【摘要】 目的 为了探讨皮肤鳞状细胞癌（简称鳞癌）中核因子-κB(Nuclear factor kappaB, NF-κB)、基质金属蛋白酶2（MMP-2）的表达及其与肿瘤分化程度和淋巴结转移的关系。 方法 用ABC免疫组化技术观察50例皮肤鳞癌（高分化鳞癌34例，低分化鳞癌16例,其中14例淋巴结转移）。 结果 NF-κB 与MMP-2表达于瘤细胞和癌旁间质细胞、内皮细胞质中。NF-κB表达阳性率为80%，在低分化鳞癌中的表达明显高于高分化者（P=0.007），且表达越高淋巴结转移率越高（P=0.004）。MMP-2表达阳性率为76%，在低分化鳞癌中的表达明显高于高分化者（P=0.021），且表达越高淋巴结转移率越高（P=0.003）。 结论 皮肤鳞癌中NF-κB和MMP-2表达与其分化程度及淋巴结转移有关。
【关键词】 皮肤鳞状细胞癌 核因子-κB 基质金属蛋白酶2
NF-κB为抗凋亡因子，在其家族中绝大部分成分为p50/p65，肿瘤的浸润转移的生物学行为与NF-κB激活有关［1］。基质金属蛋白酶（Matrix metalloproteinase, MMP）是一组在结构与功能上极为相似的含锌内肽酶，依其结构与底物特异性分为5个亚型：胶原酶（MMP-1、-8、-13）、明胶酶（MMP-2、-9）、间质溶素（MMP-3、-7、-10、-11、-12）、膜型MMP（MT1-、MT2-、MT3-、MT4-MMP或称MMP-14、-15、-16、-17）和其它MMP；MMP几乎能降解细胞外基质和基底膜的所有成分，在肿瘤侵袭和转移中起重要作用，可抑制肿瘤侵袭和转移［2,3］。近年研究表明NF-κB和MMP-2在肿瘤侵袭和转移中起重要作用［1～3］。本研究采用ABC免疫组化技术观察皮肤鳞癌及其淋巴结转移灶中NF-κB和MMP-2表达，以探讨NF-κB和MMP-2在皮肤鳞癌侵袭和转移中的作用。
1 材料与方法
1.1 材料 选取本院和广东医学院附属医院1997～2002年间的皮肤鳞癌手术切除标本50例，其中伴淋巴结转移者14例，无转移者36例。男36例，女12例，年龄32～90岁（平均61.3岁），病程1个月～30年（平均1.8年）。所有患者均按WHO分类标准将鳞癌分为Ⅰ～Ⅳ级，Ⅰ、Ⅱ级为高分化鳞癌（34例），Ⅲ、Ⅳ级为低分化鳞癌（16例）。另取10例正常皮肤标本作对照及10例皮肤鳞癌患者淋巴结转移标本作对照。
1.2 方法
1.2.1 试剂 兔抗人NF-κBp65、兔抗人MMP-2和兔抗人LN抗体及即用型SABC免疫组化染色试剂盒均购自武汉博士德生物工程公司。
1.2.2 ABC法 石蜡切片常规处理后，依次加入适当稀释的一抗（NF-κB p65、MMP-2、LN的工作浓度均为1:100），生物素标记二抗、SABC，DAB显色、苏木素复染。用已知阳性片作阳性对照，PBS代替一抗为阴性对照。
1.3 结果判断标准 NF-κB p65与MMP-2以胞质着色为阳性。按着色强弱分为：无着色为0，浅黄色为1，深黄色为2，棕黄色为3。着色范围以在10×40高倍镜下随机选取5个视野（每个视野100个细胞），计数阳性细胞所占的百分比均值：<25%为1，25%～50%为2，≧50%为3。上述两项相加为NF-κB p65或MMP-2的表达强度积分：<2为阴性，<3为弱阳性（+），3～4为阳性（++），>4为强阳性（+++）。
1.4 统计学分析 采用SAS 8.01 for windows分析，组间比较采用卡方检验。
2 结果
2.1 NF-κB p65与MMP-2表达模式 NF-κB p65在正常表皮、毛囊及小汗腺导管，其中以基底细胞表达最强。鳞癌中NF-κB p65的阳性表达率为80%（40/50），表达较弱者均匀分布于癌巢中，表达较强者以癌巢中心为著，癌旁间质中成纤维样细胞及血管内皮细胞亦有阳性着色。淋巴结转移灶中阳性表达强度及分布方式与原发灶相似。
MMP-2在正常表皮、毛囊均有弱表达，以基底细胞为著。鳞癌中MMP-2的阳性表达率为76%（38/50），表达较弱者均匀分布于癌巢中，表达较强者以癌巢中心为著，癌旁间质中成纤维样细胞及血管内皮细胞亦有阳性着色。淋巴结转移灶中阳性表达强度及分布方式与原发灶相似。
2.2 NF-κB p65、MMP-2表达与鳞癌分化及转移的关系 NF-κB p65、 MMP-2表达在高分化与低分化鳞癌组中的差异均有显著性（表1），与鳞癌分化程度亦有显著相关。
表1 NF-κB p65、MMP-2表达与鳞癌分化程度及淋巴结转移的关系（略）
3 讨论
