三种视网膜病视网膜前膜中基质金属蛋白酶及其天然抑制分子的表达

目的 研究增殖性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)、增殖性玻璃体视网膜疾病(proliferative vitreoretinopathy,PVR)和急性视网膜坏死(acute retinalnecrosis,ARN)患者视网膜前膜中基质金属蛋白酶(matrixmetalloproteinases:MMPs)及其天然抑制物(tissueinhibitorsofmetalloproteinages,TIMPs)的表达情况.方法 玻璃体手术中剥取PVR、PDR和ARN患者的视网膜前膜,同供体眼视网膜作为正常对照,冰冻切片后进行免疫组织化学染色,包括:MMP-1,MMP-2,MMP-3,MMP-7,MMP-9,TIMP-1和TIMP-2.结果 正常视网膜中能够观察到MMP-1,MMP-3,TIMP-1和TIMP-2的表达,在PVR、PDR和ARN患者标本中各种分子的表达都增强,尤以MMP-2,MMP-3和MMP-7明显.结论 正常视网膜中存在MMPs和TIMPs分子维持着细胞外基质动态的平衡,在PVR,PDR和ARN患者中MMP-2,MMP-3和MMP-7等MMPs分子表达增强,在其病变过程中可能起重要作用.     

增殖性糖尿病视网膜病变基质金属蛋白酶的表达

目的:研究增殖性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)视网膜增殖膜纤维化和新生血管形成与MMP-2和MMP-9异常表达的关系.方法:采用免疫组织化学方法,检测PDR患者20例视网膜前纤维血管膜MMP-2和MMP-9的表达,并与增殖性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)10例非血管性增殖膜进行对比研究.结果:PDR增殖膜MMP-2和MMP-9染色阳性率分别为70%和85%,PVR增殖膜MMP-2和MMP-9染色阳性率分别为80%和60%.在PDR增殖膜上可见血管周围基质MMP-9染色阳性,而且与PVR增殖膜相比,PDR增殖膜MMP-9表达有显著提高.结论:PDR增殖膜MMP-2和MMP-9均有表达,在PDR新生血管形成和纤维化的病理过程中起重要作用.     

Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells

PURPOSE: Pterygia are a common, benign, fibrovascular, and infiltrative process of the corneal-conjunctival junction of unknown pathogenesis. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes active against all components of the extracellular matrix, whose activity is specifically neutralized by tissue inhibitors of MMPs (TIMPs). In the current study the hypothesis was that MMPs and TIMPs may actively participate in the formation and progression of pterygia. METHODS: In this study, 25 pterygium specimens and 15 normal conjunctival biopsies obtained from subjects undergoing surgery for glaucoma and cataract, were processed for immunohistochemistry or in situ hybridization. Pterygium epithelial cells (PECs) were cultured under serum-free conditions and exposed to proinflammatory cytokines to determine both the mRNA and protein expression profiles of MMPs and TIMPs. RESULTS: Collagenase-1 and gelatinase A were expressed in all pterygia examined, specifically localized to the epithelium (directly adjacent to collagen type III), with gelatinase B expression exclusively associated with neutrophils. No collagenase-1 or gelatinase A was detected in normal conjunctiva. TIMP-1 and -3 were localized to epithelial cells with additional TIMP-3 immunoreactivity detected in the extracellular matrix, endothelial cells and leukocytes of all diseased tissue. TIMP-3 protein was evident in 4 of 15 normal conjunctiva. Induction of collagenase-1, gelatinase A, and TIMP-1 mRNA and protein was demonstrated in epithelial cells treated with tumor necrosis factor-alpha and interleukin-1alpha, whereas TIMP-3 expression was unaltered. CONCLUSIONS: This is the first study to document the cellular expression of MMPs and TIMPs in pterygia and cultured human PECs. MMPs and TIMPs may contribute to the inflammation, tissue remodeling, and angiogenesis that characterize pterygia. Understanding the role these proteins play may lead to novel therapies intended to reduce the progressive nature of pterygia.     

