in many cancer types. Due to the existence of multiple vascularization mechanisms and angiogenic signaling pathways, inhibition of just a single pathway will presumably trigger alternative vascularization mechanisms and additional growth factor pathways. Consequently, application of antivascular therapy in cancer patients requires the identification of the individual vascularization profile and the molecular machinery behind the vascularization process and, furthermore, the individualization of antivascular therapy to realize any potential benefits.42,43 In the second part of this review, we will briefly summarize the antivascular therapies that are currently being tested in the clinic. Subsequently, we will give an overview of how these classes of agents can be incorporated in the current multimodality of anticancer strategies. Finally, we will discuss potential novel approaches that enforce tumor regression by exploiting the emerging basic knowledge of tumor vascularization.
Antivascular Strategies in Cancer Therapy: Current Status of the Clinical Development
Any classification of antivascular strategies is difficult, with overlap in several features. However, the main categories of these approaches that have been developed are angiosuppressive (anti-angiogenic agents) and vascular-targeting therapies (vascular-disrupting agents).44 Although metronomic chemotherapy (MCT) uses conventional cytotoxic drugs, the main targets of this strategy are the tumor ECs. This is the reason that Browder et al45 coined the term "anti-angiogenic chemotherapy" to describe this treatment and why MCT is discussed here.
It is beyond the scope of this review to discuss all drugs that affect tumor capillaries. Therefore, we concentrate here on the agents that are at a more advanced stage of clinical development.
Angiosuppressive Therapy (Antiangiogenic Agents)
This approach is motivated by the fact that neoangiogenesis in cancer requires the induction of EC proliferation by specific or nonspecific mitogens. These agents target the production of endothelial mitogens, the mitogens themselves, their endothelial receptors, the associated signaling pathways, the endothelial integrins and the MMPs46 (Table 1) . Consequently, it is most probable that angiosuppressive therapy can only be applied when cancer vascularization involves EC sprouting and/or postnatal vasculogenesis (Table 2) .
Table 1. Examples of Antivascular Agents in Clinical Development
Table 2. Theoretical Strategy of Antivascular Therapy of Cancer According to the Stage of Tumor Progression and to the Mechanisms of Vascularization
Despite the promising preclinical results with these agents, in the early clinical trials positive responses in patients were rarely seen. The clinical breakthrough for angiosuppressive therapy came from a phase III trial demonstrating a significantly prolonged survival when bevacizumab, an anti-VEGF antibody, was used with chemotherapy in metastatic colorectal cancer patients.47 Based on these results, bevacizumab became the first antiangiogenic agent to be approved by the United States Food and Drug Administration (FDA) for cancer treatment. In subsequent phase III trials, bevacizumab in combination with standard chemotherapy improved overall survival in lung cancer patients and progression-free survival in breast cancer patients.42 In addition, it has been reported to be active in patients with metastatic renal-cell cancer as monotherapy (benefit in progression-free survival but not in overall survival).48
Further clinical success was obtained recently with broad-spectrum multitargeted agents that target VEGF receptors and other tyrosine kinases present in endothelial and cancer cells (Table 1) . Phase III trials have demonstrated the efficacy of SU11248/sunitinib and chemotherapy in nonsmall cell lung cancer patients. Interestingly, replacing bevacizumab with similar tyrosine kinase (TK) inhibitors, such as PTK787/ZK 222584/vatalanib (targeting VEGFR-1, -2, -3; PDGFR-ß, and c-Kit), in the combined regimen did not result in similar efficacy in chemotherapy-naive or previously treated colorectal cancer patients.49 However, the clinical success of bevacizumab, sunitinib, and sorafenib as novel medicines for the treatment of cancer patients has confirmed the relevance of angiogenesis research and has stimulated the search for novel and more effective antiangiogenic approaches. Accordingly, various angiosuppressive strategies are being actively investigated, most of which are registered with the clinical trials database of the National Cancer Institute (http://www.nci.nih.gov/clinicaltrials).
