Cyclic stretch and hypertension increase retinal succinate: potential mechanisms for exacerbation of ocular neovascularization by mechanical stress.

Abstract

We investigated succinate metabolism in cells undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. We seeded ARPE-19 cells on 6-well BioFlex collagen I-coated, silicone elastomer-bottomed culture plates. Cells then were subjected to pulsatile stretch using a computer-controlled vacuum stretch apparatus. A physiologic stretch frequency of 60 cycles per minute and 5% to 15% prolongation of the elastomer-bottomed plates were used. Succinate concentration was assessed by enzymatic analysis and high-performance liquid chromatography-mass spectrometry. The VEGF was measured using enzyme-linked immunosorbent assays. The 12-week-old male SHRs and weight-matched Wistar-Kyoto (WKY) control rats were treated with or without 100 mg·kg(-1)·day(-1) captopril for 1 week. The vitreous body and retina of each rat were extracted after 1 week of therapy, and the vitreoretinal succinate concentration was measured. Cells exposed to cyclic stretch accumulated intracellular succinate in a time- and magnitude-dependent manner, and also accumulated VEGF protein levels. Moreover, BAPTA/AM, an intracellular calcium chelate reagent, significantly inhibited the stretch-induced succinate increase. After cyclic stretch, levels of intracellular fumarate, a citric acid cycle intermediate, also were significantly increased compared to controls. The BAPTA/AM inhibited this increase. For the in vivo experiments, hypertension increased vitreoretinal succinate and fumarate in SHRs compared to the normotensive WKY controls. When hypertension was reduced using captopril, vitreoretinal succinate returned to baseline levels. These findings suggest that cyclic stretch and hypertension increased intracellular succinate in cultured retinal pigment epithelial cells and the vitreoretinal succinate of SHRs through a calcium-dependent pathway.