Abstract
Purpose:
Increased neovascularization triggered by hyperglycemia-mediated induction of VEGF, VEGFR, and NF-kB is a key mechanistic pathological abnormality in diabetic retinopathy (DR). Curcumin (diferuloylmethane), a polyphenolic compound, having its anti-inflammatory, antioxidant, and antiangiogenic activities has been reported to block diabetes-induced elevation of VEGF and NF-kB in the retina. However, the mechanisms underlying the antiangiogenic effect of curcumin is not clearly known. We investigated the effect of curcumin on protecting hyperglycemia-mediated induction of angiogenic and inflammatory molecules in hRECs through modulation of miR-152 and miR-155, negative regulators of prorenin (PRR) and angiotensin II Type 1 (AT1R) receptors, respectively.
Methods:
hRECs were grown in microvascular endothelial cell growth complete medium (Cell Applications, Inc., San Diego, CA) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (LONZA, Walkersville, MD) in a humidified incubator at 37°C with 5% CO2. Cells incubated with 5.5 mM glucose with or without curcumin (20 µM) were maintained as controls. Exposure to hyperglycemia was accomplished by incubating cells with 33 mM glucose in the presence or absence of curcumin (20 µM) for 48 hrs. Cells were treated with curcumin for 6 hrs. The expressions of mRNAs/ miRNAs and proteins were analyzed by qRT-PCR and immunoblotting, respectively.
Results:
In hRECs, hyperglycemia was found to significantly induce the expression of AT1R and PRR receptors, and their downstream signaling molecules VEGF, VEGFR, and NF-kB(p65) both at mRNA and protein levels (p<0.05), as compared to controls. Curcumin treatment significantly attenuated the hyperglycemia-induced elevation in the levels of AT1R, PRR, VEGF, VEGFR, and NF-kB (p<0.05). On the contrary, curcumin markedly up-regulated the hyperglycemia-mediated reduction of miR-152 and miR-155 expression (p<0.05), when compared with the controls. As measured by CellTiter-Blue Assay (Sigma, St Louis, MO), hyperglycemia and curcumin had no effect on hREC viability.
Conclusions:
Curcumin inhibited the glucose-mediated induction of VEGF, VEGFR, and NF-kB expression in hRECs through modulation of miRNAs that negatively regulate PRR and AT1R receptors. Our results suggest the therapeutic potential of curcumin by targeting specific miRNAs in hRECs.
Keywords: 499 diabetic retinopathy •
424 antioxidants •
700 retinal neovascularization