Vascular Targeting Therapy (Vascular Disrupting Agents; VDAs)
Vascular targeting therapy (including anti-EC antibodies and ligand based and small molecule VDAs; Table 1 ) recognizes the fact that clinical diagnosis of cancer frequently occurs when the tumor tissue has already established its vasculature.44,46 This strategy relies on ability of VDAs to distinguish the ECs of tumor capillaries from normal ones based on their different phenotype, increased proliferative potential and permeability, and inherent dependence on the tubulin cytoskeleton. VDAs cause selective and rapid shutdown of the established tumor capillaries, resulting in extensive cancer cell death in the central areas of tumors, although they leave the perfusion in peripheral tumor regions relatively intact.44,50 It is evident from the mechanism of VDAs that the effects of these drugs do not depend on the type of vascularization occurring in a given cancer. Based on promising preclinical developments, several VDAs have entered clinical development.51
MCT and Its Antivascular Effects
Among the different antivascular strategies, MCT merits particular mention. MCT refers to the close, even daily, administration of chemotherapeutic drugs in doses below the maximum tolerated dose, over prolonged periods, and with no extended drug-free breaks. Phase II trials of MCT, sometimes applied in combination with antiangiogenic drugs, have yielded promising results in adult patients with advanced cancer.52,53 Furthermore, pediatric oncologists successfully use a metronomic-like modality of chemotherapies called "maintenance chemotherapy" to treat various pediatric malignancies such as acute lymphoblastic leukemia, neuroblastoma, or Wilms?? tumor; however, the anti-angiogenic background of maintenance chemotherapy is poorly described.54
Although cytotoxic effects of MCT in the tumor parenchyma could still contribute to the observed efficacy of metronomic regimens, preclinical studies suggest that the primary targets of MCT are the tumoral ECs. Low-dose chemotherapy affects tumor capillaries directly (growth arrest and apoptosis of activated ECs) but also induces the production of an angiogenesis inhibitor thrombospondin-1 and suppresses the mobilization of EPCs.52
As mentioned above, several phase I and II studies were performed involving low, continuous doses of cytotoxic drugs, with encouraging results.53 However, the clinical benefits of MCT remain to be validated in randomized prospective phase III trials. There is also a need for surrogate markers to help define the optimal dose of this approach. Circulating ECs55 and EPCs56 have been used successfully as markers in preclinical and early clinical studies but have not yet been validated clinically. Further challenges are the definition of valid clinical endpoints, the confirmation of long-term safety of MCT, and the identification of suitable antiangiogenic agents and VDAs to be combined with MCT. Finally, it will be important to determine the types of vascularization that might be the most responsive to this therapy. MCT is probably more effective in EC sprouting, postnatal vasculogenesis, IMG, and vasculogenic mimicry (Table 2) . However, detailed clinicopathologic analysis is needed to confirm this hypothesis.
Considerations for Combination Treatment Strategies
Because antivascular agents and traditional anticancer strategies have distinctive target cells and mechanisms of action, it should be possible to achieve an increase in therapeutic efficacy with little or no increase in toxicity. In fact, although some antivascular agents have demonstrated activity as monotherapies, most human trials to date indicate that they are most effective when combined with conventional antitumor strategies, especially chemotherapy.42,43
Combination of Angiosuppressive and Chemo- and/or Radiation Therapy
Angiosuppressive therapy reduces cancer growth by suspending the blood supply, resulting in hypoxia. Because hypoxia itself is a major cause of ineffective chemo-irradiation therapy,57 one would expect that a further decrease in intratumoral oxygen levels would deteriorate the efficacy of a cytotoxic regime, but experimental and clinical data do not support this scenario. In several preclinical models, a combination of cytotoxic drugs (taxanes, cisplatin, or 5-fluorouracil) with angiogenesis inhibitors (TNP470, endostatin, SU11248) produced at least additive but in certain cases synergistic antitumoral effects.46 Thalidomide, a still ill-defined angiogenesis inhibitor, has also been shown successful preclinically in combination with standard anticancer regimes in solid tumors.58 In addition to experimental data, there are now clinical examples of the improved efficacy of chemotherapy in combination with an angiosuppressive agent. As mentioned above, bevacizumab in combination with chemotherapy improved overall survival in colorectal and lung cancer patients and progression-free survival in breast cancer patients (see review42 ). In addition, the combination of bevacizumab and chemotherapy was found to be active in pancreatic59 and ovarian60 cancer patients.