基质金属蛋白酶在孔源性视网膜脱离视网膜下液的定量研究

目的 研究孔源性视网膜脱离(RRD)视网膜下液(SRF)基质金属蛋白酶(MMPs)的表达，探讨MMPs与增生性玻璃体视网膜病变(PVR)的关系。方法 采用明胶酶谱分析法定量检测88例RRD患者的SRFMMP-2和MMP-9的活性水平，PVR A组21例，PVRB组37例，PVR C组30例，其中包括伴有脉络膜脱离RRD8例，术后随访PVR复发11例。结果 88例RRD患者的SRF均有MMP-2活性水平升高，33例有MMP-9的表达，8例RRD伴有脉络膜脱离，有6例(75％)MMP-9活性水平升高，术后PVR复发11例SRF均有MMP-9活性水平升高。结论RRD患者的SRF有MMP-2和MMP-9的表达，MMP-9活性水平升高可能与PVR的发生有关，可能成为预测术后PVR复发的指标之一。     

Expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in inflammation-induced corneal neovascularization.

PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. METHODS: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. RESULTS: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. CONCLUSION: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.     

小鼠单纯疱疹病毒性角膜炎中基质金属蛋白酶的表达及活性研究

目的 探讨角膜感染Ⅰ型单纯疱疹病毒(HSV-1)后基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)在角膜中的分布及酶活性表达。方法 BALB／c小鼠眼角膜接种HSV-1(KOS株)以诱发单纯疱疹病毒性角膜炎(HSK)。分别收集正常眼球及感染后第2、7、14及28天的感染眼球。应用免疫组织化学法和Western blot方法检测MMP-2、-8、-9及TIMP-1、-2在角膜组织中的表达,并应用酶谱(Zymography)技术检测MMPs的酶活性。结果 感染后第2天,感染眼的MMP-2、-9及TIMP-1、-2表达比未感染眼增加且表达主要位于浅表基质层及上皮下的炎性细胞中。感染后第14和28天可见坏死性角膜炎及角膜溃疡形成,同时角膜基质和浸润的炎性细胞中尤其溃疡处,可见MMP-2、-9及TIMP-1、-2表达显著增加。溃疡区域有大量MMP-8阳性染色的中性粒细胞。角膜感染HSV-1后,明胶酶(MMP-2、-9)活性和胶原酶(MMP-8)活性均增强。结论 HSV-1角膜感染后,由角膜细胞和浸润的炎性细胞分泌产生的MMPs可能对上皮性角膜炎与溃疡形成过程起重要的促进作用。MMPs与TIMPs的相互作用可能对HSK的坏死性病变起重要调节作用。（中华眼科杂志,2004,40：395-399)     

增生型糖尿病视网膜病变纤维化相关因子的研究进展

增生型糖尿病视网膜病变（PDR）纤维血管膜的形成严重损害患者的视力,其发生、发展与许多细胞因子的相互作用有关,如转化生长因子（TGF）、结缔组织生长因子（CTGF）、碱性成纤维细胞生长因子（bFGF）、血管内皮生长因子（VEGF）、血小板源性生长因子（PDGF）、肿瘤坏死因子（TNF）、细胞外基质（ECM）、基质金属蛋白酶（MMPs）和其他如内皮素、趋化因子等。因此,PDR纤维血管膜的形成机制非常复杂。就PDR纤维化的发生与细胞因子、ECM、MMPs及其他因素关系的研究进展进行总结。     

Endothelin-1-mediated signaling in the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in astrocytes

