Abstract
Purpose :
Purpose: We previously observed elevated plasma levels of both arginine and citrulline in patients with diabetic retinopathy compared to diabetic controls. Intracellularly, citrulline is converted to arginine, which serves as a substrate for both arginase and endothelial nitric oxide synthase (eNOS). In the current study, we tested whether arginine and citrulline could induce an angiogenic response in human retinal microvascular endothelial cells (HRMEC) and if arginine and citrulline activate arginase and/or eNOS in HRMEC.
Methods :
Methods: HRMEC were treated with 30 µM citrulline, 70 µM arginine, or citrulline + arginine, and the angiogenic response was measured with cell proliferation, cell migration, and tube formation assays. To determine if citrulline and arginine activate arginase or eNOS in HRMEC, arginase activity was quantified by measuring urea, a product of arginase, and DAF-FM DA was used to stain for intracellular nitric oxide, a product of eNOS. Western blots were used to measure protein expression of arginase-1, arginase-2, total eNOS, and phosphorylated eNOS.
Results :
Results: Individually, neither citrulline nor arginine resulted in an angiogenic response in HRMEC, but citrulline + arginine induced proliferation by 39.6% (p = 0.018), cell migration by 57.7% (p = 0.011), and resulted in tubes that were 36.2% longer (p = 0.0042) compared to untreated controls. There was no change in arginase activity or arginase-2 protein expression in HRMEC treated with citrulline and/or arginine, but citrulline + arginine resulted in lower protein expression of arginase-1 compared to untreated cells (p = 0.0046). Citrulline (p = 0.012), arginine (0.0029), and citrulline + arginine (p = 0.025) all increased NO production compared to controls. Arginine (p = 0.028) and citrulline + arginine (p = 6.3 x 10-4) led to greater total eNOS expression than in controls, and citrulline + arginine resulted in higher protein expression of phosphorylated eNOS compared to controls (p = 0.029).
Conclusions :
Conclusions: These data demonstrate that arginine and citrulline together can induce an angiogenic response, increased nitric oxide production, and increase eNOS expression and phosphorylation with no effect on arginase activity in HRMEC. This suggests that elevated plasma arginine and citrulline could potentially contribute to progression of diabetic retinopathy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.