Type VII Collagen Expression in the Human Vitreoretinal Interface, Corpora Amylacea and Inner Retinal Layers

Type VII collagen, as a major component of anchoring fibrils found at basement membrane zones, is crucial in anchoring epithelial tissue layers to their underlying stroma. Recently, type VII collagen was discovered in the inner human retina by means of immunohistochemistry, while proteomic investigations demonstrated type VII collagen at the vitreoretinal interface of chicken. Because of its potential anchoring function at the vitreoretinal interface, we further assessed the presence of type VII collagen at this site. We evaluated the vitreoretinal interface of human donor eyes by means of immunohistochemistry, confocal microscopy, immunoelectron microscopy, and Western blotting. Firstly, type VII collagen was detected alongside vitreous fibers6 at the vitreoretinal interface. Because of its known anchoring function, it is likely that type VII collagen is involved in vitreoretinal attachment. Secondly, type VII collagen was found within cytoplasmic vesicles of inner retinal cells. These cells resided most frequently in the ganglion cell layer and inner plexiform layer. Thirdly, type VII collagen was found in astrocytic cytoplasmic inclusions, known as corpora amylacea. The intraretinal presence of type VII collagen was confirmed by Western blotting of homogenized retinal preparations. These data add to the understanding of vitreoretinal attachment, which is important for a better comprehension of common vitreoretinal attachment pathologies

Human retinal Müller cells synthesize collagens of the vitreous and vitreoretinal interface in vitro

Purpose: To investigate the capacity of cultured Muller cells to synthesize collagens, since previous studies indicated that Muller cells could be involved in collagen remodeling at the vitreoretinal border in adult human eyes. Methods: Spontaneously immortalized cultured human Muller cells were analyzed for the presence of mRNA of types I-VII, IX, XI, and XVII collagen by RT-PCR. Furthermore, Muller cells were immunocytochemically stained for light microscopic (LM) evaluation of these collagens and their main characteristics. Finally, cell extracts and culture medium were evaluated by western blot (WB) analysis using anticollagen antibodies. Results: Cultured Muller cells contained mRNA for types I-VII, IX, and XI collagen, but not for type XVII collagen. LM and WB confirmed the intracellular expression of all the above-mentioned collagens with the exception of type XVII. Collagen secretion into the medium was established for types I-VII, IX, and XI collagen. Conclusions: Cultured Muller cells can synthesize internal limiting lamina and vitreous collagens. Possible collagen production by Muller cells could explain and expand on previous in vivo morphological findings in the embryonic and postnatal period and in pathologic conditions

Enzymatic Breakdown of Type II Collagen in the Human Vitreous

PURPOSE. To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS. Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in the vitreous. Western blot and slot blot analyses were used to further identify vitreal type II collagen breakdown products in three age groups with average ages of 25, 45, and 65 years. Purified type II collagen was cleaved by MMPs that are known to occur naturally in the vitreous to elucidate what possible type II collagen breakdown products could thus be formed in the human vitreous. RESULTS. By means of both immunohistochemistry and slot blot analysis, col2-3/4C-short was detected in the vitreous. Using Western blot analysis, a range of type II collagen breakdown products was found, mostly in younger eyes, but none of these products contained the neoepitope that characterizes the col23/4C-short molecule. Digestion of purified type II collagen by MMPs did not give the same breakdown products as found in the vitreous. CONCLUSIONS. The presence of collagen degradation products in the human vitreous supports the hypothesis that enzymatic breakdown is most likely an active process in this extracellular matrix. Based on the size of the degradation products found by Western blot analysis, it is likely that in addition to MMPs, other proteolytic enzymes able to digest type II collagen are also active. (Invest Ophthalmol Vis Sci. 2009; 50: 4552-4560) DOI:10.1167/iovs.08-312

Collagen distribution in the human vitreoretinal interface

PURPOSE. To evaluate the presence of collagen types I to VII, IX, XI, and XVIII at the posterior pole, the equator and the preequatorial area in human donor eyes, since collagens are important macromolecules that contribute to vitreoretinal adhesion at the vitreoretinal interface. METHODS. Freshly isolated human retinectomy samples from the equator were used for reverse transcription-polymerase chain reaction to detect mRNA of the above-mentioned collagens. In addition, human donor eyes and equatorial retinectomy samples were embedded in paraffin, stained with antibodies against the collagens and evaluated by light microscopy (LM). RESULTS. Retinectomy samples expressed mRNA of all tested collagen types. By LM, vitreous cortex was positive for collagen types II, V, IX, and XI. In all three regions within the donor eyes and in the retinectomy samples, the internal limiting membrane (ILM) showed types IV, VI, and XVIII; the retinal vasculature was positive for types I to VI and XVIII in most specimens; and the retinal layers showed condensed spots of type VII. In addition, type VII increased in density and in distribution over the retinal layers toward the posterior pole. CONCLUSIONS. Staining patterns of collagen types I to V, IX, XI, and XVIII confirmed previous observations. Important new findings include the presence of type VI in the ILM and type VII in several layers of the retina. Both collagens can anchor matrix components, and type VI could be involved in vitreoretinal attachment. Furthermore, the presence of collagen mRNA in human retinectomy samples may be an indication of postnatal collagen production by retinal cells

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and alpha-Smooth Muscle Actin Expression of Retinal Muller Cells

