3-nitrotyrosine and fasudil

Systemic sclerosis (SSc) is a connective-tissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin and multiple internal organs. Recent reports found that RhoA/Rho-kinase (ROCK) pathway is implicated in various fibrogenic diseases. Intradermal injection of hypochlorous acid (HOCl)-generating solution induced inflammation, autoimmune activation, and fibrosis, mimicking the cutaneous diffuse form of SSc in humans. Our study aimed firstly to describe pulmonary inflammation and fibrosis induced by HOCl in mice, and secondly to determine whether fasudil, a selective inhibitor of ROCK, could prevent lung and skin fibroses in HOCl-injected mice.. Female C57BL/6 mice received daily intradermal injection of hypochlorous acid (HOCl) for 6 weeks to induce SSc, with and without daily treatment with fasudil (30 mg·kg(-1)·day(-1)) by oral gavage.. HOCl intoxication induced significant lung inflammation (macrophages and neutrophils infiltration), and fibrosis. These modifications were prevented by fasudil treatment. Simultaneously, HOCl enhanced ROCK activity in lung and skin tissues. Inhibition of ROCK reduced skin fibrosis, expression of α-smooth-muscle actin and 3-nitrotyrosine, as well as the activity of ROCK in the fibrotic skin of HOCl-treated mice, through inhibition of phosphorylation of Smad2/3 and ERK1/2. Fasudil significantly decreased the serum levels of anti-DNA-topoisomerase-1 antibodies in mice with HOCl-induced SSc.. Our findings confirm HOCl-induced pulmonary inflammation and fibrosis in mice, and provide further evidence for a key role of RhoA/ROCK pathway in several pathological processes of experimental SSc. Fasudil could be a promising therapeutic approach for the treatment of SSc.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Animals; Disease Models, Animal; Female; Hypochlorous Acid; Lung; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; rho-Associated Kinases; Scleroderma, Systemic; Skin; Smad2 Protein; Smad3 Protein; Tyrosine

Activation of RhoA/Rho-kinase (ROCK) is increasingly implicated in acute vasospasm and chronic vasoconstriction in major organ systems. Therefore we aimed to ascertain whether an increase in ROCK activity plays a role in the deterioration of coronary vascular function in early stage diabetes.. Synchrotron radiation microangiography was used to determine in vivo coronary responses in diabetic (3 weeks post streptozotocin 65 mg/kg ip) and vehicle treated male Sprague-Dawley rats (n = 8 and 6). Changes in vessel number and calibre during vasodilator stimulation before and after blockade of nitric oxide synthase and cyclooxygenase were compared between rats. Acute responses to ROCK inhibitor, fasudil (10 mg/kg iv) was evaluated. Further, perivascular and myocardial fibrosis, arterial intimal thickening were assessed by histology, and capillary density, nitrotyrosine and ROCK1/2 expressions were evaluated by immunohistochemical staining.. Diabetic rats had significantly elevated plasma glucose (P < 0.001 vs control), but did not differ in fibrotic scores, media to lumen ratio, capillary density or baseline visible vessel number or calibre. Responses to acetylcholine and sodium nitroprusside stimulation were similar between groups. However, in comparison to control rats the diabetic rats showed more segmental constrictions during blockade, which were not completely alleviated by acetylcholine, but were alleviated by fasudil. Further, second order vessel branches in diabetic rats were significantly more dilated relative to baseline (37% vs 12% increase, P < 0.05) after fasudil treatment compared to control rats, while visible vessel number increased in both groups. ROCK2 expression was borderline greater in diabetic rat hearts (P < 0.053).. We found that ahead of the reported decline in coronary endothelial vasodilator function in diabetic rats there was moderate elevation in ROCK expression, more widespread segmental constriction when nitric oxide and prostacyclin production were inhibited and notably, increased calibre in second and third order small arteries-arterioles following ROCK inhibition. Based on nitrotyrosine staining oxidative stress was not significantly elevated in early diabetic rats. We conclude that tonic ROCK mediated vasoconstriction contributes to coronary vasomotor tone in early diabetes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Coronary Angiography; Coronary Circulation; Coronary Vessels; Cyclooxygenase Inhibitors; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Epoprostenol; Fibrosis; Male; Microcirculation; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Tyrosine; Vasoconstriction; Vasodilation; Vasodilator Agents