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Nicholas Sitaras, Jean-Sébastien Joyal, Karine Zaniolo, Przemyslaw Sapieha, Sylvain Chemtob; Activation Of Protease-activated Receptor Type 2 (par-2) Prompts A Rapid Revascularization By Down-regulating Semaphorin 3a In A Mouse Oxygen-induced Retinopathy Model. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4866.
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In the retina, a highly regulated vascular meshwork irrigates neurons and supplies necessary metabolic requirements. During ischemic conditions, as in Proliferative Retinopathies (PRs), neurons play a pivotal role in reinstating the compromised vascular network by specifically modulating angiogenic cues. We have previously demonstrated the role of Protease-activated receptor type 2 (PAR-2), a G-protein coupled receptor, in modulating pro-angiogenic cues in the developing retina. Here, we investigate the mechanisms by which PAR-2 modulates angiogenic cues in PRs and reveal its ability to suppress the anti-angiogenic neuronal guidance cue, semaphorin 3A (Sema3A).
Protein and mRNA expression of PAR-2 was determined in mouse whole retina and localization determined by immunohistochemistry on coronal sections. Sema3A mRNA expression was assessed following intravitreal administration of PAR-2 agonist peptide (SLIGRL) in WT and PAR-2 null mice exposed to oxygen-induced retinopathy (OIR) model (75% O2 from P7-P12). The RGC-5 cell line served as an ex vivo model of retinal ganglion cells. Microvascular growth was assessed on aortic rings treated with conditioned media (CM) from SLIGRL-treated RGC.
Our results reveal a pronounced expression of PAR-2 specifically in RGCs. WB analysis showed reduced levels of PAR-2 expression during the VO phase (P12) but elevated levels during the NV phase (P16). Activation of PAR-2 in vivo reduced Sema3A levels specifically in the avascular zones. Stimulation of RGC-5 with SLIGRL translated to robust increases in VEGF, Ang-2 and TNF-α while down-regulating Sema3A. Aortic rings exposed to SLIGRL-primed RGC-5 CM showed significant increases in vascular growth; this was abrogated by shRNA-based knockdown of PAR-2. Pertinently, treatment with SLIGRL in vivo triggered a prompt revascularization following oxygen-induced vaso-obliteration; this was not observed in the PAR-2 null mice.
This study underscores the importance of neurovascular coupling whereby RGC-specific PAR-2 plays a pivotal role in modulating angiogenesis in a pathological context. This was observed by increases in pro-angiogenic factors, VEGF, Ang-2 and TNF-α as well as the suppression of the vaso-repulsive cue, Sema3A. These data shed light on a novel approach to promote revascularization in PRs using PAR-2 agonists.
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