There are several explanations for the improved efficacy. An obvious effect of angiogenesis inhibitors is the decrease in interstitial pressure in cancer tissue improving the delivery of cytotoxic agents. Furthermore, a hypothesis called "normalization of tumor vasculature" was put forth by Jain and colleagues recently to explain the clinical effects of antiangiogenic agents.42 According to this theory, tumor vasculature is structurally insufficient to provide maximal blood supply for cancer cells as a result of capillary leakiness and tortuosity. Because the key regulator cytokine family of tumoral vessels is the VEGF/VEGFR system, targeting it could potentially help in the "normalization" of tumor vasculature and in the improvement of the delivery of chemotherapeutic agents.42 Accordingly, recent experimental data indicate that anti-VEGF therapy induces rapid alterations in tumor vasculature. Within a few hours, EC proliferation is halted, luminal stability vanishes, and circulation ceases in tumor capillaries. Some ECs undergo apoptosis and disappear. Remaining capillaries lack endothelial fenestrations and have reduced VEGFR-2 and VEGFR-3 expression.61 Thus, inhibition of VEGF signaling devastates some tumor capillaries and transforms others into a more normal phenotype.42
Further mechanisms for the additional benefits experienced for combined chemo- and angiosuppressive therapy might be the direct killing of proliferating ECs and/or the inhibition of the mobilization/viability of EPCs by cytotoxic drugs. Results of preclinical studies support this hypothesis. On the other hand, VEGF inhibition might have direct cytotoxic effects on tumor cells that aberrantly express VEGF receptors and depend to some extent on VEGF for their survival. Finally, it has also been suggested recently that antiangiogenic agents prevent rapid cancer cell repopulation during the break periods between courses of chemotherapy (see review43 ).
Experimental studies indicate that antiangiogenic therapy in combination with irradiation is an encouraging concept for the improvement of the radiation response of tumors.62 In addition, recent discoveries show that the EC layer of the tumor vessels is one key target of radiotherapy.63 In fact, the antivascular effect of radiotherapy predicts its anti-cancer effect.64 Thus far, although early phase human trials have also yielded promising results, there are no large phase III trials known in which such combinations were successfully applied. Nevertheless, the discovery of the "normalization window" of angiosuppressive agents when combined with radiotherapy in preclinical models65 suggests that it would be as difficult to design a successful combination strategy with radiation as with chemotherapy.
In this normalization window (the time period during which the vasculature normalizes and hypoxia decreases), the antiangiogenic drugs improve the efficacy of chemoradiotherapy.42 Although these studies were performed in experimental tumor systems, one may expect a similar effect on the human tumor vasculature and oxygenation. However, intratumoral hypoxia, responsible for chemo- and radiotherapy resistance and triggering molecular pathways that promote cancer progression, is due not only to the inefficient blood supply by the abnormal tumor vessels but to the systemic anemia of the host as well.66 Unfortunately, although the oxygen tension of experimental tumors tends to rise with increasing Hb levels67 and treatment with recombinant human erythropoietin (rHuEpo) significantly reduces the risk for red blood cell transfusions in cancer patients, correction of anemia with rHuEpo does not necessarily improve survival of cancer patients.66 The issue of Epo/EpoR co-expression in tumor cells and EpoR expression in ECs is critical in this perspective. The expression of EpoR in tumor cells has raised the possibility that exogenous rHuEPO may directly influence cancer cell proliferation, apoptosis, or sensitivity to chemoradiation therapy. In addition, the EpoR expression in ECs has suggested potential effects of Epo on the tumor capillaries, such as the stimulation of angiogenesis.68 However, as it has been suggested by experimental studies, the overall direct effect of Epo-EpoR signaling on tumor progression and therapy is not a straightforward one. For instance, rHuEpo administration has recently been shown to be associated with decreased intratumoral VEGF expression, remodeling of tumor capillaries, and increased chemosensitivity to 5-fluorouracil treatment of human tumor xenografts.69 In a preclinical myeloma model, rHuEpo induced tumor regression and antitumor immune responses.70 In addition, human kidney carcinoma and myelomonocytic leukemia cell lines treated with rHuEpo exhibited an increase in apoptosis in response to chemotherapy.71 Overall, these findings warrant additional experimental and clinical research of rHuEpo to clarify further the risks of its use as well as optimize its known or potential benefits.
