Abstract
Purpose: :
Proliferative Retinopathies are characterized by an initial micro-vascular degeneration followed by ischemia-driven aberrant pre-retinal neovascularization, which predisposes the retina to blinding retinal detachment. Accordingly, a prompt normal revascularization prevents the exaggerated intravitreal neovascularization. Selective targets to accelerate revascularization of the retina are not known. We have previously demonstrated that G protein-coupled receptor protease-activated receptor type 2 (PAR-2) induces angiogenesis in the developing retina. Herein, we investigate the role of PAR-2 in accelerating revascularization in oxygen-induced retinopathy (OIR).
Methods: :
PAR-2 expression was measured in mouse whole retina by Western blot and localization determined by immunohistochemistry on coronal sections. Retinal vasculature was assessed subsequent to intra-vitreal administration of PAR-2 agonist peptide (SLIGRL, 100μM) or lentiviral-based shRNA knockdown of PAR-2 (LV shPAR-2) in wild-type mice exposed to a model of OIR (75% O2 from P7-P12). Expression profiles of pro- and anti-angiogenic cues were assessed in whole retina of mice subjected to OIR treated with SLIGRL or LV shPAR-2. The RGC-5 cell line served as an ex-vivo model of retinal ganglion cells (RGC). Microvascular growth was assessed using the aortic explant sprouting assay.
Results: :
Our results reveal a prominent expression of PAR-2 in RGCs. Levels of PAR-2 significantly increased during the initial phases of VO (P8) and during revascularization (P16) compared to normoxic controls. Treatment with SLIGRL in vivo prior to and during hyperoxia accelerated revascularization of the retina, which in turn decreased VO at P12. Similarly, SLIGRL treatment following hyperoxia reduced avascular area and pathological neovascularization at P17. Conversely, knockdown of PAR-2 using LV shPAR-2 increased VO at P12 and P17 while augmenting avascular zones and pathological neovascularization at P17. Activation of PAR-2 with SLIGRL in vivo reduced anti-apoptotic cues while increasing pro-angiogenic factors. Similar gene regulation was observed in SLIGRL-treated RGC-5. Aortic ring explants exposed to SLIGRL-primed RGC conditioned media exhibited significant increases in vascular growth; this was abrogated by LV shPAR-2.
Conclusions: :
This study highlights the role of PAR-2 in triggering revascularization during and following the vaso-obliterative phase by modifying pro- and anti-angiogenic cues. These data demonstrate a novel approach to curtail vessel loss and promote normal revascularization in proliferative retinopathies using PAR-2 agonists.
Keywords: neovascularization • oxygen • receptors: pharmacology/physiology