title: A key role for ROCK in TNF-α-mediated diabetic microvascular damage.
abstract: OBJECTIVE
  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.
  
  
  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.
authors: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Higashi-Ku, Fukuoka, Japan.
authors: Ryoichi
authors: R
authors: Arita
authors: Shintaro
authors: S
authors: Nakao
authors: Takeshi
authors: T
authors: Kita
authors: Shuhei
authors: S
authors: Kawahara
authors: Ryo
authors: R
authors: Asato
authors: Shigeo
authors: S
authors: Yoshida
authors: Hiroshi
authors: H
authors: Enaida
authors: Ali
authors: A
authors: Hafezi-Moghadam
authors: Tatsuro
authors: T
authors: Ishibashi
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D003925:Diabetic Angiopathies / Q000378:metabolism*; D003930:Diabetic Retinopathy / Q000378:metabolism; D042783:Endothelial Cells; D004730:Endothelium, Vascular / Q000378:metabolism; D005260:Female; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D047428:Protein Kinase Inhibitors; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D052250:Nitric Oxide Synthase Type III; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human