Combination of VDAs and Chemo- and/or Radiation Therapy
VDAs work best in the poorly perfused hypoxic central tumor areas, leaving a viable rim of well-perfused cancer tissue at the periphery, which rapidly regrows.50 Consequently, responses of tumors to VDAs given as single agents have been poor; however, combination therapy with chemoradiotherapy, which targets cancer cells at the tumor periphery, has produced promising responses in preclinical models. Nevertheless, the timing and sequencing of VDAs and chemo-irradiation therapies are important in such treatments. By far the greatest enhancement was observed when the VDA was administered within a few hours after chemo- and/or irradiation therapy. Based on these experimental results, the VDA compounds 5,6-dimethylxantlenone-4-acetic acid (DMXAA) and combretastatin A4 phosphate (CA4P) are being evaluated in human phase II trials in combination with conventional anticancer therapies.51
Combination of Angiosuppressive and Vascular Disrupting Agents
Because both angiogenesis and the integrity of the existing vasculature are critical to tumor progression and survival, dual targeting of the tumor vasculature would seem to have considerable promise. Preclinical results demonstrated that this strategy could significantly enhance therapeutic response beyond that achieved with either antivascular agent alone.51 One example of this strategy is the combination of the inhibitor of VEGFR2-associated TK ZD6474 with the microtubulin-disrupting VDA ZD6126.72 Further combinations that are under preclinical testing include the combination of OXi-4503, CA4P, and DMXAA with bevacizumab. Clinical testing of combined antivascular therapy has started with the recent initiation of a phase I human trial combining CA4P with bevacizumab.51
Theoretical Considerations for Designing Antivascular Therapy of Cancer
From the discussion above it is clear that the combination of either angiosuppressive or the vascular disrupting therapies with conventional chemoradiotherapy of cancer is highly problematic and must be carefully designed in cases where the sequence of the multiple types of agents might be critical. The molecular machinery behind the vascularization process and type of tumor vascularization are further issues that have to be taken into account. Thus, an efficient antivascular cancer therapy could be designed based on the identification of the molecular targets of the angiogenic geno-/phenotype (molecular pathway-based approach) or on the vascularization mechanism (vascular mechanism-based approach). However, it is most probable that the two approaches would have to be combined. We propose below a rationale for the design of antivascular strategies with the aim that such consideration may help to improve the clinical efficacy of these novel therapies.
Molecular Pathway-Based Antivascular Therapy of Cancer
Because of its pivotal role in neovascularization, the VEGF/VEGFR axis has been a major target of basic and clinical research. It is, therefore, not surprising that most of the antivascular strategies currently in clinical development focus on inhibition of VEGF signaling.46,73 However, the development of the angiogenic phenotype of cancer is characterized by several interconnected pathways. One of the major triggers of this phenotype is tissue hypoxia, which is responsible for the activation of gene expression of angiogenic cytokines through up-regulation of the transcription factor hypoxia inducible factor-1 (HIF1-). Nevertheless, HIF-1 may already be active in particular cancers due to hyperactive growth factor signaling or genetic alteration of the HIF1 gene itself or its regulators .74 Because HIF-1 plays such a central role in triggering numerous pathways responsible for cancer progression, disruption of the HIF-1-mediated pathways is expected to cause cancer cell death due to a combination of metabolic dysregulation and reduced microvessel growth. The aim of anti-HIF-1 therapy (used as an antivascular modality) therefore might be to cause the angiogenic phenotype of cancer to revert to a less angiogenic one, thereby preventing the production of the major angiogenic cytokines.75 HIF1 can be inhibited by guanyl cyclase or HSP90 inhibitors and even by the targeting of topoisomerase-1, and several of such agents are in clinical trials (Table 1) . However, none of the currently available inhibitors seems to disrupt the HIF-1 pathway as their exclusive target.75 If the additional targets of nonselective HIF-1 inhibitors are also involved in cancer progression, these agents could be therapeutically beneficial, but inhibition of the pathways involved in normal cellular homeostasis could result in an unacceptable toxicity profile. Therefore, the design of more specific HIF-1 targeting agents is the focus of current research efforts. However, it is also important to note that HIF-1 targeting alone may not be enough to halt angiogenesis and tumor progression, as HIF-independent pathways may bypass or overcome HIF inhibition. Consequently, a combination of anti-HIF agents with conventional anticancer modalities or other molecular-targeted drugs may be required.
