Asymmetric dimethylarginine contributes to retinal neovascularization of diabetic retinopathy through EphrinB2 pathway

Abstract

Diabetic retinopathy (DR) is a leading cause of vision loss with retinal neovascularization. This study aims to investigate whether Asymmetric dimethylarginine (ADMA) impacts the pathogenesis of DR via focusing on promoting retinal neovascularization and its underlying molecular mechanisms. Diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ) for 20 weeks. ADMA levels in aqueous and the influence of hypoxia on ADMA and angiogenesis in RF/6A cells were examined. The effects and underlying molecular mechanisms of ADMA on neovascularization of RF/6A cells were further evaluated by administration of ADMA, DDAH siRNA or ephrinB2 siRNA. Results showed that ADMA levels were elevated in both aqueous from diabetic rats and culture medium in RF/6A cells pretreated with hypoxia. Administration of ADMA directly promoted proliferation, migration, adhesion and tube formation of RF/6A cells, which was further confirmed by DDAH1 siRNA or DDAH2 siRNA. In addition, ephrinB2 expression was increased under diabetic conditions, and the angiogenic effects of ADMA were blocked by ephrinB2 siRNA. In conclusion, ADMA contributes to the neovascularization of retina in diabetic mellitus, which is regulated by ephrinB2.

Introduction

Diabetic retinopathy (DR), one of the most common microvascular complications of diabetes, is characterized by the formation of new blood vessels on the retina which causes irreversible damage to retina and even tractional retinal detachment [1]. Pathological angiogenesis is the results of a complex interplay of molecular mediators, cellular interactions and extracellular matrix modulation, and is also the target of novel therapeutic approaches [2]. Vascular endothelial growth factor (VEGF) is a critical factor in the development of angiogenesis diseases, and selective inhibition of VEGF has improved management of the retinal neovascularization but some patients do not respond, indicating that other vascular mediators may also contribute to retinal angiogenesis [3]. Therefore, searching more specific therapy targeting molecular mechanisms underlying retinal angiogenesis may lead to better treatment results.

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is synthesized by type 1 protein arginine N-methyltransferase (PRMT1) and mainly metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) pathway. There are two DDAH isoforms, DDAH1 and DDAH2 [4]. The significance of ADMA in angiogenesis has been demonstrated in numerous studies. Exogenous ADMA depressed fibroblast growth factor-induced angiogenesis or VEGF-induced tube formation in the fibrovascular disc system or cultured human umbilical vein endothelial cells (HUVECs) [[5], [6], [7]]. DDAH1 global KO or endothelial specific KO decreased Matrigel angiogenesis in vivo [8,9]. DDAH1 transgenic mice showed increased blood vessel formation in a mouse model of hind limb ischemia as well as in the fibrovascular disc system [10]. However, these studies about DDAH/ADMA mainly focused on macrovascular diseases, until now very few studies have tried to explore the role of ADMA in retinal angiogenesis in the background of diabetes. ADMA has been reported to play an important role in the pathogenesis of diabetes [[11], [12], [13]]. Recently, a serial of clinical studies have shown that ADMA was related to diabetic retinopathy. ADMA levels in diabetic patients with retinopathy were markedly higher than that in the individuals without retinopathy [14]. Elevated ADMA was also detected in aqueous humor in diabetic patients, especially those with severe retinopathy [15]. Therefore, in the present study, we investigated the influence of diabetic conditioning induced alteration in ADMA levels on retinal neovascularization.

EphrinB2, a transmembrane ligand for Eph receptors, plays an important role in retinal vascular formation. Elevated ephrinB2 was detected in retinal tissue in oxygen-induced retinopathy (OIR) mouse and patients with diabetic retinopathy, and blockade of ephrinB2 could decrease pathological retinal neovascularization [[16], [17], [18], [19]]. Recently, it has been reported that downregulation of VEGF expression could markedly reduce ephrinB2 [20]. While some evidence showed that DDAH/ADMA pathway regulated angiogenesis by targeting VEGF. Based on above studies [21,22], we investigated whether ADMA regulated retinal angiogenesis through ephrinB2 pathway.