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Muller cell expression of genes implicated in proliferative vitreoretinopathy is influenced by substrate elastic modulus.

ABSTRACT: Matrix stiffness is recognized increasingly as a significant factor in cell and tissue function. To understand better the mechanosensitivity of Müller cells and its association with vitreoretinal disorders, we examined morphology, propagation, and expression of genes in Müller cells that were cultured on substrates of varying elastic moduli.A conditionally immortalized mouse Müller cell line was cultured on laminin-coated polyacrylamide substrates with calibrated Young's moduli. Glass was used as a control. Phase contrast, fluorescence, and atomic force microscopy were used to study cell morphology and propagation. Expression of extracellular matrix (ECM) genes was analyzed using quantitative reverse-transcription PCR.The adherent area, stiffness, and propagation of Müller cells all are affected by matrix stiffness, but to different extents and with different ranges of sensitivity. Of 85 ECM genes tested 11 showed a continuous >4-fold increase or decrease in mRNA expression as a function of the substrate elastic modulus. The changes were statistically significant in four genes: connective tissue growth factor (Ctgf, P = 0.04), tenascin C (Tnc, P = 0.035), Collagen I?1 (Col1a1, P = 0.0001), and Collagen IV?3 (Col4a3, P = 0.05), with all showing increased expression on softer substrates.There are significant changes in morphology, cytoskeletal integrity, and gene regulation in Müller cells as a function of the stiffness of the substrate. Changes in local tissue elastic modulus may have a role in vitreoretinal disorders. These findings also may have implications for strategies for improved integration of retinal prosthetics, and for stem cell therapies, particularly targeting the transcriptional regulators YAP and TAZ.

SUBMITTER: Davis JT

PROVIDER: S-EPMC3378084 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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Muller cell expression of genes implicated in proliferative vitreoretinopathy is influenced by substrate elastic modulus.

Davis Joshua T JT Wen Qi Q Janmey Paul A PA Otteson Deborah C DC Foster William J WJ

Investigative ophthalmology & visual science 20120517 6

<h4>Purpose</h4>Matrix stiffness is recognized increasingly as a significant factor in cell and tissue function. To understand better the mechanosensitivity of Müller cells and its association with vitreoretinal disorders, we examined morphology, propagation, and expression of genes in Müller cells that were cultured on substrates of varying elastic moduli.<h4>Methods</h4>A conditionally immortalized mouse Müller cell line was cultured on laminin-coated polyacrylamide substrates with calibrated ...[more]

PMID: 22447866

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Project description:PurposeThe purpose of the study was to evaluate the efficacy and safety of intravitreal pirfenidone for inhibition of proliferative vitreoretinopathy (PVR) in a model of penetrating ocular injury.Patients and methodsPenetrating trauma was induced on the retina of rabbit and treated either with 0.1 ml of phosphate-buffered saline (PBS) or 0.1 ml of 0.5% pirfenidone, and development of PVR was evaluated clinically and graded after 1 month. Histopathology and immunohistochemistry with transforming growth factor beta (TGFβ), alpha smooth muscle actin (αSMA), and collagen-1 were performed to assess the fibrotic changes. Expression of cytokines in the vitro-retinal tissues at different time points following pirfenidone and PBS injection was examined by RT-PCR. Availability of pirfenidone in the vitreous of rabbit at various time points was determined by high-performance liquid chromatography following injection of 0.1 ml of 0.5% pirfenidone. In normal rabbit eye, 0.1 ml of 0.5% pirfenidone was injected to evaluate any toxic effect.ResultsClinical assessment and grading revealed prevention of PVR formation in pirfenidone-treated animals, gross histology, and histopathology confirmed the observation. Immunohistochemistry showed prevention in the expression of collagen-I, αSMA, and TGFβ in the pirfenidone-treated eyes compared to the PBS-treated eyes. Pirfenidone inhibited increased gene expression of cytokines observed in control eyes. Pirfenidone could be detected up to 48 h in the vitreous of rabbit eye following single intravitreal injection. Pirfenidone did not show any adverse effect following intravitreal injection; eyes were devoid of any abnormal clinical sign, intraocular pressure, and electroretinography did not show any significant change and histology of retina remained unchanged.ConclusionThis animal study shows that pirfenidone might be a potential therapy for PVR. Further clinical study will be useful to evaluate the clinical application of pirfenidone.

Project description:Purpose: The development of vitreoretinal scars remains an unsolved challenge in clinical practice and often leads to repeated revision surgery and blindness due to lack of efficient therapy. The aim of this study was to characterize the cellular and molecular environment in vitreoretinal scar tissue from patients with proliferative vitreoretinopathy (PVR) in comparison to membranes of the vitreoretinal junction, in order to subsequently identify potential drug treatment options using bioinformatics techniques. Methods: A total of 23 patients undergoing vitrectomy for retinal detachment due to proliferative vitreoretinopathy (PVR, n = 15), idiopathic macular hole (MH, n = 10) or idiopathic macular pucker (MP, n = 8), were included in this study. Vitreoretinal samples were analysed by RNA sequencing, MS-CyTOF proteomic and by conventional immunohistochemistry. The cellular microenvironment was assessed by bioinformatics cell type enrichment analysis. Drug target screening was performed by using a transcriptome-based drug-repurposing approach using drug-exposure transcriptome data from public available databases. Results: RNA sequencing of vitreoretinal tissue samples showed distinct transcriptional differences of PVR, ERM and ILM samples allowing an accurate clustering according to the tissue types. The cellular microenvironment of PVR membranes was characterized by the enrichment of melanocytes, astrocytes and various immune cell types, such as M2 macrophages. Differential gene expression (DEG) analysis revealed XX up- and XX downregulated genes in PVR compared to ILM. Among them, FN1 (fibronectin1), SPARC (osteonectin) and various collagens were among the most upregulated factors in PVR, contributing to biological processes such as the organization of extracellular structures, the regulation of cell adhesion and the morphogenesis of blood vessels. Finally, we identified 13 drugs that induce a gene expression profile complementary to the PVR signature and thus represent potential therapeutic options for PVR, including aminocaproic acid and various topoisomerase 2A inhibitors such as doxorubicin, etoposide and mitoxantrone. Conclusion: The present study reveals significant differences in the transcriptional signature of vitreoretinal scar tissue including PVR, MP and ILM tissue. Among a plethora of differentially expressed genes, FN1 and SPARC appeared to be central mediators underlying PVR development. Based on the transcriptome analyses, aminocaproic acid and topoisomerase-2 inhibitors, such as doxorubicin, etoposide and mitoxantrone, were identified as compatible drugs for the treatment of PVR.

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Muller cell expression of genes implicated in proliferative vitreoretinopathy is influenced by substrate elastic modulus.

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Muller cell expression of genes implicated in proliferative vitreoretinopathy is influenced by substrate elastic modulus.

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