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Hirofumi Kinoshita, Kiyoshi Suzuma, Toshihide Maki, Makiko Matsumoto, Mao Kusano, Masafumi Uematsu, Takashi Kitaoka; Cyclic Stretch And Hypertension Increase Retinal Succinate And Fumarate: Potential Mechanisms For Exacerbation Of Ocular Neovascularization By Mechanical Stress. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5314.
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Recently it is suggested that posterior vitreomacular adhesion is a potential risk factor for exudative age-related macular degeneration (AMD) and the succinate receptor GPR91 in neurons has a major role in retinal angiogenesis. We evaluated the expression of succinate in retinal pigment epithelial (RPE) cells undergoing clinically relevant cyclic stretch to reveal role of succinate and mechanical stress in AMD.
Succinate extracted from ARPE19 cells, a human RPE cell line, in culture undergoing mechanical stretch was evaluated. Cells were seeded on six-well flexible-bottom plates coated with collagen1. When cells were cultured to near confluency, cells were subjected to a 5, 10 or 15% cyclic stretch maintained for 1-48 hours using a computer-controlled vacuum stretch apparatus (Flexcer Cell Strain Unit; Flexcel). The medium was decanted and cells lysed directly in the culture plates with TNE buffer. Succinate concentration was assessed by enzymatic analysis of succinate and high performance liquid chromatography mass spectrometry (HPLC/MS), and normalized with total protein quantity. Moreover succinate concentration after treatment with pharmacological inhibitors was evaluated to speculate the pathway of succinate activity in cells. And fumarate concentration, Krebs cycle intermediate, was also assessed by HPLC/ MS. In vivo, Succinate concentration in retina and vitreous body of SHR; Spontaneously Hypertensive rat (hypertensive model; non-treatment group and captopril treated group), WKY; Wistar Kyoto rat (normal model) were also evaluated.
10% cyclic stretch maximally increased succinate (mg)/ total protein (g) after 2 hours (25.3 ± 5.7mg/g; P<0.05 Tukey test). BAPTA/AM, an intracellular calcium chelator, inhibited stretch-induced succinate increase in cells significantly (17.2 ± 6.2mg/g) by 86.9%. Succinate concentration in retina and vitreous body of SHR (non-treatment group) were significantly increased compared to that in WKY rat, SHR (captopril treated group)(16.5 ± 3.3mg/g versus 12.7 ± 3.3mg/g, 12.4 ± 1.7mg/g respectively; P<0.05 Tukey test). Fumarate concentration in retina and vitreous body of SHR (non-treatment group) was significantly increased compared to that in WKY rat (P<0.05 Tukey test), as well succinate concentration.
Cyclic stretch increased intracellular succinate concentration in cultured RPE cells through a calcium dependent pathway. Our data suggest that succinate has a role in pathology of AMD, and may be a target for medical treatment.
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