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Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

ABSTRACT: Purpose:This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR. Methods:Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-?. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-?1 stimulation and the PKA pathway. The effect of H89 on the TGF-?-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment. Results:PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-?-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6. Conclusions:PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

SUBMITTER: Lyu Y

PROVIDER: S-EPMC7325625 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

Lyu Yali Y Xu Wei W Zhang Jieping J Li Mengwen M Xiang Qingyi Q Li Yao Y Tan Tianhao T Ou Qingjian Q Zhang Jingfa J Tian Haibin H Xu Jing-Ying JY Jin Caixia C Gao Furong F Wang Juan J Li Weiye W Rong Ao A Lu Lixia L Xu Guo-Tong GT

Investigative ophthalmology & visual science 20200201 2

<h4>Purpose</h4>This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR.<h4>Methods</h4>Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in ...[more]

PMID: 32031573

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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.

Dataset Information

Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

Publications

Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

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