PURPOSE: Endothelin (ET)-1 levels are increased in aqueous and vitreous humor in patients with glaucoma and animal models of glaucoma. Whether the elevated ET-1 induces extracellular matrix (ECM) remodeling in the optic nerve head is still unknown. In the present study, the regulation of matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases (MMPs/TIMPs) and ECM remodeling in ET-1-activated human optic nerve head astrocytes (hONAs) were determined. METHODS: Primary hONAs were exposed to ET-1 for 1 day and 4 days. Incubation media were subjected to zymography and Western blot to detect activity and expression of MMPs and TIMPs. Fibronectin (FN) was monitored by Western blot and immunofluorescent staining. RESULTS: ET-1 increased the activity of MMP-2 and the expression of TIMP-1 and -2 in hONAs. The expression of TIMP-1 and -2 induced by ET-1 was abolished by application of inhibitors of mitogen-activated protein kinase (MAPK) or PKC, leading to enhanced activity of MMP-2. Knockdown of MMP-2, by using small interfering (si)RNA, not only decreased the activity of MMP-2 but also decreased the expression of TIMP-1 and -2. ET-1 increased the soluble (s)FN expression as well as FN matrix formation. However, the accumulation of sFN did not enhance FN matrix formation. Unlike ET-1's effects on MMP-2, blockade of MAPK and PKC did not alter the expression and deposition pattern of FN in hONAs. CONCLUSIONS: ET-1 increased the expression and activity of MMP-2 and TIMP-1 and -2. The ERK-MAPK and PKC pathways are involved in the regulation of expression of MMP-2 and TIMP-1 and -2. ET-1's effects on MMPs/TIMPs may be important, not only in regulating the expression of MMPs and TIMPs, but also in influencing ECM remodeling.     

Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-1 in the Retinal Pigment Epithelium and Interphotoreceptor Matrix: Vectorial Secretion and Regulation

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) play an essential role in both normal and pathological extracellular matrix degradation, and a TIMP has been associated with at least one type of retinal degeneration. We have studied expression of MMP-2 and TIMP-1 by zymography, immunocytochemistry, and immunoblotting in the retinal pigment epithelium (RPE) from normal, aged and diseased retinas. MMPs and TIMPs were found in the rat RPE, interphotoreceptor matrix (IPM), and in media conditioned by human and rat RPE in culture. In other polarized cells, MMPs and TIMP-2 are secreted vectorially towards the basal lamina. In the RPE, however, MMP-2 and TIMP-1 were secreted preferentially from the apical surface, the surface bordering the IPM. These findings provide new evidence that MMPs and TIMPs could play a role in the turnover of IPM components.Cell homogenates and conditioned media from RPE isolated from mutant Royal College of Surgeons (RCS) rats with inherited retinal dystrophy had similar amounts of MMP-2 and TIMP-1 as those from congenic control rats. The secretion of MMP-2 and TIMP-1 from RPE cell cultures isolated from young and aged human donors varied widely. However, with increasing cell passage number, secretion of MMPs and TIMPs from human RPE increased dramatically. Also, growing human RPE on bovine corneal endothelial cell-generated extracellular matrix instead of plastic reduced the secretion of both MMPs and TIMPs. These data suggest that the integrity of Bruch's membrane may serve to regulate RPE functions in MMP and TIMP secretion and that extracellular matrices contain signals that regulate MMP and TIMP synthesis and/or secretion by the RPE.     

Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.     

基质金属蛋白酶在实验性单纯疱疹性角膜炎中的分布表达

目的 明确角膜感染Ⅰ型单纯疱疹病毒(HSV—1)后基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)在角膜中的分布。方法 HSV—1(KOS株)接种于BALB／c小鼠角膜上。分别收集正常眼球及感染后第2、7、14及28d的感染眼球行石蜡包埋，并应用抗MMP—2、—8、—9及TIMP—1、—2的抗体免疫染色角膜切片。结果 感染后第2d，MMP—2、—9及TIMP—1、—2的表达比未感染眼增加且表达主要位于浅表基质层及上皮下的炎性细胞中。感染后第14d及28d可见坏死性角膜炎及角膜溃疡形成，角膜基质中及浸润的炎性细胞中尤其是溃疡处可见MMP—2、—9及TIMP—1、—2表达显著增加。溃疡区域可见大量MMP—8阳性染色的中性粒细胞。结论 HSV—1角膜感染后由角膜细胞及浸润的炎性细胞分泌产生的MMPs对于上皮性角膜炎及溃疡形成过程可能起重要作用。     

rd1 mouse retina shows imbalance in cellular distribution and levels of TIMP-1/MMP-9, TIMP-2/MMP-2 and sulfated glycosaminoglycans

