Substrate Stiffness Influences the Time Dependence of CTGF Protein Expression in Müller Cells

Following ocular trauma and retinal detachment, gliotic changes in the retina may develop over the subsequent month, a process known as PVR (proliferative vitreoretinopathy). There have been no successful therapeutic interventions to inhibit PVR. The protein CTGF (Connective Tissue Growth Factor) has been associated with retinal PVR and other fibrotic diseases of the retina in clinical studies but the mechanistic link between different pathologies and retinal gliosis has not been determined. In addition, CTGF has been previously noted to be associated, in some cases, with YAP/TAZ (Yes-associated protein and Tafazzin protein complex), transcriptional regulatory proteins that change subcellular localization in response to mechanical cues, such as the stiffness of the underlying material. We have previously shown that the mRNA for CTGF is markedly (100-fold) upregulated in retinal Müller cells grown on soft substrates. In order to evaluate if the mechanism by which mechanotransduction modulating CTGF production in retinal Müller cells involves the YAP/TAZ complex, this study tests the influence of substrate stiffness on the time dependence of CTGF protein expression, as well as subcellular localization of YAP/TAZ using a conditionally-immortalized mouse retinal Müller cell line plated on laminin-coated, polyacrylamide substrates of varying elastic modulus. Changes were assayed using immunohistochemistry and ELISA (Enzyme-Linked ImmunoSorbent Assay). In retinal Müller cells, the relationship between elastic modulus and the pattern of CTGF protein expression was bimodal, with CTGF levels rising more rapidly for cells on hard substrates and more slowly for cells grown on soft substrates. In addition, nuclear localization of YAP/TAZ corresponded directly to the maximum CTGF expression.

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Fenofibrate Inhibits Subretinal Fibrosis Through Suppressing TGF‐β—Smad2/3 signaling and Wnt signaling in Neovascular Age‐Related Macular Degeneration

Frontiers in Pharmacology ◽

10.3389/fphar.2020.580884 ◽

2020 ◽

Vol 11 ◽

Author(s):

Qian Chen ◽

Nan Jiang ◽

Yuhan Zhang ◽

Sihao Ye ◽

Xu Liang ◽

...

Keyword(s):

Protein Expression ◽

Wnt Signaling ◽

Macular Degeneration ◽

Müller Cells ◽

Tissue Growth ◽

Age Related Macular Degeneration ◽

Collagen Deposition ◽

Muller Cells ◽

Age Related ◽

Subretinal Fibrosis

Subretinal fibrosis is a common pathological change that causes vision loss in neovascular age-related macular degeneration (nAMD). Treatment modalities for subretinal fibrosis are limited. In the present study, the effects of fenofibrate, a specific peroxisome proliferator–activated receptor alpha agonist, on subretinal fibrosis of nAMD were tested, and its molecular mechanisms of action were delineated. Collagen deposition and protein expression of fibrotic markers, such as vimentin, collagen-1, alpha-smooth muscle actin, and fibronectin, were increased in very low–density lipoprotein receptor (VLDLR) knockout mouse, indicating Vldlr−/− mice can be used as a model for subretinal fibrosis. Fenofibrate suppressed subretinal fibrosis of Vldlr−/− mice by reducing collagen deposition and protein expression of fibrotic markers. Two fibrotic pathways, TGF-β—Smad2/3 signaling and Wnt signaling, were significantly up-regulated, while inhibited by fenofibrate in Vldlr−/− retinas. Moreover, fenofibrate significantly reduced the downstream connective tissue growth factor (CTGF) expression of these two pathways. Müller cells were a major source of CTGF in Vldlr−/− retinas. Fenofibrate was capable of suppressing Müller cell activation and thus reducing the release of CTGF in Vldlr−/− retinas. In cultured Müller cells, fenofibrate reversed TGF-β2–induced up-regulation of Wnt signaling and CTGF expression. These findings suggested that fenofibrate inhibits subretinal fibrosis by suppressing TGF-β—Smad2/3 signaling and Wnt signaling and reducing CTGF expression, and thus, fenofibrate could be a potential treatment for nAMD with subretinal fibrosis.

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Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy

Frontiers in Immunology ◽

10.3389/fimmu.2020.601639 ◽

2021 ◽

Vol 11 ◽

Author(s):

Ahmed M. Abu El-Asrar ◽

Mohd Imtiaz Nawaz ◽

Ajmal Ahmad ◽

Alexandra De Zutter ◽

Mohammad Mairaj Siddiquei ◽

...

Keyword(s):

Diabetic Retinopathy ◽

Blood Vessels ◽

Proliferative Diabetic Retinopathy ◽

Müller Cells ◽

Intercellular Adhesion Molecule ◽

Enzyme Linked Immunosorbent Assay ◽

Diabetic Patients ◽

Muller Cells ◽

Epiretinal Membranes ◽

Positive Correlations

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.

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Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7Receptor Expression and Retinal Ganglion Cell Death

BioMed Research International ◽

10.1155/2016/9020715 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Ying Xue ◽

Yuting Xie ◽

Bo Xue ◽

Nan Hu ◽

Guowei Zhang ◽

...