VEGF expression is not only associated with hypoxia or VHL mutations but also is influenced by a broad spectrum of onco- and tumor suppressor genes. A growing body of evidence suggests that inactivation of tumor suppressor genes such as p53 and PTEN and activation of oncogenes such as Ras, c-Src, EGFR, human epidermal growth factor receptor 2 (HER-2), FBJ murine osteosarcoma viral oncogene homolog (FOS), neurotrophic receptor tyrosine kinase B (trkB), V-p3K, and Bcl-2 are connected to the up-regulation of VEGF. Consequently, molecular targeting of these regulators is also a potential strategy for indirectly modulating the VEGF/VEGFR axis.73 For example, based on the results of recent clinical trials, cetuximab (a monoclonal antibody that binds to EGFR with high specificity) induces a significant decrease in circulating VEGF levels in colon cancer patients,76 or likewise, imatinib mesylate (a specific inhibitor of Bcr/Abl protein TK activity) reduces VEGF plasma concentration77 and bone marrow microvessel densities78 in patients with chronic myeloid leukemia. However, preclinical and early phase clinical data demonstrate that the addition of anti-VEGF therapy to anti-EGFR therapies generates further beneficial effects on angiogenesis inhibition and tumor reduction.42 This suggests that inhibiting upstream signaling of VEGF does not necessarily provide the same benefit as the direct targeting of it and, more importantly, that the dual targeting of cancer and endothelial cells might become a successful practice in clinical oncology.
Mechanism-Based Antivascular Therapy of Cancer
A proposal for the application of antivascular therapies according to the alternative vascularization mechanisms in cancer is summarized in Table 2 . Probably the most important aspect of mechanism-based antivascular therapy is its strict dependence on the stage of tumor progression. Interestingly, antivascular therapy may have an effect at the very early stages of tumor growth. This idea was put forward by Li et al,79 who analyzed the earliest events that take place during the onset of tumor neovascularization and found that individual tumor cells exhibited a chemotaxis-like growth pattern toward the host vasculature. When the tumor cell population reached approximately 60 to 80 cells, clear evidence of perivascular tumor cell migration (ie, vessel co-option), and host vessel dilation was observed. Moreover, in a mouse model of glomeruloid angiogenesis, our group found that even single tumor cells can induce radical changes in the host tissue vasculature24 (Figure 4) . These observations are important in two ways. First, they suggest that anti-invasive agents (which are not yet available clinically) may have a therapeutic effect on the interaction between cancer and endothelial cells and, consequently, on the processes of vessel co-option and glomeruloid angiogenesis. Second, the finding that single tumor cells can induce increased capillary permeability/tortuosity highlights the need for application of angiosuppressive/antiangiogenic therapy at the very early stages of cancer progression. These considerations may be true for the next step of tumorigenesis (pre-angiogenic phase) as well.
After the onset of "angiogenic switch," elevated serum levels of angiogenic growth factors in cancer patients may activate and mobilize EPCs to support local microvessel growth.26 If we accept this assumption, then, in addition to angiogenesis inhibitors and metronomic chemotherapy,52 ligand-based, EPC-specific VDAs may also be useful in eliminating circulating EPCs throughout the further stages of tumor progression (Table 2) . Furthermore, because IMG can be effective only in tumor capillary networks already built by other vascularization mechanisms (mainly sprouting and vessel co-option), steps should be taken to impede the additional increase in the density of the tumor tissue capillary bed following the angiogenic switch. This could be achieved by the use of VDAs and/or "metronomic chemotherapy," which both target the cytoskeleton of ECs responsible for the remodeling of capillary walls.
