A key role for ROCK in TNF-α-mediated diabetic microvascular damage

Ryoichi Arita, Shintaro Nakao, Takeshi Kita, Shuhei Kawahara, Ryo Asato, Shigeo Yoshida, Hiroshi Enaida, Ali Hafezi-Moghadam, Tatsuro Ishibashi

Research output: Contribution to journalArticlepeer-review

34 Citations (Scopus)

Abstract

PURPOSE. Leukocyte adhesion releases tumor necrosis factor (TNF)-a 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 TNFa- mediated microvascular damage is investigated. METHODS. 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. RESULTS. 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. CONCLUSIONS. 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.

Original languageEnglish
Pages (from-to)2373-2383
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume54
Issue number3
DOIs
Publication statusPublished - 2013

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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