Type VII Collagen in the Human Accommodation System: Expression in Ciliary Body, Zonules, and Lens Capsule.

Purpose: To investigate intraocular expression of COL7A1 and its protein product type VII collagen, particularly at the accommodation system. Methods: Eyes from 26 human adult donors were used. COL7A1 expression was analyzed in ex vivo ciliary epithelium by microarray. Type VII collagen distribution was examined by Western blot analysis, immunohistochemistry. and immuno-electron microscopy. Results: COL7A1 is expressed by pigmented and nonpigmented ciliary epithelia. Type VII collagen is distributed particularly at the strained parts of the accommodation system. Type VII collagen was associated with various basement membranes and with ciliary zonules. Anchoring fibrils were not visualized. Conclusions: Type VII collagen distribution at strained areas suggests a supporting role in tissue integrity.

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.

Immunohistochemical Evaluation of Idiopathic Epiretinal Membranes and In Vitro Studies on the Effect of TGF-ß 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-ß in the expression of collagens and α-smooth muscle actin (α-SMA) in retinal Müller cells. METHODS: Idiopathic ERM samples from vitrectomy were analyzed for glial acidic fibrillary protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), α-SMA, and type VI collagen using flat-mount immunohistochemistry. To study intracellular collagen expression in relation to cellular phenotype, spontaneously immortalized human Müller cells (MIO-M1) were treated with TGF-ß1 for 48 hours, and the expression of α-SMA and intracellular type I, II, IV, and VI collagens was studied by using immunocytology. Findings in Müller cells were compared with those in fetal lung fibroblasts and newborn skin fibroblasts. RESULTS: A colocalization of GFAP/CRALBP and GFAP/α-SMA was found in iERM, indicating a dynamic process of activation of retinal Müller cells in vivo. Transforming growth factor-ß1 induced up-regulation of α-SMA stress fibers in retinal Müller cells and both types of fibroblasts in vitro. The intracellular staining intensity of type I, II, and VI collagens was decreased in retinal Müller cells containing α-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 Müller cells are present in iERM. Transforming growth factor-ß1 induces an up-regulation of α-SMA stress fibers in retinal Müller cells and fibroblasts and appears to have a cell-specific effect on intracellular collagen expression.

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

PURPOSE: The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller 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 Müller cell morphology and differentiation state in relation to matrix stiffness. METHODS: A spontaneously immortalized human Müller 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 α-smooth muscle actin induced by transforming growth factor ß (TGF-ß [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, Müller cells treated with TGF-ß1 and TGF-ß2 and cultured on stiff substrates showed an increased incorporation of α-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 Müller 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.

The Ultrastructural Localization of Type II, IV, and VI Collagens at the Vitreoretinal Interface.

BACKGROUND: The vitreoretinal interface is the border of the cortical vitreous and the inner surface of the retina. The adhesion of the cortical vitreous to the ILM, namely vitreoretinal adhesion, involves a series of complex molecular adhesion mechanisms and has been considered as an important pathogenic factor in many vitreoretinal diseases. The presence of type VI collagen at the vitreoretinal interface and its possible interaction with collagens and glycoproteins indicates that type VI collagen may contribute to the vitreoretinal adhesion. PURPOSE: To clarify the ultrastructural location of type VI collagen and its relationship to type II and IV collagens at the vitreoretinal interface. METHODS: The ultrastructural localization of type II, IV and VI collagens in the adult human vitreoretinal interface of five donor eyes was evaluated by transmission electron microscopy using immunogold labeling. RESULTS: In the pre-equatorial region, we observed densely packed vitreous lamellae with a partly intraretinal course containing type II and VI collagens, reticular structures containing type IV and VI collagens and a thin inner limiting membrane (ILM) containing type IV and VI collagens in a linear distribution pattern. From the anterior to the posterior retina, the linear pattern of type IV and VI collagen labeling gradually became more diffusely present throughout the entire thickness of the ILM. CONCLUSIONS: The presence of type VI collagen in vitreous lamellae penetrating the ILM into the superficial retina suggests that type VI collagen may be involved in the organization of vitreous fibers into lamellae and in the adhesion of the vitreous fibers to the retina. The close relation of type VI to type IV collagen in the ILM suggests that type VI collagen is an important collagen type in the ILM. The topographic variations of type IV and VI collagens in the different regions of the ILM suggest a regional heterogeneity of the ILM. The reticular labeling pattern of type IV and VI collagens observed in the anterior vitreous are highly similar to labeling patterns of blood vessel walls. In the anterior vitreous, they may represent remnants of the regressed embryonic hyaloid blood vessel system. Their presence is in support of the theory on interactive remodeling of the developing vitreous as opposed to the main stream theory of displacement and compression of the primary by the secondary vitreous.

