Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis

Clemens Lange,Freya Mowat,Haroon Sayed,Manjit Mehad,Lucie Duluc,Sophie Piper,Ulrich Luhmann,Manasi Nandi,Peter Kelly,Alexander Smith,Robin Ali,James Leiper,James Bainbridge

doi:10.1016/j.exer.2016.05.007

Abstract

Ischemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1+/− and DDAH2−/− deficient mice by in vivo fluorescein angiography, immunohistochemistry and electroretinography revealed normal neurovascular function compared with wildtype control mice. Finally, DDAH1+/− and DDAH2−/− deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1+/− exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis.

Highlights

Adaptive tissue responses to ischemia promote blood flow and angiogenesis that are critical for 53 normal development, tissue repair and regeneration
Having established that heterozygous loss of dimethylarginine dimethylaminohydrolases-1 (DDAH1) or homozygous loss of DDAH2 has no effect on the adult retinal vasculature, we investigated the role of DDAH1 and DDAH2 in murine oxygen-induced retinopathy (OIR), a model of retinal ischemia-induced neovascularization
L-NMMA is present in the normal retina at higher levels than Asymmetric dimethylarginine (ADMA), we identified no measurable impact of OIR or DDAH2-deficiency on local L-NMMA (Fig. 4L)

Summary

Introduction

Adaptive tissue responses to ischemia promote blood flow and angiogenesis that are critical for 53 normal development, tissue repair and regeneration. Local therapeutic inhibition of vascular endothelial growth factor (VEGF) attenuates pathological neovascularization (Avery et al, 2006) but this strategy fails to promote effective revascularization of ischemic retina. Asymmetric methyl-arginines are endogenously produced on degradation of proteins containing asymmetrically methylated L72 arginine residues, and are metabolized to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolases-1 (DDAH1) and -2 (DDAH2) (Fig. 1) (Leiper et al, 1999)(Ogawa et al., 1987). These two DDAH isoforms have distinct tissue distributions (Leiper et al, 1999) suggesting isoform-specific regulation of NOS. DDAH1 is predominantly found in tissues that express nNOS whereas DDAH2 is found in high levels in tissues expressing eNOS, which has a 77 role in promoting angiogenesis in the retina (Fukumura et al, 2001)(Brooks et al, 2001)

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