The cystathionine gamma-lyase/hydrogen sulfide pathway is involved in pathological retinal neovascularization in ischemic retinopathy

Abstract

Purpose: Retinal neovascularization is a major cause of blindness in ischemic retinopathy including proliferative diabetic retinopathy and retinopathy of prematurity. Cystathionine gamma-lyase (CSE) is one of the key enzymes that generates hydrogen sulfide (H₂S), which has been shown to promote angiogenesis in vitro and ex vivo. However its role in pathological neovascularization in animal models is unknown. The purpose of this study was to investigate the role of CSE in retinal neovascularization (NV).

Methods: Studies were performed in a mouse model of oxygen-induced retinopathy (OIR) by maintaining wild type mice, mice with a heterozygous disruption of CSE (^+/-), and mice with a homozygous disruption of CSE (^-/-) in 70% oxygen from postnatal day (P)7 to P12 and room air from P12 to P17. AOAA, an inhibitor for CSE and cystathionine beta synthase (CBS), was injected (i.p., 3 mg/kg qday) in WT mice exposed to OIR from P12 to P16. Retinas were prepared for analyses of CSE, CBS and 3-mercaptopyruvate sulfurtransferase (3-MST). Whole mounted retinas were labeled with isolectin B4 and the vasculature was examined by fluorescence microscopy. Morphometric data were quantified using NIH ImageJ.

Results: Western blot analysis revealed a significant increase (2.1 fold) of CSE protein in OIR retinas at P17 compared to room air control mice. In contrast, levels of CBS and 3-MST, the other two H₂S-producing enzymes, were only slightly changed in OIR. CSE mRNA was not changed, indicating that OIR induces up-regulation of CSE via post-transcriptional modification. To investigate the role of CSE in OIR, we generated CSE-deficient mice. Compared to CSE^+/- littermates, CSE^-/- mice exhibited a 19% reduction in retinal NV and a 31% increase in vaso-obliteration during OIR (p<0.05). Similarly, inhibition of CSE/CBS by AOAA decreased retinal NV while accelerating vaso-obliteration.

Conclusions: The CSE/H₂S pathway is critically involved in the development of pathological retinal neovascularization during retinal ischemia. Up-regulation of CSE in OIR likely occurs at the post-transcriptional modification level.