PURPOSE. The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Muller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Muller cell morphology and differentiation state in relation to matrix stiffness. METHODS. A spontaneously immortalized human Muller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of alpha-smooth muscle actin induced by transforming growth factor beta (TGF-beta [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. RESULTS. MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Muller cells treated with TGF-beta 1 and TGF-beta 2 and cultured on stiff substrates showed an increased incorporation of alpha-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. CONCLUSIONS. Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Muller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings

Immunohistochemical Evaluation of Idiopathic Epiretinal Membranes and In Vitro Studies on the Effect of TGF-beta on Müller Cells

PURPOSE. The purpose of this study was to investigate the presence of type VI collagen and glial cells in idiopathic epiretinal membrane (iERM) and the role of TGF-beta in the expression of collagens and alpha-smooth muscle actin (alpha-SMA) in retinal Muller cells. METHODS. Idiopathic ERM samples from vitrectomy were analyzed for glial acidic fibrillary protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), alpha-SMA, and type VI collagen using flat-mount immunohistochemistry. To study intracellular collagen expression in relation to cellular phenotype, spontaneously immortalized human Muller cells (MIO-M1) were treated with TGF-beta 1 for 48 hours, and the expression of alpha-SMA and intracellular type I, II, IV, and VI collagens was studied by using immunocytology. Findings in Muller cells were compared with those in fetal lung fibroblasts and newborn skin fibroblasts. RESULTS. A colocalization of GFAP/CRALBP and GFAP/alpha-SMA was found in iERM, indicating a dynamic process of activation of retinal Muller cells in vivo. Transforming growth factor-beta 1 induced up-regulation of alpha-SMA stress fibers in retinal Muller cells and both types of fibroblasts in vitro. The intracellular staining intensity of type I, II, and VI collagens was decreased in retinal Muller cells containing alpha-SMA stress fibers, whereas the intracellular staining intensity of type I and VI collagens in both types of fibroblasts was not affected. CONCLUSIONS. Type VI collagen and activated retinal Muller cells are present in iERM. Transforming growth factor-beta 1 induces an up-regulation of alpha-SMA stress fibers in retinal Muller cells and fibroblasts and appears to have a cell-specific effect on intracellular collagen expression

Age-related liquefaction of the human vitreous body: LM and TEM evaluation of the role of proteoglycans and collagen

PURPOSE. To evaluate morphologic aspects of age-related liquefaction of the human vitreous body by fight and electron microscopy to provide a basis from which future studies directed at the pathogenesis of this phenomenon can be undertaken. The study focuses on changes in fibrillar Collagen and proteoglycans; (PGs). METHODS. Morphologic aspects of intravitreal liquefied spaces and matrix areas surrounding them were examined in 13 adult human donor eyes (aged 21-80 years) by light (LM) and transmission electron microscopy (TEM). Collagen fibrils were visualized by using standard contrasting methods. PGs were specifically stained by cupromeronic blue (CB). RESULTS. Eyes from older donors contained larger spaces than eyes from younger ones. Transitions between matrix and spaces were abrupt or gradual. In transition areas of all specimens, a gradual decrease in the number of collagen fibers, and to a lesser extent of PGs was observed. In addition, a fragmentation of collagen fibers and an aggregation of PG-molecules around these fragments were found. Neither cells nor their fragments were observed in these areas. CONCLUSION. This is the first study to evaluate vitreous liquefaction at the fight and electron microscopic level. A breakdown of collagen fibrils into smaller fragments seems to be crucial to the pathogenesis of age-related liquefaction of. the human vitreous body. The mechanism inducing fragmentation of vitreous fibrils has yet to be elucidated. From the absence of cells and cellular reimnants in all specimens, it is tentatively concluded that an extracellular process is involved. (Invest Ophthalmol Vis Sci. 2003;44:2828-2833) DOI:10.1167/ iovs.02-0588

Western blot confirms type VII collagen content in retinal substrates.

<p>Western blot stained for type VII collagen. A 290 kDa band appeared in samples (directly) placed in reducing agent buffer (group/lane <b>A</b>). Omitting the use of reducing agents resulted in fading of the 290 kDa band (group/lane <b>B</b>). When samples were incubated in 30 units/ml collagenase, the 290 kDa band disappeared completely, while a 145 kDa band appeared (group/lane <b>C</b>).</p

Retinal type VII collagen distribution.

<p>Immunohistochemical analysis at the vitreoretinal junction of a paraffin-embedded, serially sectioned (3–4 <i>μ</i>m) human donor eye evaluated by light microscopy. Anti-type VII collagen labeling with monoclonal LH7.2 (sections 1, 3 & 4) and periodic acid-Schiff (sections 2 & 5). Type VII collagen is present in astrocytic corpora amylacea. At least three corpora amylacea were sliced in this series, consecutively visualized in sections 1–2 (white arrows), sections 2–3 (black arrows) and sections 3–5 (black arrowheads). Corpora amylacea reside in the ganglion cell layer and inner plexiform layer (IPL). Type VII collagen is also present in small vesicles, clustered near large nuclei in the inner retina (white arrowhead). The vesicles reside outside the nucleus, and within the cytoplasm (sections 1, 2 & 3 compared). The cytoplasm of the type VII collagen yielding cell type seems more extensive than that of most other cells residing in the ganglion cell layer (GCL). Furthermore, their nuclei are larger and appear less dense in PAS (or hematoxylin) stains. The inner limiting membrane (ILM) does not label visibly for type VII collagen. Negative control section (NC) shows no labeling. (200 x)</p