Keyword(s):

Protein Expression ◽

Ganglion Cell ◽

Intravitreal Injection ◽

Retinal Ganglion Cell ◽

Cell Activation ◽

Müller Cells ◽

Müller Cell ◽

Retinal Ganglion ◽

Muller Cells ◽

Muller Cell

P2X7receptor (P2X7R), an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC) apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.

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

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.14-15969 ◽

2015 ◽

Vol 56 (10) ◽

pp. 5974 ◽

Cited By ~ 6

Author(s):

Shao-Chong Bu ◽

Roel Kuijer ◽

Roelofje J. van der Worp ◽

Sander M. van Putten ◽

Olaf Wouters ◽

...

Keyword(s):

Smooth Muscle ◽

Elastic Modulus ◽

Smooth Muscle Actin ◽

Focal Adhesions ◽

Müller Cells ◽

Muller Cells ◽

Muscle Actin ◽

Actin Expression

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Cyanin Chloride Inhibits Hyperbaric Pressure-Induced Decrease of Intracellular Glutamate-Aspartate Transporter in Rat Retinal Müller Cells

Journal of Ophthalmology ◽

10.1155/2018/6128470 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6

Author(s):

Xiaomin Chen ◽

Yue Wang ◽

Fangfang Han ◽

Min Ke

Keyword(s):

Protein Expression ◽

Survival Rates ◽

Müller Cells ◽

Control Group ◽

Pressure Group ◽

Muller Cells ◽

Sprague Dawley ◽

Additional Pressure ◽

Hyperbaric Pressure ◽

Mrna And Protein Expression

Purpose. Glaucoma is the leading cause of irreversible blindness throughout the world. The pathogenesis of glaucoma is complex, and neuroprotection is a crucial aspect of therapy. High concentrations of extracellular glutamate are toxic to the optic nerve. The glutamate-aspartate transporter (GLAST) in retinal Müller cells is involved in the development of glaucoma. Anthocyanin has been reported to protect retinal neurons. We hypothesize that cyanin chloride, a type of anthocyanin, can inhibit hyperbaric pressure-induced GLAST decreases in cultured rat retinal Müller cells and may serve as a potential neuroprotective agent in glaucoma treatment. Materials and Methods. Sprague Dawley rat Müller cells were cultured in a hyperbaric pressure device at 60 mmHg additional pressure and treated with cyanin chloride (10 μmol/L, 30 μmol/L, or 50 μmol/L) or vehicle for 2 hours. Cell survival rates (SRs) were evaluated by an MTT assay. GLAST mRNA and protein expression were determined by western blot and RT-PCR analyses, respectively. Results. Cell SR was significantly decreased in the 60 mmHg additional hyperbaric pressure group compared to the control group (P<0.01). Cyanin chloride treatment significantly improved SR under 60 mmHg additional pressure (P<0.01). GLAST mRNA and protein expression levels in Müller cells were significantly reduced in the 60 mmHg hyperbaric pressure group compared to the control group (P<0.01), but cyanin chloride significantly inhibited hyperbaric pressure-induced decreases in GLAST expression (P<0.01). Conclusion. Our results support our hypothesis and demonstrate that cyanin chloride can protect rat retinal Müller cells from hyperbaric pressure-induced decreases of GLAST.

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Faculty Opinions recommendation of Muller cells are living optical fibers in the vertebrate retina.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1083850.535980 ◽

2007 ◽

Author(s):

Roger Hardie

Keyword(s):

Optical Fibers ◽

Müller Cells ◽

Muller Cells ◽

Vertebrate Retina

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K+ Channels of Müller Glial Cells in Retinal Disorders

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527317666180202114233 ◽

2018 ◽

Vol 17 (4) ◽

pp. 255-260 ◽

Cited By ~ 2

Author(s):

Feng Gao ◽

Lin-Jie Xu ◽

Yuan Zhao ◽

Xing-Huai Sun ◽

Zhongfeng Wang

Keyword(s):

Müller Cells ◽

Major Type ◽

Delayed Rectifier ◽

Muller Cells ◽

Bkca Channels ◽

K Channels ◽

Functional Changes ◽

Physiological Properties ◽

Retinal Disorders ◽

Inwardly Rectifying

Background & Objective: Müller cell is the major type of glial cell in the vertebrate retina. Müller cells express various types of K+ channels, such as inwardly rectifying K+ (Kir) channels, big conductance Ca2+-activated K+ (BKCa) channels, delayed rectifier K+ channels (KDR), and transient A-type K+ channels. These K+ channels play important roles in maintaining physiological functions of Müller cells. Under some retinal pathological conditions, the changed expression and functions of K+ channels may contribute to retinal pathogenesis. Conclusion: In this article, we reviewed the physiological properties of K+ channels in retinal Müller cells and the functional changes of these channels in retinal disorders.

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Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

Journal of Translational Medicine ◽

10.1186/s12967-021-02823-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Qilu Wei ◽

Ning Kong ◽

Xiaohui Liu ◽

Run Tian ◽

Ming Jiao ◽

...

Keyword(s):

Protein Expression ◽

Cell Counting ◽

Enzyme Linked Immunosorbent Assay ◽

Fast Green ◽

Expression Levels ◽

Tnf Α ◽

Anti Inflammatory ◽

Safranin O ◽

Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

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