NF-κB是近年来发现的一种转录因子，其表达水平与某些肿瘤的浸润、粘附、转移行为密切相关。NF-κB引起肿瘤细胞浸润、粘附、转移的原因是由于NF-κB激活后导致许多粘附分子、血管生长因子、基质蛋白酶的瀑布样释放［1］。肿瘤细胞和ECM的相互作用在肿瘤的浸润与转移过程的各个阶段均存在，MMP-s能降解ECM和基底膜（BM），与肿瘤的浸润和转移密切相关，BM的降解意味着肿瘤的浸润与转移，而在良性病变过程中BM是完整的。ＩＶ型胶原在维护BM的完整性起重要作用，ＩＶ型胶原酶可分为MMP-2 和MMP-9。MMP-2分子量为72KD是一种依赖锌离子的蛋白水解酶，由多种细胞（如成纤维细胞、巨噬细胞和内皮细胞）分泌；通常以酶原的形式存在，须激活后才能酶解蛋白，能降解细胞外基质（ECM）和基底膜成分，破坏局部组织结构、增加肿瘤诱导的血管形成，故其在肿瘤发展和侵袭的各个阶段均起重要作用［2～5］，其作用的底物主要是ＩＶ型胶原和明胶。
研究证实NF-κB在许多肿瘤中被激活，其活性程度与许多肿瘤的转移能力密切相关；药物或基因转染抑制NF-κB的活化，则肿瘤细胞的转移能力明显降低； NF-κB与肿瘤转移的关系是间接的，MMPs，IL-8，VEGF等分子可能起桥梁作用［6～8］。本研究证实了NF-κBp65蛋白表达较强者以癌巢中心为著，在低分化皮肤鳞癌的表达明显高于高分化皮肤鳞癌的表达，表明NF-κB激活在皮肤鳞癌的发展中有重要作用；NF-κBp65蛋白表达在淋巴结转移组高于非转移组，表明NF-κB参与了皮肤鳞癌的浸润转移过程。本研究亦发现MMP-2在皮肤鳞癌的癌细胞与间质细胞中均有表达，其在淋巴结转移灶中的表达强度与分布方式类似于原发灶；MMP-2表达随鳞癌分化程度降低而增加，其与淋巴结转移有关。结果提示低分化鳞癌可产生较多的MMP-2来降解细胞外基质，从而有助于肿瘤的局部侵袭。因此，NF-κB 和MMP-2在鳞癌的浸润转移过程中共同发生作用。
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作者单位：东莞市慢性病防治院，广东 东莞 523008;广东医学院附属医院皮肤科,广东 湛江 524001
【摘要】 目的 研究基质金属蛋白酶-2（MMP-2）和组织基质金属蛋白酶抑制剂-1（TIMP-1）在银屑病皮损组织中的表达情况及其与微血管密度（MVD）的关系。 方法 采用免疫组化ABC法检测银屑病和正常皮肤组织MMP-2、TIMP-1蛋白的表达情况，用血管内皮细胞特异性标志CD34抗体检测MVD。 结果 银屑病患者皮损MVD明显高于对照组(P<0.01)；患者皮损中既可表达MMP-2，又可表达TIMP-1，而在正常皮肤不表达；MMP-2表达强度随着MVD的增高而增强（P<0.01），TIMP-1表达程度随着MVD的增高而减弱（P<0.01）。 结论 银屑病患者皮损组织中MMP-2、TIMP-1的表达与MVD有关。
【关键词】 银屑病 微血管密度 基质金属蛋白酶-2 组织基质金属蛋白酶抑制剂-1
Expression of MMP-2 and TIMP-1 in psoriasis patients and their relationships with microvessel density.
CHEN Ji-guang, REN Xiao-li.
(Taizhou Municipal People’s Hospital, Taizhou 318000, Zhejiang, P. R. China)
Abstract：Objective To observe the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-1(TIMP-1)in patients with psoriasis, and their relationships with microvessel density（MVD）. Methods Immunohistochemical technique-Avidin Biotin comple(ABC) was employed to observe the expression of MMP-2、TIMP-1 in the lesions of patients; MVD was detected by immunohistochemical method with monoclonal antibody specific for the endothelial marker CD34 and healthy subject. Results MVD in the lesions of psoriasis patients was higher than that of normal control (P<0.01)；MMP-2、TIMP-1 were expressed in the lesions of patients，not in normal skin；The expression level of MMP-2 increased and TIMP-1 decreased with the increasing of MVD. Conclusion The expression of MMP-2 、TIMP-1 protein is closely correlated with the MVD of psoriasis patients.
