Localization of extracellular proteins of the human trabecular meshwork by indirect immunofluorescence

We used monospecific antibodies on semithin frozen sections to identify and localize the major tissue constituents of the nonglaucomatous human trabecular meshwork. The trabecular beams (sheets and cords) consist of a basement membrane (subendothelial extracellular matrix) surrounding an interstitial central core of connective tissue (substantia propria). The basement membrane contains collagen types III, IV, and V, the glycoproteins laminin and fibronectin, and the basement membrane-associated heparan sulfate proteoglycan. The trabecular basement membrane is unlike most subendothelial basement membranes because it contains collagen type III and a relatively disorganized structure. The central core contains collagen types I and III, and elastin. The closely linked juxtacanalicular meshwork contains collagen type III, but no collagen type I or elastin. The connective tissue composition of the trabecular meshwork appears similar to other highly compliant and resilient tissues, such as lung, blood vessels, and conjunctiva.     

Collagen,Factor VIII Antigen,and Immunoglobulins in the Human Aqueous Drainage Channels

Twenty-five trabeculectomy specimens from patients with primary open angle glaucoma and chronic angle closure glaucoma, and 11 age-matched controls were examined by immunofluorescence and immunoperoxidase techniques to determine the types of collagen, immunoglobulins, and the presence of factor VIII-related antigen in the human aqueous drainage channels. In the glaucoma cases and in controls, we demonstrated that the electron dense basement membrane-like material in the peripheral portion of the trabecular beams and in the juxtacanalicular meshwork, consists at least in part, of type IV collagen, a noncollagenous protein (“laminin”) and fibronectin. Factor VIII-related antigen was demonstrated in conjunctival vessels of the control eyes. Schlemm's canal and the trabecular endothelial cells did not stain for factor VIII-related/antigen in any of the specimens examined. No deposits of IgA, IgM, IgG, and the C3 component of complement were detected in the aqueous drainage channels.     

Immunofluorescence studies of the human trabecular meshwork

Accumulation of extracellular material in the trabecular meshwork is responsible for increased resistance to outflow in the human eye. By means of the indirect immunofluorescence technique the authors detected extracellular collagen type IV, fibronectin and laminin in the trabecular meshwork and the wall of Schlemm's canal of glaucoma patients and controls. Collagen type IV is the so-called "basement membrane collagen" present in the lens capsule, Bowman's and Descemet's membranes. Fibronectin is a glycoprotein with many biologic activities; it can promote cell attachment and forms complexes with collagen. Laminin was isolated in 1979 and is present in the lamina lucida of basement membranes. The increased production and pathological metabolism of these three substances might be of some importance in the pathogenesis of primary open-angle glaucoma.     

Distribution of myocilin and extracellular matrix components in the corneoscleral meshwork of human eyes

PURPOSE: To examine ultrastructurally the composition of major extracellular matrix (ECM) components and the distribution of myocilin in the trabecular lamellae of corneoscleral (CS) meshwork in normal human eyes. The codistribution of myocilin with ECM components was also investigated. METHODS: Postembedding immunoelectron microscopic studies were performed with antibodies against myocilin and other ECM components, including fibronectin, laminin, vitronectin, tenascin, elastin, fibrillin-1, microfibril-associated glycoprotein (MAGP)-1, decorin, versican, hyaluronic acid, and five types of collagen (I, III, IV, V, and VI). Double labeling of myocilin with other ECM components was performed with different sized gold particles. RESULTS: In the trabecular beams of CS meshwork, fibronectin, laminin, and collagen type IV were associated with basement membranes, whereas elastin was specifically localized to the core of elastic-like fibers. Several types of collagens, glycoproteins, proteoglycans, and hyaluronic acid were detected both in the collagen fibers and ground substances. Myocilin predominantly localized in the long-spacing collagens and sheath materials surrounding elastic-like fibers, codistributed with fibronectin, fibrillin-1, MAGP-1, decorin, and type VI collagen. CONCLUSIONS: This study illustrated the composition of ECM materials in the trabecular lamellae of CS meshwork. Myocilin was specifically localized to long-spacing collagens and the surrounding sheath of elastic-like fibers interacting with microfibril-associated elements where changes have been documented to occur in glaucomatous and aging eyes.     