Glial cells and collagens in epiretinal membranes associated with idiopathic macular holes.

PURPOSE: To investigate the identity of collagens and cellular components in the epiretinal membrane (ERM) associated with full-thickness idiopathic macular hole and their clinical relevance. METHODS: Pars plana vitrectomy with the peeling of internal limiting membrane and ERM was performed by 2 surgeons in 40 eyes with idiopathic macular holes. The clinical data were reviewed and the surgical specimens were processed for flat-mount and immunohistochemical analysis. RESULTS: Epiretinal membrane is a GFAP-positive gliotic and fibrotic scar which contains newly formed Type I, III, and V collagens. Type VI collagen was not observed. Colocalization studies found cells coexpressing GFAP/CRALBP, GFAP/α-SMA, and α-SMA/CRALBP, which are consistent with transdifferentiation of Müller cells into fibroblasts and myofibroblasts. The clinically significant ERMs can be divided into two groups according to the amount of cells in ERM: sparse cellular proliferation and dense cellular proliferation. The latter group is associated with a higher chance of surgical difficulty during internal limiting membrane peeling (P = 0.006). Preoperative and postoperative visual function were not affected by the density of the cellular proliferation. CONCLUSION: Retinal glial cells, probably transdifferentiated Müller cells, are involved in the formation of full-thickness macular hole-associated ERMs by a gliotic and fibrotic process. Such ERMs contain newly formed Type I, III, and V collagen depositions. The cell density of ERM affects its biomechanical properties and determines the difficulty of ERM peeling.

Intraocular degradation behavior of crosslinked and linear poly(trimethylene carbonate) and poly(D,L-lactic acid).

The intraocular degradation behavior of poly(trimethylene carbonate) (PTMC) networks and poly(D,L-lactic acid) (PDLLA) networks and of linear high molecular weight PTMC and PDLLA was evaluated. PTMC is known to degrade by enzymatic surface erosion in vivo, whereas PDLLA degrades by hydrolytic bulk degradation. Rod shaped specimens were implanted in the vitreous of New Zealand white rabbits for 6 or 13 wk. All materials were well tolerated in the rabbit vitreous. The degradation of linear high molecular weight PTMC and PTMC networks was very slow and no significant mass loss was observed within 13 wk. Only some minor signs of macrophage mediated erosion were found. The fact that no significant enzymatic surface erosion occurs can be related to the avascularity of the vitreous and the limited number of cells it contains. PDLLA samples showed more evident signs of degradation. For linear PDLLA significant swelling and a large decrease in molecular weight in time was observed and PDLLA network implants started to lose mass within 13 wk. Of the tested materials, PDLLA networks seem to be most promising for long term degradation controlled intravitreal drug delivery since this material degrades without significant swelling. Furthermore the preparation method of these networks allows easy and efficient incorporation of drugs.

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 col2-3/4C-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.

Mature enzymatic collagen cross-links, hydroxylysylpyridinoline and lysylpyridinoline, in the aging human vitreous.