Key words：Psoriasis；Microvessel density；Matrix; Metalloproteinase-2；Tissue inhibitors of metalloproteinase-1
皮肤由角质形成细胞、成纤维细胞等实质细胞及细胞外基质（ECM）构成。基质金属蛋白酶（MMPs）是一组锌离子依赖的蛋白水解酶, 是ECM降解主要酶类，可以水解ECM的多种成分。其中MMP-2属于明胶酶类,可以水解胶原成分,是RRW7中研究较广泛的一种,而基质金属蛋白酶组织抑制剂(TIMPs)家族成员中又以TIMP-1研究较为深入，它是MMP-2的选择性抑制剂。目前有关MMPs/ TIMPs的研究多集中在乳腺、膀胱等肿瘤方面，在银屑病中MMPs与MVD的关系尚未见报道。笔者应用免疫组化法对银屑病患者皮损区MMP-2、TIMP-1的表达进行检测，探讨二者与微血管密度（MVD）的关系。
1 资料和方法
1.1 病例 寻常型银屑病患者17例，男10例，女7例，年龄26～60岁，平均43岁；病程6月～17年，平均11年，均为本院门诊及住院病人。10例对照组系非皮肤病外科手术人群，男6例，女4例，年龄25～60岁，平均41岁。皮肤标本用4mm环钻常规取材，石蜡包埋，4um厚连续切片。
1.2 方法
1.2.1 试剂 兔抗人MMP-2、兔抗人TIMP-2和即用型SABC免疫组化染色试剂盒均购自武汉博士德生物工程公司，鼠抗人CD34单抗(DAKO公司)。
1.2.2 ABC法 石蜡切片按常规处理后，微波炉抗原修复，依次加入适当稀释的一抗（MMP-2/TIMP-2的工作浓度均为1：100，CD34工作浓度1:40）、生物素标记二抗、SABC，DAB显色、苏木素复染，用已知阳性片作阳性对照，PBS代替一抗作阴性对照。
1.2.3 MMP-2、TIMP-2结果判断标准 MMP-2和TIMP-2以胞质内出现黄色颗粒状物质为阳性，按着色强弱分级：无着色为0，浅黄色为1；深黄色为2，棕黄色为3；着色范围以×40倍镜下随机选取5个视野（每个视野计数100个细胞）计数阳性细胞所占的百分比均值：<25%为1，25%～50%为2，>50%为3。上述两项相加即为MMP-2或TIMP-2表达强度积分：<2为阴性，<3为弱阳性（+），3～4为阳性（++），>4为强阳性（+++）。
1.2.4 MVD结果判断 在低倍镜(100)下观察真皮浅层CD34阳性染色的微血管(毛细血管、小动脉、小静脉)，选择染色最多的区域，在高倍镜(400)下进行观察，任何被CD34抗体染成棕黄色的内皮细胞或内皮细胞簇，不论管腔内有无红细胞，均作为一个微血管计数。计算5个高倍镜(400)视野下的微血管数，取其平均值作为MVD。
2 结果
2.1 MMP-2、TIMP-1结果分析 阳性信号为胞浆内出现黄色颗粒，以PBS代替一抗的空白对照切片中，无任何阳性反应。
17例患者中, 有15例患者皮损表皮中MMP-2表达，主要位于基底层细胞胞浆(见图1)。14例患者的角质形成细胞及真皮浅层血管周围有TIMP-1的表达(见图2)。正常人皮肤中MMP-2、TIMP-1表达均为阴性。详见表1。
图1 皮损中MMP-2阳性染色(×40)（略）
图2 皮损中TIMP-2阳性染色(×40)（略）
正常人与患者皮损中MMP-2、TIMP-1表达强比较用Ridit分析。MMP-2：R正常组=0.533,R银屑病组=0.426,U=3.83>2.58,P<0.01; TIMP-1：R正常组=0.481,R银屑病组=0.383,U=3.57>2.58,P<0.01。
2.2 MVD结果分析 在正常对照组及银屑病患者的皮损真皮层血管内皮细胞均有CD34表达，银屑病患者MVD（18.93±9.46）显著高于正常对照组的MVD（4.34±1.67），差异有统计学意义(t=8.36,P<0.01) (见图3)。Spearman等级相关分析，银屑病患者皮损部位的MVD和MMP-2呈正相关(r=0.603,P<0.05)，与TIMP-1表达负相关（r=-0.524, P＜0.01)。
图3 皮损中CD34阳性染色（×20）（略）
3 讨论
MMP-2为MMPs成员之一，是ECM降解主要酶类之一，几乎可降解所有的细胞外基质成分,包括各种胶原、蛋白多糖和基质蛋白。TIMPs结合于活性MMPs的催化部位，抑制ECM的降解。二者具有精确的调控机制，保证了体内生理状态下ECM的重建与稳定，如果调节失控，MMPs/TIMPs平衡破坏，则可引发各种病理过程。ECM在维持正常组织结构与功能及细胞的生长和分化过程中起重要作用。ECM中的胶原、纤维连结蛋白、层黏素的结构在MMPs作用下降解,使血管内皮细胞增殖，原有血管形成新的分支，并在血管形成后期重塑基底膜和管腔过程中发挥重要的作用，研究发现［1］MMPs在促进肿瘤血管新生的过程中也具有重要的作用。银屑病皮损病理学显示真皮乳头层微血管扩张迂曲、通透性增高、血管数量增多。CD34为目前公认的血管内皮细胞标记物，通过染色血管内皮细胞，计数血管数量，反映血管增生的情况［2］。本研究显示，银屑病患者皮损的MVD明显高于正常对照组，与病理机制相符。有学者报道［3］，PUVA可能系通过抗血管新生作用达到治疗银屑病作用。
表1 MMP-2、TIMP-1在正常人与患者皮损中表达强度比较（略）
