Our previous studies identified dysfunctional

Our previous studies identified dysfunctional sphingolipid metabolism, due to pathological activation of ASM in diabetic retina and bone marrow, as an important metabolic insult contributing to pro-inflammatory changes in the retina and development of diabetic retinopathy (Opreanu et al., 2011; Tikhonenko et al., 2013; Busik et al., 2012; Chakravarthy et al., 2015; Opreanu et al., 2010). Endothelial quinacrine are the major source of ASM and we observed the highest activation of ASM in retinal endothelial cells in diabetes compared to other retinal cells (Opreanu et al., 2011; Tikhonenko et al., 2013; Opreanu et al., 2010). VEGF is another well-known factor that is increased in the diabetic retina leading to increased retinal vascular permeability and ultimately neovascularization in some species. Intravitreal anti-VEGF treatments provide the most successful diabetic retinopathy therapy to date. Both ASM and VEGF-A are, however, essential for normal retinal function and their inhibition may lead to serious consequences, such as lysosomal storage disease (Opreanu et al., 2011; Chen et al., 2014; Kaarniranta et al., 2013) for ASM and damage to cone photoreceptors and choroidal vasculature for VEGF. Indeed, complete ASM deficiency leads to neurodegeneration, activation of microglia and loss of retinal function in ASM−/−mice (Dannhausen et al., 2015; Horinouchi et al., 1995). Based on the ability of miRNAs to function more like a rheostat than an on/off switch as they work by “dimming”, rather than complete silencing of gene expression, we reasoned that miRNAs would provide a feasible therapeutic approach, particularly for targets such as ASM and VEGF-A where partial reductions rather than complete inhibition, is desirable. In search of miRNAs that have combined anti-inflammatory and anti-angiogenic potential, we first performed the miRNA array to determine differentially expressed miRNAs in the HRECs isolated from control and diabetic donors. Among all the miRNAs downregulated in diabetic donors, miR-15a was the only one that had the predicted anti-inflammatory and anti-angiogenic targets, ASM and VEGF-A. Moreover, miR-15a was the most significantly downregulated miRNA in the blood of diabetic patients and T2D hyperglycemic Lepob mice (Zampetaki et al., 2010). Importantly, miR-15a was reported to directly target 3′UTR and inhibit VEGF-A and FGF2 (Yin et al., 2012) providing an important novel mechanism for control of angiogenesis. ASM regulation by miR-15a had not been previously confirmed, thus we performed 3′UTR of ASM mutation studies. These studies demonstrated that miR-15a negatively regulates ASM expression through direct targeting the 3′UTR of ASM mRNA. Angiogenesis is strictly controlled by a balance between pro-angiogenic and anti-angiogenic factors. Regulation of key angiogenic factors, VEGF-A and FGF2, by miR-15a is important in maintaining this delicate balance. Disruption of this balance could favor pathological angiogenesis, such as seen in diabetic retinopathy. Indeed, miR-15a was shown to be reduced in pathological angiogenesis in hindlimb ischemia model (Yin et al., 2012). Our data confirm this finding and demonstrate that miR-15a could thus be used to simultaneously control both sphingolipid metabolism and VEGF-A production. Reduction in miR-15a expression can affect retinal vascular pathology at two different levels. First, decrease in miR-15a has a direct effect on the retina leading to ASM activation, low-grade chronic inflammation and VEGF-A production, resulting in increased retinal endothelial permeability and cell injury. In addition, low miR-15a leads to high ASM expression and ceramide production in bone marrow-derived CACs in diabetes. Previously, we demonstrated that CACs with high ASM and ceramide levels have diminished migration, homing to the injured tissue and repair function (Opreanu et al., 2011; Tikhonenko et al., 2013; Busik et al., 2012; Chakravarthy et al., 2015; Opreanu et al., 2010). Furthermore, the combination of retinal endothelial cell injury and failed attempts by CACs to repair injured retinal capillaries, eventually results in progression to the vaso-degenerative stage of diabetic (Bhatwadekar et al., 2010; Chakravarthy et al., 2016; Abu El-Asrar et al., 2011; Li Calzi et al., 2010; Tan et al., 2010; Sukmawati and Tanaka, 2015; Caballero et al., 2013; Chakravarthy et al., 2015).