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

Idiopathic epiretinal membrane

Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling. Methods: Analysis of the current literature regarding the epidemiology, clinical features, and pathogenesis of iERM and fibrotic tissue contraction. Results: Epidemiologic studies report a relationship between iERM prevalence, increasing age, and posterior vitreous detachment. Clinically, iERM progresses through different stages characterized by an increased thickness and wrinkling of the membrane. Pathophysiologically, iERM formation is a fibrotic process in which myofibroblast formation and the deposition of newly formed collagens play key roles. Anomalous posterior vitreous detachment may be a key event initiating the formation of iERM. The age-related accumulation of advanced glycation end products may contribute to anomalous posterior vitreous detachment formation and may also influence the mechanical properties of the iERM. Conclusion: Remodeling of the extracellular matrix at the vitreoretinal interface by aging and fibrotic changes, plays a significant role in the pathogenesis of iERM. A better understanding of molecular mechanisms underlying this process may eventually lead to the development of effective and nonsurgical approaches to treat and prevent vitreoretinal fibrotic diseases

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

<div><p>Background</p><p>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.</p><p>Purpose</p><p>To clarify the ultrastructural location of type VI collagen and its relationship to type II and IV collagens at the vitreoretinal interface.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

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/alpha-SMA, and alpha-SMA/CRALBP, which are consistent with transdifferentiation of Muller 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 Muller 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

Transmission electron microscopic images of the pre-equatorial, equatorial and posterior ILM stained with an antibody against type IV collagen.

<p>A. Pre-equatorial ILM of the eye of the 56 year-old donor. Note the linear distribution pattern of the labeling. B. Equatorial ILM of the eye of the 77 year-old donor. Note the more diffuse staining pattern. C. Posterior vitreoretinal interface of the 56 year-old donor eye. Type IV collagen appears to be distributed throughout the entire thickness of the ILM. A and B, bar = 1 μm; C, bar = 5 μm. V = vitreous; R = retina.</p

Complete set of transmission electron microscopic images of the pre-equatorial, equatorial and posterior vitreoretinal interface of one donor (male, aged 66 years).

<p>Type II collagen staining of vitreous fibrils at the pre-equatorial (A), equatorial (B) and posterior (C) vitreoretinal interface. Type IV collagen staining of the ILM at the pre-equatorial (D), equatorial (E) and posterior (F) vitreoretinal interface. Type VI collagen staining in the pre-equatorial (G), equatorial (H) and posterior (I) vitreoretinal interface. Note the lineair labeling pattern at the pre-equatorial location, and the more diffuse distribution of the labeling at the equator and posterior sites. D and G: note reticular pattern (arrows). Also, the ILM thickens towards the posterior retina, in this older donor, the ILM at the posterior pole is thicker than that in the younger donor (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134325#pone.0134325.g004" target="_blank">Fig 4</a>). Bar = 2μm. V = vitreous; R = retina; ILM = inner limiting membrane.</p

Summary of the collagens present at the vitreoretinal interface.

<p>ILM: inner limiting membrane;</p><p>*type IV collagen is attached to reticular structures in the anterior vitreous.</p><p>Summary of the collagens present at the vitreoretinal interface.</p