本研究发现银屑病患者皮损中MMP-2、TIMP-1呈强表达，而在正常皮肤不表达，与Fleisehmajer［4］等研究结果一致。而且患者皮损中MMP-2表达强度随着MVD的增高而增强，TIMP-1表达程度随着MVD的增高而减弱，提示MMP-2、TIMP-1介导银屑病新生血管形成中发挥重要的作用。笔者认为, TIMP-1作为MMP-2的天然抑制物在多个环节产生抑制血管形成的作用，通过诱导TIMP-1的表达，阻碍MMP-2介导的内皮细胞移动，抑制基质中ECM的降解和微血管生成，有望成为治疗银屑病的新途径，国外研究显示［5］，使用MMP-2抑制剂治疗银屑病具有较好的疗效。因此，对MVD的相关研究不仅为进一步探索银屑病发病机制提供新亮点，而且也为抗银屑病新药开发提供新的药物靶位。
（致谢：本文蒙第四军医大学西京医院皮肤科刘玉峰教授审阅。）
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作者单位：台州市立医院皮肤科,浙江 台州 318000.
肿瘤细胞及其基质细胞分泌的明胶酶B（matrix metalloproteinase-9。MMP-9）和明胶酶A（matrix metalloproteinase-2，MMP-2）在促血管生成、肿瘤细胞侵袭和转移灶形成中的作用已日益引起人们的关注。有资料显示，在非小细胞肺癌患者的体液（如尿液、血清）中可检测到MMP-9（相对分子量92 000）和MMP-2（相对分子量72 000）蛋白，且与侵袭转移密切相关。然而，在淋巴瘤患者血清中MMP-9、MMP-2蛋白检测及其意义的文献报道极少.
非霍奇金淋巴瘤患者血清中MMP-9和MMP-2蛋白的检测及临床意义(全文)
【摘要】
Objective- Although it has been reported that matrix metalloproteinase (MMP)-2 is a major proteinase in atherosclerotic plaque lesions, there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using apolipoprotein E-deficient (apoE -/- ) mice.
Methods and Results- To generate MMP-2-deficient, apoE-deficient mice (MMP-2 -/- :apoE -/- ), MMP-2 -/- mice were crossed with apoE -/- mice. After 8 weeks of feeding with a lipid-rich diet, morphological and biochemical studies of the aortic sinus and arch were conducted. A significant reduction of the atherosclerotic plaque in the aortic sinus and arch with the decrease in smooth muscle cell-positive area was observed in MMP-2 -/- :apoE -/- mice compared with that of MMP-2 +/+ :apoE -/- mice. Macrophage- and collagen-positive areas were less in aortic sinus but not in aortic arch in MMP-2 -/- :apoE -/- mice. There was no difference of MMP-9 mRNA expression in the plaque lesion between the 2 genotypes. A much lower level of mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesions of MMP-2 -/- :apoE -/- mice than in those of MMP-2 +/+ :apoE -/- mice.
Conclusions- MMP-2 contributes to the development of atherosclerosis in apoE -/- mice.
We determined the role of MMP-2 in atherosclerosis plaque development using the MMP-2 deficient, apolipoprotein E-deficient (MMP-2 -/- :apoE -/- ) mice. After 8 weeks of feeding with a lipid-rich diet, a significant reduction of the atherosclerotic plaque in the aortic sinus was observed in MMP-2 -/- :apoE -/- mice compared with that of MMP-2 +/+ :apoE -/- mice.
