Hypercapnia Inhibits Intra-retinal Angiogenesis via a NO-Dependent Pathway: Implications for Ischemic Retinopathies

Abstract

Abstract: : Purpose: Ischemic proliferative retinopathy is a main cause of visual impairment and blindness in the industrialized world. While hypoxia has been shown to be involved in this process, mechanisms triggered by accompanying local high CO₂ levels and acidosis are unclear. Endothelial nitric oxide synthase (eNOS) expression is increased during moderately prolonged hypercapnia. Thus, we investigated the impact of hypercapnia on retinal angiogenesis and local NOS expression in vivo, and assessed if CO₂'s actions could be independent of those mostly attributed to hemodynamics by studying its effects on cultured neuro-retinovascular endothelial cells. Methods: Rats were exposed to 10% CO₂/21% O₂ from within 24 h of birth to day 6. Retinas were assessed for vascularization, 3-nitrotyrosine, neuronal (n), inducible (i) and endothelial (e) NOS (immunofluorescence), and for dot blot quantification of 3-nitrotyrosine levels. Effects of 10% CO₂ on primary neuro-retinovascular endothelial cell viability was tested (MTT assay and electron microscopy) in the presence or absence of NO-donor DETA NONOate. Results: The 10% CO₂/21% O₂ exposure retarded vascular growth to the periphery in accordance with previous findings. All three NOS isoforms (n, i, eNOS) increased in the retinal vasculature. In vitro, neuro-retinovascular endothelial cells exhibited increased viability in 10% CO₂ compared to controls at 5% CO₂. In contrast, DETA NONOate dose-dependently induced cell death to a greater extent in 10% versus 5% CO₂. Electron microscopy disclosed both apoptotic and necrotic cell death with DETA NONOate and 5% CO₂, while mostly necrotic features were observed with 10% CO₂. In vivo, hypercapnia resulted in augmented 3-nitrotyrosine levels within the retinal endothelial cell vasculature as detected by immunohistochemistry, with increases confirmed by dot blot. Conclusions: Data demonstrate that high CO₂ increases NOS expression in the rat retinal vasculature, and that alone CO₂ increases cell viability, but with NO, necrosis is induced. In vivo, CO₂ couples with NO to promote nitration in endothelial cells, a marker of NO-mediated toxicity, which may explain the vasoattenuation illustrated in CO₂ exposed rats. This novel mechanism furnishes an explanation for how hypercapnia leads to inhibition of intra-retinal vascularization, and unveils previously undescribed dual actions of CO₂ independent of hemodynamic effects, notably cytotoxic when NO levels are high and cytoprotective when NO is diminished.

Keywords: vascular cells • cell death/apoptosis • nitric oxide