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Ryoichi Arita, Shintaro Nakao, Takeshi Kita, Shuhei Kawahara, Ryo Asato, Shigeo Yoshida, Hiroshi Enaida, Ali Hafezi-Moghadam, Tatsuro Ishibashi; A Key Role for ROCK in TNF-α–Mediated Diabetic Microvascular Damage. Invest. Ophthalmol. Vis. Sci. 2013;54(3):2373-2383. doi: 10.1167/iovs.12-10757.
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Leukocyte adhesion releases tumor necrosis factor (TNF)–α that contributes to endothelial damage in early diabetic retinopathy (DR). Rho/Rho-kinase (ROCK) signaling mediates retinal endothelial damage in early DR. However, whether ROCK regulates TNF-α–mediated diabetic vascular damage is unknown. Here, the contribution of ROCK to TNF-α–mediated microvascular damage is investigated.
In DR patients and nondiabetic control subjects, the levels of membranous (m) TNF-α on neutrophils, soluble (s) TNF-α and its receptors in sera, were measured. In cultured microvascular endothelial cells, phosphorylation of myosin phosphatase target protein (MYPT)-1, a downstream target of ROCK, was investigated with TNF-α or DR sera pretreatment. TNF-α–induced intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) phosphorylation were measured with and without ROCK inhibition by fasudil or ROCK-specific small-interfering RNA (siRNA). In isolated neutrophils from control subjects, MYPT-1 phosphorylation was investigated in the presence of TNF-α. The impact of ROCK inhibition by fasudil on TNF-α–induced integrin (CD18, CD11a, CD11b) and intracellular cytoskeletal changes were investigated.
The serum levels of mTNF-α, sTNF-α, and its receptors were significantly elevated in DR patients. TNF-α as well as DR sera promoted MYPT-1 phosphorylation in endothelial cells, which was significantly reduced by anti–TNF-α neutralizing antibody. TNF-α–induced ICAM-1 expression, eNOS dephosphorylation, cytoskeletal changes, and CD11b/18 expression in neutrophils were significantly suppressed by fasudil as well as ROCK-specific siRNA.
ROCK is a key mediator of TNF-α signaling in diabetic microvessels. The important role of TNF-α in early DR provides a new rationale for ROCK inhibition beyond the previously shown mechanisms.
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