PURPOSE: The vitreous body of the human eye undergoes progressive morphologic changes with aging. Since the enzymatic collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are known to be important for the integrity of the collagen matrix, the presence in the vitreous on aging was studied. METHODS: Vitreous bodies (VBs; n = 143) from 119 donors (age 4-80 years; mean +/- SD, 54.3 +/- 17.0 years) were carefully dissected. After weighing and freeze-drying, all samples were analyzed by high performance liquid chromatography. Left and right eyes of 24 donors were compared and, for age-related phenomena, 119 single eyes were used. RESULTS: Within one donor, no significant differences were found between left and right eyes. On aging, VB wet weight (4.42 +/- 0.84 g) accumulates until 35 years and decreases thereafter. Collagen content (0.30 +/- 0.14 mg), HP per triple helix (TH; 0.55 +/- 0.18), and (HP plus LP)/TH (0.61 +/- 0.19) increase until 50 years followed by a decrease, whereas LP/TH (0.057 +/- 0.018) accumulates until 50 years and remains constant thereafter. The ratio between HP and LP (range, 0.42-31.0; median, 10.0) is constant over time. CONCLUSIONS: The accumulation of enzymatic collagen cross-links until 50 years is consistent with collagen maturation and possible collagen synthesis in the human vitreous body. The decline of collagen cross-links after 50 years is consistent with collagen breakdown.

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 pre-equatorial 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.

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

PURPOSE: To investigate the capacity of cultured Müller cells to synthesize collagens, since previous studies indicated that Müller cells could be involved in collagen remodeling at the vitreoretinal border in adult human eyes. METHODS: Spontaneously immortalized cultured human Müller cells were analyzed for the presence of mRNA of types I-VII, IX, XI, and XVII collagen by RT-PCR. Furthermore, Müller 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 Müller 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 Müller cells can synthesize internal limiting lamina and vitreous collagens. Possible collagen production by Müller cells could explain and expand on previous in vivo morphological findings in the embryonic and postnatal period and in pathologic conditions.

Packages of vitreous collagen (type II) in the human retina: an indication of postnatal collagen turnover?

The purpose of this study was to evaluate the vitreoretinal border in the (pre-)equatorial area in nonpathologic human donor eyes, because the majority of retinal defects induced by posterior vitreous detachment (PVD) are located there. Nine eyes (24-80 years) were fixed and embedded in Technovit 8100. After evaluation by light microscope, areas of interest were selected for immunotransmission electron microscope. Anti-type II collagen antibody was used to stain vitreous fibrils and lamellae; anti-type IV collagen antibody was used to identify the internal limiting lamina (ILL); anti-vimentin and anti-CD-68 antibodies stained retinal Muller cells and macrophages, respectively. Observations included fusing of lamellae with the ILL, an intravitreal course of the ILL, and clear focal interruptions in the ILL. In addition, an obvious finding was the presence of intraretinal packages of type II collagen. Interestingly these collagen packages were closely related to Muller cells and, in several eyes, also to macrophages, cell debris and interruptions in the ILL. In our opinion, the collagen packages can reflect the net result of a process of interactive remodelling, in which both breakdown and synthesis of vitreous and ILL collagens take place. Connections between vitreous and intraretinal collagen networks can make the (pre-)equatorial area more vulnerable to tearing and retinal detachment in the case of liquefaction and PVD.

Presence of collagen IV in the ciliary zonules of the human eye: an immunohistochemical study by LM and TEM.

The ciliary zonules of the eye are composed of fibrillar and non-fibrillar components. Fibrils provide tensile strength and elasticity, whereas non-fibrillar components serve as a coating surrounding the fibrils. This coating behaves as a barrier to macromolecules. The present light and transmission electron microscopic (LM and TEM) study identified collagen IV as a novel component of this coating. Collagen IV was demonstrated by pre-embedding and postembedding immunohistochemical (IHC) techniques using monoclonal and polyclonal antibodies. The specificity of the polyclonal anticollagen IV antibody was verified by ELISA.

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 light 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. CONCLUSIONS: This is the first study to evaluate vitreous liquefaction at the light 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 remnants in all specimens, it is tentatively concluded that an extracellular process is involved.