BACKGROUND: The rd1 mouse retina displays fast degeneration of photoreceptors resulting in a depletion of almost all rod photoreceptors by postnatal day 21 (PN21). To evaluate the role of proteinases in the pathophysiology of this animal model of retinitis pigmentosa, C3H rd1 and congenic wild-type (wt) mice retinas were analyzed. MATERIAL AND METHODS: The cellular localization and levels of proteins, matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), total sulfated glycosaminoglycans (sGAG) and nature of saccharides in rd1 and wt retinal extracts were compared. RESULTS: MMP-2/TIMP-2 and MMP-9/TIMP-1 were predominantly localized in the interphotoreceptor matrix (IPM) of both genotypes, but MMP-2/TIMP-2 also appeared in the Muller cell fibers of rd1 retina. In rd1 retinal extracts the levels of total proteins were lower and those of active MMP-9, MMP-2, TIMP-1 and total sGAG were higher than those of wt extracts. Despite an increase in TIMP-1, active MMP-9/MMP-2 were disproportionately elevated in rd1 compared to wt retina. With increasing age, MMPs in wt retinas were decreased but were increased in rd1. The sialylation of proteoglycans in PN2 and PN7 rd1 retinas was lower, and galactosylation was higher than that in wt retinas. CONCLUSIONS: MMP-9/MMP-2 and TIMP-1/TIMP-2 are associated with IPM, possibly after secretion by retinal pigmented epithelial cells. In degenerating rd1 retina, MMP-2/TIMP-2 are associated with the Muller cell fibers, which apparently play a central role in modifying the balance between MMPs and TIMPs. Elevated sGAG and proteolysis due to an imbalance in the levels of TIMPs and active MMP-9/MMP-2 in rd1 retina possibly contribute to retinal degeneration in the rd1 mouse.     

Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in the human lens: implications for cortical cataract formation

PURPOSE: To characterize the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in human cortical cataract and to determine whether there is a correlation with the localization of cortical cataract. To evaluate the expression and activity of MMPs and TIMPs after cytokine and UV-B exposure in a human lens epithelial cell line. METHODS: Twenty-eight human donor eyes with cortical cataract and 21 normal human donor eyes were photographed. Thirteen cortical cataract and six normal lenses were immunohistochemically analyzed for MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3. Twelve fresh cortical cataract and 12 normal lenses were divided into quadrants to quantify, by ELISA, the expression of MMP-1, -2, -3, and -9 and TIMP-1. Three fresh cortical cataract and three control lenses were assessed for MMP-1, -2, and -9 activity by SDS-PAGE zymography. Human lens epithelial cells (HLE-SRA-01/04) were exposed to proinflammatory cytokines and UV-B radiation to determine the protein expression profiles of MMP-1, -2, -3, and -9 and TIMP-1 and -2. RESULTS: Immunohistochemical analysis revealed specific localization of MMP-1 within lens epithelium and cortical lens fibers of cortical cataract. Normal lenses had equally low MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3 immunoreactivity, expression, and activity in all lens quadrants. IL-1 and TNF-alpha upregulated the expression of MMP-2, -3, and -9, and UV-B upregulated the expression of MMP-1 in the SRA-01/04 HLE cell line. CONCLUSIONS: This is the first study to localize the expression of MMP-1 in cataracts with clinically observed opacification in vivo and to examine the expression induced by UV-B, in vitro.     

基质金属蛋白酶/基质金属蛋白酶组织抑制剂系统与糖尿病眼部并发症

基质金属蛋白酶/基质金属蛋白酶组织抑制剂（MMPs/TIMPs）系统是降解和重塑细胞外基质（ECM）最重要的酶系统,它参与调节眼的一些生理和病理过程,如眼的发育、炎症、伤口愈合、新生血管形成、肿瘤转移等,与多种眼病,如增生性玻璃体视网膜病变、增生型糖尿病视网膜病变、糖尿病性白内障、孔源性视网膜脱离、视网膜母细胞瘤、年龄相关性黄斑变性、眼外伤、青光跟等的发生密切相关.就MMPs/TIMPs系统的生物学特性及其在糖尿病眼部并发症研究方面的应用进行综述.     

Expression and distribution of MMPs and TIMPs in human uveal melanoma

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).