【关键词】 atherosclerosis collagen metalloproteinases plaque
Introduction
In human or animal models of atherosclerosis, varying matrix metalloproteinases (MMPs) have been demonstrated to increase in atherosclerotic lesions, including MMP-1, -2, -3, -7, -9, -12, -13, and MT-MMPs. 1-3 MMPs have been believed to contribute to the development and progression of atherosclerosis. 1-3 However, there are only limited data providing direct evidence of the contribution of MMPs to the development of atherosclerotic lesions. Although MMP activity is commonly considered instrumental to the development of atherosclerotic lesions, this notion has been challenged by recent studies in gene-targeting mice. It has been reported that overexpression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) reduced atherosclerotic lesions in apolipoprotein E-deficient (apoE -/- ) mice. 4 The deletion of the TIMP-1 gene resulted in either a reduction of or no change in the plaque size in the apoE -/- mice. 5,6 Atherosclerotic lesions were significantly larger in mice with a combined deficiency of apoE and MMP-3 than in apoE -/- mice. 7 Overexpression of MMP-1 in apoE -/- mice, which is not normally expressed in mice, decreased the extent of atherosclerosis. 8 A more recent study demonstrated that MMP-9 deficiency but not MMP-12 deficiency reduced the atherosclerotic lesion growth in apoE -/- mice. 9 MMP-13 deficiency had no effect on atherosclerotic plaque formation with similar accumulation of plaque macrophages and smooth muscle cells, but contained more interstitial collagen in apoE -/- mice. 10 These results seemingly contradict a central tenet in our understanding of atherosclerosis-that increased MMP activity leads to the formation of a thicker neointima-suggesting that the role of individual MMPs in plaque lesions may actually have a different effect on the development of atherosclerotic plaque formation. The contribution of individual MMPs to plaque formation is only beginning to be investigated by the genetic manipulation of the expression of individual MMPs. Further work is necessary to determine the full spectrum of antiatherogenic or proatherogenic activities of the many MMPs that are expressed in atherosclerosis.
We previously demonstrated that MMP-2 deficiency significantly reduces neointimal lesion development in the carotid artery after ligation compared with control mice. 11 Although it has been reported that MMP-2 is a major proteinase in atherosclerotic plaque lesions not only in human but also in animal models, 12,13 there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using the apoE -/- mice.
Methods
Animals and the Experimental Protocol
All animal studies were conducted in accordance with the Animal Care and Use Committee guidelines of the Nagoya University School of Medicine. The generation of MMP-2-deficient (MMP-2 -/- ) mice with the genetic background of C57BL/6 was described previously. 14 ApoE -/- mice with the C57BL/6 genetic background were obtained from the Jackson Laboratory (Bar Harbor, Me). MMP-2 -/- mice were intercrossed with apoE -/- mice to generate breeding pairs with heterozygous deficiency of MMP-2 and apoE (MMP-2 +/- :apoE +/-, which sired MMP-2 -/- /apoE -/- and MMP-2 +/+ :apoE -/- littermate offspring. Genomic DNA was extracted from the tail tips for genotyping of offspring by Southern blotting for MMP-2 and by polymerase chain reaction for apoE (data not shown). At 8 weeks of age, male mice were started on a Western-type diet containing 21% fat and 0.15% cholesterol without sodium cholate, and they were maintained on this diet for 8 weeks.
Histological Analysis
Mice were euthanized by intraperitoneal pentobarbital injection, and blood samples of mice were collected into syringes that contained EDTA just before perfusion with isotonic saline from the left cardiac ventricle. The mice were perfused through the left cardiac ventricle with isotonic saline and 4% paraformaldehyde in 0.01 mol/L phosphate buffer (pH 7.4) under physiological pressure. The heart with 1 mm of proximal aorta attached and aortic arch were removed. The top half of the heart containing the aortic root or aortic arch was embedded and frozen in Tissue-Tek O.C.T. media. Sequential 20-µm sections were cut until the aortic valve leaflets appeared. From this point on, serial 6-µm sections were collected and stained routinely with hematoxylin and eosin (H&E), oil red O for lipid, and Masson?s trichrome for collagen. The corresponding sections on separate slides were used for immunohistochemical staining. The sections were preincubated with 5% serum and then incubated with antibodies against smooth muscle actin ( SM actin; 1:50, Sigma Aldrich), macrophages (Mac-3, 1:40, BD Pharmingen), MMP-2 (MMP-2; 1:200, Fuji Chemical Co), and MMP-9 (MMP-9; 1:100, Chemicom). Immunohistochemical staining was visualized using an ABC kit (Vector Laboratories) according to the manufacturer?s instructions. Levamisole (Vector Laboratories) was used as the inhibitor of endogenous alkaline phosphatase. The counterstaining for the nucleus was performed with Mayer?s hematoxylin.
For the quantification of atherosclerotic lesions, all images were captured and analyzed by National Institutes of Health Image software. The images showed the area of intimal atherosclerotic plaque, lipid lesion assessed by the positive for oil red O staining, region containing macrophage as assessed by Mac-3 stating, and smooth muscle cell (SMC)-positive area as assessed by -SM actin staining. The atherosclerotic plaque area of the aortic sinus or aortic arch was reported as the net area and the proportion of total intimal plaque lesion area to total cross-sectional vessel wall area, which was defined by the external elastic lamina. For quantification by image analysis, we set a threshold to automatically compute the areas positive for each antibody or histochemical stain and then computed the ratio of positively stained area to the total cross-sectional vessel wall area and intimal plaque lesion area studied.