Human trabecular meshwork cells in culture: morphology and extracellular matrix components

This study demonstrates the presence of laminin and collagen type IV in the extracellular space of human trabecular meshwork (HTM) cells in culture and its absence in cultures of fibroblasts from sclera adjacent to the outflow pathway. These basement membrane components can be detected by standard immunohistochemical techniques. Positive staining for these macromolecules is found only after the HTM cells have reached confluence. The presence of laminin and human collagen type IV in the early passages can serve as additional criteria for identification of HTM cells in culture. These cells provide a useful experimental system for studying the effects of drugs and other factors on the synthesis of components of the extracellular matrix.     

Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats

PURPOSE: To understand the pathophysiology of the corneal basement membrane in diabetes, we compared the localization of laminin and type IV collagen in the epithelial basement membrane during corneal epithelial wound healing in diabetic and nondiabetic rats. METHODS: Streptozotocin was used to induce diabetes in half the rats. Two weeks later, the whole corneal epithelium was debrided. Diabetic and healthy rats (3-5 per group) were sacrificed before debridement and 1, 3, and 7 days and 1 month afterwards. The localization of laminin and type IV collagen was observed in cryosections by epifluorescence microscopy. RESULTS: In unwounded corneas of both diabetic and normal rats, laminin and type IV collagen were localized in the corneal epithelial basement. The intensity of fluorescence, however, was clearly stronger in the diabetic rats. In normal rats, wounding initially removed laminin and type IV collagen, but during healing these two proteins reappeared beneath the resurfacing corneal epithelium. Although similar results were observed in diabetic rats, the expression of laminin and type IV collagen was delayed, and their deposition was fragmented and irregular. CONCLUSIONS: These results suggest that delayed corneal epithelial wound healing in diabetes might involve delayed reappearance and abnormal reformation of epithelial basement membrane proteins.     

Immunocytochemical localization of laminin, type IV collagen and fibronectin in rat retinal vessels

Laminin, type IV collagen and fibronectin have been identified as major components of the basement membrane (basal lamina) in various tissues. These antigens have also been identified in retinal vessels by light microscopic immunofluorescence but their precise location could not be determined at this level of resolution. In this study, we examined the localization of these constituents at the ultrastructural level using the protein A-immunoperoxidase technique. The basal lamina of all retinal capillaries, arterioles and venules was immunostained after exposure to antisera against laminin, type IV collagen and fibronectin. Staining was localized to the lamina densa, which appeared as a single or double layer. Immunostaining for fibronectin showed the weakest activity. The reaction was also seen in discrete patches between endothelial cells and pericytes. The inner limiting membrane of the retina was also reactive for laminin and type IV collagen but not for fibronectin. The results indicate that laminin, type IV collagen and fibronectin are components of the basal lamina in all types of retinal vessels. The presence of fibronectin at the endothelial-pericyte interface suggests that this protein may promote adhesion between cells and thus help to maintain the integrity of the vessel wall.     

Extracellular matrix of the optic nerve lamina cribrosa in the normal monkey eyes

The presence and distribution of collagen type I through VI, laminin, fibronectin and alpha-elastin were demonstrated in the normal monkey optic nerve lamina cribrosa, using a immunohistochemical method, the biotin-streptavidin (B-SA) system. Interstitial collagens, collagen type I and III, were detected diffusely within laminar beams continuously with surrounding sclera. The adventitia around the central artery and vein, and pial septa were also stained with collagen type I and III. Collagen type IV and laminin, which were basement membrane materials, were localized linearly along the margin of the laminal beams, pial septa and adventitia. They were also stained in all vascular walls. Collagen type V, VI and fibronectin were similar with collagen type I and III, but their linear staining was more intense onto the margin of the laminar beams and the pial septa. Rich immunoreactivity for alpha-elastin was found in the glial columns and the laminal beams. The presence and distribution of the structural protein components of the ECM of the monkey optic nerve lamina cribrosa was the same as that previously reported in human.     