Because ischemic milieu is what forces aggressive tumor cells to express endothelial genes and form vascular channels,33,80 the initiation of this mechanism is most likely simultaneous with the angiogenic switch. Therefore, when vasculogenic mimicry plays a role in the nutrient supply in cancer, besides the use of ligand-based VDAs against cancer cells with endothelial phenotype, targeting those pathways responsible for the development of this mechanism such as Eph2A, PI3K, or FAK seems to be an appropriate strategy. On the other hand, metronomic scheduling of chemotherapy52 may also effectively target cancer cells with vasculogenic geno/phenotype when both physiological angiogenesis inhibitors and angiosuppressive drugs are unable to modify this vascularization mechanism.80
The next stage of malignant progression is when tumor tissue reaches macroscopic size detectable by simple or sophisticated imaging techniques. As we know, for cancer survival "the edge is the future and the center is history,"81 because active tumor vascularization processes, resulting in vascular networks built by defective new capillaries, occur mainly, though not exclusively, at the tumor periphery. Consequently, at this stage the main target of antivascular therapies is the invading front of the cancer tissue. However, since in addition to causing chemo- and radiotherapy resistance, reduction of vascularity in the center of tumors can lead to the appearance of more aggressive/highly metastatic hypoxia-resistant cancer cells and to the induction of vasculogenic mimicry, when designing antivascular strategies central tumor areas cannot be neglected. We should emphasize, therefore, that in the case of clinically detectable tumors the whole range of antivascular weapons should be used theoretically. Although antiangiogenic agents targeting proliferating ECs could possibly be the key drugs at the tumor boundary, established tumor vasculature might well be attacked by VDAs and/or metronomic chemotherapy in the central tumor areas. Altogether, it seems feasible that antivascular therapy in tumors can only be successful if the entire vascular network and all of the possible vascularization mechanisms are targeted and, furthermore, if the phenotypic analysis of tumor capillaries/vascular channels is adequately performed.
Although tumors, as other tissues, require a vessel network supplying them with blood, tumor vasculature is not necessarily derived by EC proliferation and sprouting of new capillaries. In addition to alternative vascularization mechanisms, the novel antivascular strategies must be harmonized with the stage of tumor progression and with the molecular mechanism responsible for the angiogenic phenotype. A further challenge is to combine antivascular strategies with the existing therapeutic regimes in at least an additive manner. We have provided here proposals for a rational application of antivascular agents with the notion that these therapies have to be individually tailored in a given cancer type. Better understanding of the different vascularization mechanisms of the various cancer types will certainly help to fine-tune these novel anti-cancer strategies.
We are grateful to Sirpa Jalkanen (Medicity, University of Turku, Turku, Finland) and to S?ndor Eckhardt (National Institute of Oncology, Budapest, Hungary) for critique and suggestions.
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作者单位：Bal?zs Döme*, Mary J.C. Hendrix, S?ndor Paku, J?zsef T?v?ri and J?zsef T?m?rFrom the Department of Tumor Biology and Thoracic Oncology,* National Koranyi Institute of Pulmonology, Budapest, Hungary; Department of Tumor Progression, National Institute of Oncology, Budapest, Hungary; First Instit
Aug. 10, 2007 -- Looking for a safe substitute for cigarettes? Smokeless tobacco isn't the way to go, according to a new report.???????
The report shows that smokeless tobacco may be as bad -- or worse -- than cigarettes, in terms of exposing users to certain cancer-causing chemicals.
"Our results raise serious questions about the strategy of using smokeless tobacco as a substitute for cigarette smoking. Long-term nicotine replacement therapy may be a better option," write the researchers, who included the University of Minnesota's Steven Hecht, PhD.
"This study lends evidence to support the notion that oral use of tobacco actually provides a more efficient means for delivering certain carcinogens into the body through the bloodstream, although cigarette smoke includes a host of carcinogenic products that aren't a major factor in smokeless tobacco," Hecht says in a news release.
Data came from six studies that together included 420 smokers and 182 smokeless tobacco users, all of whom were trying to cut down on their tobacco use.
Hecht's team compared levels of certain cancer-causing chemicals detected in the participants' urine samples.
Levels of those chemicals from the smokeless tobacco users were equal to or higher?than the levels from?the cigarette smokers.
The researchers say that while "there is no doubt that the risk for lung cancer is greater in smokers than in smokeless tobacco users," smokeless tobacco isn't harmless and can cause oral cancer.
The report didn't include details about the participants' medical history. The findings appear in the August edition of the journal Cancer Epidemiology, Biomarkers & Prevention.