Gelatin Zymography and mRNA Quantification
After perfusion with ice-cold phosphate-buffered saline, the aorta arch was dissected out and placed in ice-cold phosphate-buffered saline. After further mechanical rinsing under a dissection microscope, protein and total RNA were extracted from the tissue so that gelatin zymography and mRNA quantification could be performed.
For gelatin zymography, 20-µg protein extracts of the aortic arch were mixed with SDS sample buffer without a reducing agent and loaded onto a 10% SDS-polyacrylamide gel containing 1 mg/mL gelatin, as previously described in detail. 15 Digestion bands were quantified by an image analyzer system (NIH image 1.62) and compared with a human MMP-2 standard (Oncogene Research Products).
The mRNA levels of MMPs and TIMPs in atherosclerotic lesions were quantified by real-time reverse-transcription and polymerase chain reaction. The total RNA was extracted from carotid arteries, and then reverse-transcribed. The synthesized cDNA was quantified by using TaqMan quantitative polymerase chain reaction analysis of each gene with the ABI PRISM 7700 Detection System according to the manufacturer?s protocol. Specifically, primer and probe sequences used for mouse MMP-2 were (forward) 5'-CCCCATGAAGCCTTGTTTACC, (reverse) 5'-TTGTAGGAGGTGCCCTGGAA, (probe) 5'-AATGC-TGATGGACAGCCCTGCA; for mouse MMP-9 were (forward) 5'-AGACCAAGGGTACAGCCTGTTC, (reverse) 5'-GGCACGCTG-GAATGATCTAAG (probe) 5'-TGGCTCATGCCTATGCACCTGGAC; for mouse TIMP-1 (forward) 5'-GCCTACACCCCAGTCATGGA, (reverse) 5'-GGCCCGTGATGAGAAACTCTT, (probe) 5'-TGGATATGCCCACAAGTCCCAGAACC; and for mouse TIMP-2 (forward) 5'-GTC-CCATGATCCCTTGCTACA, (reverse) 5'-TGCCCATTGATGCTCT-TCTCT, (probe) 5'-CTCCCCGGATGAGTGCCTCTGGA. Each RNA quantity was normalized to its respective glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA quantity.
Polyacrylamide Gel Disc Electrophoresis of Serum Lipoproteins
Polyacrylamide gel disc electrophoresis of serum (100 µL) was performed according to the method of Narayan et al. 16 The pattern of electrophoresis was analyzed by densitometry.
Statistical Evaluation
Values were expressed as means±SD. Significant differences were analyzed using the Student t test. A value of P <0.05 was considered to be statistically significant.
Results
Phenotype of MMP-2 -/- :ApoE -/- Mouse
The MMP-2 -/- :apoE -/- mouse did not show any gross anatomic abnormalities, including abnormalities in the blood vessels. However, this mutant mouse showed a slower growth rate at 16 weeks than the MMP-2 +/+ :apoE -/- mouse (n=12) with body weight of 19.9±2.9 grams and the MMP-2 -/- :apoE -/- mouse (n=14) with a body weight of 18.0±2.3 grams. There were no differences in plasma total cholesterol levels between the MMP-2 -/- :apoE -/- mouse and the MMP-2 +/+ :apoE -/- mouse after 8 weeks of a Western-type diet (56.9±9.9 mmol/L, n=12 versus 64.4.±9.5 mmol/L, n=14; P <0.08). The distribution of lipoproteins in serum assessed by the densitometric pattern of polyacrylamide gel disc electrophoresis was similar between 2 mutant mice ( Figure 1 ).
Figure 1. Representative distribution of lipoproteins in serum assessed by the densitometric pattern of polyacrylamide gel disc electrophoresis of MMP-2 +/+ :apoE -/- and MMP-2 -/- :apoE -/- mice.
Effect of MMP-2 Deficiency on Atherosclerotic Lesion Formation in ApoE-Deficient Mice
Cross-sectional analysis of the aortic sinus area as well as aortic arch showed that the total intimal lesion area was significantly less in MMP-2 -/- :apoE -/- mice than in MMP-2 +/+ :apoE -/- mice, whether the lesion area was controlled for the total cross-sectional vessel wall area ( Figures 2a, 2b, 3f, 3 h; Table ). As shown in Figure 2c, 2d, 2e, 2f, 2g, and 2 h and the Table, the areas that stained positive for lipid, macrophages, SMC, and collagen in the lesion of aortic sinus were less in MMP-2 -/- :apoE -/- mice than those in MMP-2 +/+ : apoE -/- mice. However, the ratio of stained positive area for lipid, macrophage, and collagen to the intimal plaque area was similar in MMP-2 -/- :apoE -/- and MMP-2 +/+ :apoE -/-. In aortic arch there was no difference of macrophage positive area between 2 genotypes, whether the lesion area was controlled for intimal lesion area ( Figure 3g and 3 i; Table ). In contrast, SMC-positive area was less in MMP-2 -/- : apoE -/- mice than that in MMP-2 +/+ :apoE -/- mice ( Figure 3J, 3 I; Table ). As shown in Figure 3a 3b, 3j, and 3 l, a much thicker fibrous cap region stained with SMC-specific antibody was observed in MMP-2 +/+ :apoE -/- mice than that of MMP-2 -/- :apoE -/- mice both in aortic sinus and arch. The collagen-positive area was observed in thicker fibrous cap lesions in MMP-2 +/+ :apoE -/- mice but not in MMP-2 -/- :apoE -/- mice ( Figure 3k and 3 m).