Immunohistochemical light and electron microscopy of basal laminar deposit

The formation of basal laminar deposit (BLD) is one of the histopathologic changes in the aging human macula. BLD is assumed to be an early stage of age-related macular degeneration. The location of BLD, between the RPE plasma membrane and its basement membrane and in the outer collagenous zone of Bruch's membrane, and its ultrastructure suggest that it is composed of excessive amounts of basement membrane material. The main components of basement membranes are type IV collagen, heparan sulfate proteoglycans (HSPG) and laminin. Labeled antibodies against these components can therefore be used for the identification and localization of basement membrane material by means of immunohistochemical techniques. In this study the presence of type IV collagen, laminin and HSPG was determined in aged human maculae by immunohistochemistry and immunoelectron microscopy. Tests for the presence of type VI collagen and fibronectin were also performed. We obtained 76 eyes from 68 human subjects at autopsy or after surgical enucleation for anteriorly located choroidal melanomas. The finely granular component of BLD stained positive with antibodies against type IV collagen, HSPG and laminin, but the long-spacing collagen component of BED did not. Neither component of BED was stained with antibodies against type VI collagen or fibronectin. We conclude that BLD consists partly of excess basement membrane material.     

Type IV collagen and laminin in Bruch''s membrane and basal linear deposit in the human macula.

Tissue obtained from the macula in 10 human eyes (53-77 years) was used for an investigation into the extracellular matrices of the retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris. The ultrastructural distribution of type IV collagen and laminin was documented using immunogold labelling. Labelling for type IV collagen was strongly positive in all the specimens in the basement membranes of the choriocapillaris but not that of the RPE where labelling was either weak or absent. Laminin was localised to deposits of granular material in Bruch's membrane but was absent from the basement membrane of the RPE and the choriocapillaris. Basal linear deposit, observed in three cases, demonstrated labelling for laminin but not for type IV collagen. The series was too small for correlation of these morphological changes with age.     

实验性糖尿病视网膜微血管病变的病理研究

目的：观察糖尿病视网膜病变（diabetic retinopathy,DR）的组织学改变。方法：应用光镜、免疫组织化学、电镜及组织化学电镜等技术,研究在不同时间点Spregue-Dawley(SD)大鼠视网膜毛细血管基底膜中的Ⅳ型胶原蛋白及层黏蛋白和视网膜毛细胞血管基底膜的厚度,以及其负电荷位点数目的变化。结果：随着糖尿病病程的发展,视网膜毛细血管基底膜下不断增厚伴有Ⅳ型胶原蛋白及层黏蛋白的增加,同时负电荷位点数目减少。结论：视网膜毛细血管基底膜增厚,Ⅳ型胶原蛋白及层黏蛋白的增加,负电荷位点数目减少可能是导致DR渗出性病变的病理基础。     

Extracellular matrix in aged human ciliary body: an immunoelectron microscope study.

Tissue from nine human eyes (ages 52-78 yr) was used to investigate the fine structural distribution of collagens I-VI and laminin in the ciliary body using the immunogold antibody labeling technique. The anterior segments of the specimens were normal, and the eyes were removed in treatment of choroidal melanoma. The basement membranes of the ciliary epithelium contained collagens I, III, and IV. Laminin was in greater concentration in the outer part of the nonpigmented epithelial basement membrane, and the distribution suggested a washout effect. The zonular apparatus labeled intensely with laminin. In contrast, laminin was not present in the basement membrane of the myocytes in the ciliary body. These cells were sheathed in a basement membrane that contained types I, III, and IV collagen. Plaque-like structures of slightly different morphology (a, filamentous; b, granular; c, amorphous) were found in the tendinous insertions, and subtypes a and b were strongly labeled with laminin. The basement membranes of the vessels contained types I and IV collagen, but laminin labeling was inconclusive. The major finding was that the lamina densa in the basement membranes of various sites labeled for collagens I, III, and IV. Striated collagen fibrils in the stroma were labeled for types I and III. Collagen subtypes V and VI were not identified in significant quantity in any of the regions examined.     

