期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 60, 期 10, 页码 3297-3309出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.18-26344
关键词
TRPV1; TRPV4; angiogenesis
资金
- Fight for Sight, UK [1387/88]
- Biotechnology and Biological Sciences Research Council [BB/I026359/1]
- Health & Social Care R&D Division, Northern Ireland [STL/4748/13]
- Medical Research Council [MC_PC_15026]
- Biotechnology and Biological Sciences Research Council [BB/I026359/1] Funding Source: researchfish
- British Heart Foundation [PG/11/99/29207] Funding Source: researchfish
- Medical Research Council [MC_PC_15026] Funding Source: researchfish
- Public Health Agency [STL/4748/13] Funding Source: researchfish
- BBSRC [BB/I026359/1] Funding Source: UKRI
- MRC [MC_PC_15026] Funding Source: UKRI
PURPOSE. We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis. METHODS. Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording. RESULTS. TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model. CONCLUSIONS. TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.
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