Figure 2. Representative light micrographs of aortic sinus from MMP-2 +/+ :apoE -/- (a, c, e, g) and MMP-2 -/- :apoE -/- (b, d, f, h) mice. Staining was performed with hematoxylin and eosin (H&E) (a, b), oil red O (c, d), anti-Mac-3 antibody (e, f), and Masson?s trichrome (g, h). Scale bars=300 µm.
Figure 3. Light micrographs of aortic sinus (MMP-2 +/+ apoE -/- in a, c, d) (MMP-2 -/- /apoE -/- in b, e), and from aortic arch (MMP-2 +/+ apoE -/- in f, g, j, k; MMP-2 -/- /apoE -/- in h, i, l, m). Staining was performed with anti- smooth muscle actin antibody (a, b, j, l), anti-MMP-2 (c, d, e) antibody, hematoxylin and eosin (H&E) (f, h), anti-Mac-3 antibody (g, i), and Masson?s trichrome (k, m). Scale bars=300 µm. L, lumen; F, fibrous cap.
Quantification of Atherosclerotic Plaque Size of Aortic Sinus and Aortic Arch
MMPs Expression and Localization
In the gelatin zymographic analysis of protein extracts from atherosclerotic plaque lesions, the latent forms of MMP-9 (92kDa) and MMP-2 (72kDa) and the active form of MMP-2 (62kDa) were detected in MMP-2 +/+ :apoE -/- mice ( Figure 4 A). As expected, a similar level of MMP-9 activity and no MMP-2 activity were observed in MMP-2 -/- :apoE -/- mice.
Figure 4. Gelatin zymography and mRNA quantification of atherosclerotic lesion. Protein extracts (20 µg in each line) from the aortic arch of MMP-2 +/+ :apoE -/- (n=4) and MMP-2 -/- :apoE -/- mice (n=4) after 8 weeks Western diet feeding was used for gelatin zymography (A). Data of mRNA quantification were obtained from the independent aortic arches of MMP-2 +/+ :apoE -/- (n=5) or MMP-2 -/- :apoE -/- mice (n=5) for each genotype. Data were expressed as a percentage of mRNA levels from MMP-2 +/+ :apoE -/- mice after normalizing to GAPDH mRNA; means±SD, P <0.01, P <0.001.
No MMP-2 mRNA expression was found in atherosclerotic plaque lesions in MMP-2 -/- :apoE -/- mice ( Figure 4 B). Although no significant difference in MMP-9 mRNA expression in plaque was observed between MMP-2 -/- :apoE -/- and MMP-2 +/+ :apoE -/- mice, much lower mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesion of MMP-2 -/- :apoE -/- mice ( Figure 4C, 4D, 4 E). Immunohistochemical analysis revealed that the staining for MMP-2 was observed at the fibrous cap region as well as atheromatous lesion containing foam cells of the aortic sinus of MMP-2 +/+ :apoE -/- mice ( Figure 3c, 3 d). MMP-2 was not detected in the lesion of MMP-2 -/- :apoE -/- mice ( Figure 3 e). MMP-9 staining was observed in the plaque, and the staining pattern was not different between the 2 genotypes (data not shown).
Discussion
Previous observations illustrate the potential complexities of manipulating a system in which MMPs have a dual role in plaque growth by means of SMC migration, matrix deposition, and instability caused by matrix destruction. In fact, MMP-1 overexpression to macrophages reduced the progression of atherosclerosis in apoE -/- mice, because it resulted in less collagenous matrix accumulation. 8 MMP-3 deficiency was associated with increased collagen content in the plaques in apoE -/- mice, which is consistent with greater stability, although plaque size was increased overall. 7 These previous observations may suggest that the activities of MMP-1 and MMP-3 in atherosclerotic lesions may contribute to a reduction of plaque size, possibly by causing the degradation of matrix components. In contrast, a deficiency of MMP-9 reduced the plaque size, macrophage content, and collagen deposition in aortic lesion of apoE -/- mice, but MMP-12 deficiency had no effect on plaque size or on the composition of the plaque. 9 Interestingly, more recent report by Johnson et al suggested that MMP-9 deficiency increased plaque size with a increase in macrophage accumulation and a decrease in SMCs in brachiocephalic artery from apoE -/- mice. 17 These inconsistent effects of MMP-9 deficiency on the atherosclerotic lesion formation as well as the changes in cellular composition at the lesion suggest that MMP-9 has different actions to the development of atherosclerosis at different regions of artery. This is also true for MMP-12 deficiency, as Johnson et al have also demonstrated that MMP-12 deficiency reduced the plaque size as well as macrophage content in brachiocephalic artery from apoE -/- mice. 17 More recent findings suggested that MMP-13 deficiency had no effect on plaque development but decreased collagen content in the plaque in apoE -/- mice. 10 These findings indicate the different contributions of individual MMPs to atherosclerotic plaque formation as well as to matrix protein accumulation in the plaque.