Increases in collagen type IV and laminin in galactose-induced retinal capillary basement membrane thickening--prevention by an aldose reductase inhibitor

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet (P less than 0.001), or animals on a diet containing 30% galactose and 250 mg kg-1 of the aldose reductase inhibitor sorbinil (P less than 0.001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea]

PURPOSE: To understand corneal responses to holmium YAG (Ho: YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. METHODS: Rats were radiated with an Ho: YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin. RESULTS: One day after Ho: YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. CONCLUSIONS: These results suggest that Ho: YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea

Purpose: To understand corneal responses to holmium YAG (Ho:YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. Methods: Rats were radiated with an Ho:YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin.Results: One day after Ho:YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. Conclusions: These results suggest that Ho:YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.     

Extracellular matrix of the human retrobulbar optic nerve

The presence and distribution of laminin, fibronectin and type I through V collagens were determined in the human retrobulbar optic nerve using a immunohistochemical method, the biotin-streptavidin (B-SA) system. Type I and type III collagens, which were interstitial collagens, were detected diffusely within pial septa, pia mater, arachnoid membrane, dura mater and adventitia around the central retinal artery and vein. Type IV collagen and laminin, which were basement membrane materials, were localized linearly along the borders between optic nerve fascicles and pial septa, adventitia, pia mater. Also They were also stained in all vascular walls and the arachnoid membrane. Type V collagen and fibronectin were detected both in the interstitial connective tissues, diffusely, and in the basement membranes, linearly. Fibronectin was stained more intensively in the basement membranes than type V collagen.     

In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.

PURPOSE: To determine whether trabecular meshwork-inducible glucocorticoid response/myocilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS: The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/MYOC. Solid phase binding assays using 125I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS: TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cDNA. In solid phase binding assays, 125I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS: TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the TM and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.     

正常成人视乳头筛板细胞外基质的免疫组化研究

目的 用免疫组化法研究正常成人视乳头筛板细胞外基质的结构组成和分布。方法 用免疫过氧化酶法（ＡＢＣ法）观察７例（１４只眼）正常成人眼筛板中,Ⅳ型胶原蛋白、纤维连接蛋白（ＦＮ）和层连接蛋白（ＬＮ）的分布。结果 正常成人筛板中存在大量Ⅳ型胶原蛋白和层连接蛋白,纤维连接蛋白仅在血管处呈阳性染色,筛板中无阳性染色。结论 筛板是中枢神经系统的一种特殊组织,由类似基底膜样物质组成,赋予筛板弹性和基底膜的机械稳     

猴实验性青光眼视乳头筛板细胞外基质免疫组化研究

目的 研究猴实验性青光眼视乳头筛板细胞外基质的改变,探讨眼压增高对筛板的影响。方法 用免疫过氧化物酶技术J（ABC法）观察9只猴早、中、晚期青光眼模型筛板中,Ⅳ型胶原蛋白、层连接蛋白的分布。结果 与正常猴视乳头相比,早、中期青光眼模型视乳头筛板无明显变化。晚期青光眼视乳头筛板前区和筛板部Ⅳ型胶原蛋白和层连接蛋白阳性染色物明显增多,筛板后陷、重叠及融合。结论 青光眼病理过程中的眼压增高,可引起视乳头筛板细胞外基质发生特异性改变,从而使视乳头筛板的生物力学特性发生变化。     

Colocalization of fibroblast growth factor binding sites with extracellular matrix components in normal and keratoconus corneas

Basic and acidic fibroblast growth factor (bFGF, aFGF) binding sites were determined in frozen sections of normal and keratoconus corneas. After incubation with I-125 radiolabelled growth factors, corneal binding sites were revealed by autoradiography. The growth factors were localized mainly to Descemet's membrane and to the epithelial basement membrane. FGF binding sites were generally similar in normal and keratoconus corneas. The binding specificity was demonstrated by competitive inhibition experiments with an excess of unlabelled growth factors. The binding sites were sensitive to pretreatment of the corneal sections with heparitinase. We have attributed FGF's basement membrane affinity to one of its constituents, proteoheparan sulfate. Proteoheparan sulfate, laminin, collagen type IV, and fibronectin were all revealed by immunofluorescent techniques. While keratoconus cornea stroma had less laminin but more fibronectin than normal corneas the main difference lied in type IV collagen which was overexpressed in keratoconus epithelium.