In the present study, we clearly demonstrated that MMP-2 deficiency reduces the atherosclerotic plaque lesion formation in apoE -/- mice. In aortic sinus, the reduction in plaque volume was associated with a significantly lower macrophage-positive and SMC-positive areas, as well as less collagenous matrix accumulation in the plaque lesions of MMP-2 -/- :apoE -/- mice than those of MMP-2 +/+ :apoE -/- mice. In aortic arch, the reduction of lesion was associated with a lower SMC-positive area in MMP-2 -/- :apoE -/- mice, but there were no differences in macrophage-positive and collagen-positive areas in the lesions between those genotypes. The reduction of the accumulation of SMCs in plaque lesions, common feature to aortic sinus and arch in MMP-2 -/- :apoE -/- mice, is consistent with our previous findings that the targeted deletion of the MMP-2 gene reduced neointimal lesion formation after flow cessation in the murine carotid arteries, mainly because of the attenuation of the migration of SMCs from the medial to the intimal region through the reduction of proteolytic activities resulting from MMP-2 deficiency. 11,18 In addition, a much thicker fibrous cap region that contains SMCs and collagen was observed in MMP-2 +/+ :apoE -/- mice than that of MMP-2 -/- :apoE -/- mice, suggesting MMP-2 may induce plaque stability by accumulating SMC into the fibrous cap. We showed the different effects of MMP-2 on the macrophage accumulation in the atherosclerotic lesions of different regions of artery. It is possible that MMP-2 contributes to macrophage accumulation in the atherosclerotic lesions in different ways at different regions. This might be similar to the effect MMP-9 on macrophage accumulation in the atherosclerotic lesions, because MMP-9 deficiency increased macrophage accumulation in the lesions of brachiocephalic artery but reduced its accumulation in carotid artery and descending aorta in apoE -/- mice. 9,17,21 The fact that MMP-2 colocalized with macrophage in atherosclerotic plaque lesion of aortic sinus, consistent with human atherosclerotic lesions 19,20 may suggest that MMP-2 is involved in monocyte migration into the intima or that MMP-2 may affect macrophage proliferation in the intima. Further studies will be required to evaluate the role of MMP-2 on macrophage accumulation in the atherosclerotic plaque lesion.
MMPs have overlapping substrate specificities, so the effect of the loss of MMP-2 can be compensated for to some degree by another gelatinase, MMP-9. However, the MMP-9 mRNA level observed in MMP-2 -/- :apoE -/- mice was not significantly higher than that observed in] MMP-2 +/+ :apoE -/- mice.
We also found a decreased expression of TIMPs mRNA in MMP-2-deficient mice, in agreement with our previous observation. 11 These findings strengthen our hypothesis that MMP-2 may play a role in regulating the expression of TIMPs.
There are limitations in the present study. We have evaluated the atherosclerotic lesions only after 8 weeks of high-fat diet. This relative short period may represent early developing plaques. However, we demonstrated that some plaques already contain advanced lesions with lipid core surrounded by thick fibrous cap. This rapid growth of the plaque in the present study might be due to the higher serum cholesterol levels ( 60 mmol/L) than those of others previously reported. In addition, we did not demonstrate the net proteolytic activity in the lesions. It is ultimately the balance between the MMPs and TIMPs that determines the focal proteolysis around the cellular components in the lesion. Further studies will be required to examine the effect of MMP-2 deficiency on the net proteolytic activity in the atherosclerotic lesions.
In conclusion, we demonstrated that MMP-2 deficiency reduced atherosclerotic plaque lesions in apoE -/- mice. This reduction of the plaque lesions in MMP-2 deficiency was associated with the reduction of SMC accumulation in the plaque lesions. These results suggested that MMP-2 contributes to atherosclerotic plaque development in apoE -/- mice and that MMP-2 may induce plaque stability by accumulating SMC into the fibrous cap.
Acknowledgments
This work was supported by a research grant from the Scientific Research Fund of the Ministry of Education, Science, and Cultures, Japan (No. 13671182).
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作者单位：Department of Geriatrics (M.K, K.N, T.S., X.W.C., T.I., A.I.), Nagoya University Graduate School of Medicine, Japan; and RIKEN Brain Science Institute (S.I.), Saitama, Japan.
