Roles Of Rho Kinase Signaling Pathway In Atherosclerosis And Cardiovascular Protection Of Fasudil In Rats

Atherosclerosis is the leading cause of cardiovascular morbidity and mortality in the world, and hypercholesterolemia is one of the most important risk factors for atherosclerosis and subsequent cardiovascular disease. Hypercholesterolemia-induced inflammatory response and oxidative stress has been reported within a few days of feeding a high cholesterol enriched diet, that is, long before the appearance of fatty streak lesions in large arteries. Several inflammatory factors and the products induced by oxidative stress such as oxidized low density lipoprotein (ox-LDL) could stimulate the proliferation of vascular smooth muscle cell (VSMC) through various pathways, which plays a key role in atherosclerotic plaque formation. Therefore, anti-inflammatory and antioxidant treatment have been important approaches for the prevention and treatment of atherosclerosis.Rho kinase (ROCK) has been identified as one of the effectors of the small G-protein RhoA. In addition to its effect on the regulation of actin cytoskeleton organization, ROCK plays a central role in diverse cellular functions such as vascular smooth muscle cell contraction, stress fiber formation, cell adhesion and motility, cytokinesis, and gene expressions. To date, high expression or excessive activation of ROCK has been implicated in the regulation of vascular tone, proliferation, inflammation and oxidative stress. Evidence from animal studies suggests potential involvement of ROCK signaling in the pathogenesis of cardiovascular disease such as hypertension, restenosis, atherosclerosis, pulmonary hypertension, cerebral vasospasm and myocardial ischemia reperfusion injury. The so-called pleiotropic effects of statins, which are independent of lipid lowering, are believed to be mediated by inhibition of Rho protein isoprenylation and the subsequent activation of ROCK. Thus, ROCK is an important therapeutic target in cardiovascular medicine.Fasudil was the first and only ROCK inhibitor approved for clinical use, and initially approved for the treatment of cerebral vasospasm complicating intracranial hemorrhage. In several clinical studies, fasudil is effective for the treatment of a wide range of cardiovascular disease, including cerebral and coronary vasospasm, angina, hypertension, pulmonary hypertension, heart failure and acute stroke, with a reasonable safety. In addition, fasudil, a potent and specific inhibitor of ROCK, is used to be a pharmacological tool to investigate the role of ROCK in physiology and pathophysiolgy. However, the role of ROCK pathway in inflammatory response and oxidative stress of hypercholesterolemia, and ox-LDL-induced VSMC proliferation remain unknown. Thus, the purpose of this study is to investigate the roles of ROCK pathway in the inflammatory response and oxidative stress in high cholesterol diet (HCD)-induced hypercholesterolemic rats in vivo, and ox-LDL-induced VSMC proliferation in vitro.Part 1 Roles of Rho kinase signaling pathway in inflammatory response and anti-inflammatory effects of fasudil in hypercholesterolemic ratsObjective: To investigate the role of ROCK pathway in high cholesterol diet-induced hypercholesterolemic rats.Methods: Thirty male SD rats were weighed and randomized into five groups (n = 6 in each group): (1) Normal control group (Control): rats were fed a normal rat chow only. In addition to the normal control group, the other rats were all fed a high-cholesterol diet (normal rat chow supplemented with 4% cholesterol, 1% sodium tauroglycocholate and 0.5% propylthiouracil, HCD) for 4 weeks and received different treatment 2 weeks after the HCD feeding. (2) The high-cholesterol diet group (HCD): rats received physiological saline (1 ml/100g per day) by intraperitoneal injection. (3) The simvastatin-treated group: rats were administered simvastatin (10 mg/kg per day) by oral gavage. (4) The low-dose fasudil treated group: rats received fasudil hydrochloride (10 mg/kg per day) by intraperitoneal injection. (5) The high-dose fasudil treated group: rats received fasudil hydrochloride (30 mg/kg per day) by intraperitoneal injection.On the 29th day of experiment, final body weight of each animal was recorded and overnight fasted animals were sacrificed by decapitation after sodium pentobarbital anesthesia. Blood from each animal was collected and serum was isolated for the estimation of lipid profiles and inflammatory markers. The aorta was immediately excised and rinsed in cold physiological saline. Just below the branch of the left subclavicular artery,the thoracic aorta of 3 mm length was immediately prepared for histological examination and immunohistochemical analysis. The remaining aortic samples were frozen and stored at ?86°C for reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis.Results: In hypercholesterolemic rats, we found the serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and several inflammatory markers including interleukin (IL)-8, IL-6, C-reaction protein (CRP) and soluble intercellular adhesion molecule (sICAM)-1 significantly elevated, while those of high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) decreased. Moreover, mRNA expressions of ROCK and nuclear factor-kappaB (NF-κB) and activity of ROCK in thoracic aorta were greatly up-regulated. Remarkably, administrating fasudil (10, 30 mg/kg per day) or simvastatin (10 mg/kg per day) to hypercholesterolemic rats for 2 weeks, activation of ROCK and NF-κB in thoracic aorta were suppressed, status of dyslipidemia were improved and inflammatory markers lowered. From the histopathological examination, fasudil treatment was found to lessen the thickening noted in the aortic intima and media of the hypercholesterolemic rats.Conclusions: These results suggest that activated ROCK pathway is involved in inflammatory response in hypercholesterolemic rats. Inhibition of ROCK activation may improve lipid metabolism and has anti-inflammatory effect. Furthermore, ROCK activity should be considered as a novel target in the treatment of hypercholesterolemia and preventing the development of atherosclerosis.Part 2 Roles of Rho kinase signaling pathway in oxidative stress and anti-oxidant effects of fasudil in hypercholesterolemic ratsObjective: To investigate the role of ROCK pathway in oxidative stress in hypercholesterolemic rats.Methods: Forty male SD rats weighing 170-210 g were weighed and randomized into four groups (each n = 10): (1) Normal control group (control) rats received untreated chow and drinking water. The rats of other groups were fed a high-cholesterol diet (normal rat chow supplemented with 4% cholesterol, 1% sodium tauroglycocholate and 0.5% propylthiouracil, HCD) for 4 weeks and received different treatments 2 weeks after the start of the HCD feeding. (2) The second group of rats (HCD) received physiological saline (1 ml/100 g). (3) The third group of rats (H+F10) received low-dose fasudil (10 mg/kg per day). (4) The fourth group of rats (H+F30) received high-dose fasudil (30 mg/kg per day). The saline or fasudil were administered by intraperitoneal (i.p.) injection to groups 2, 3 and 4 as split daily doses given twice a day.The experiment was terminated after 2 weeks of treatment. The final body weight of each animal was recorded, and overnight fasted animals were sacrificed by decapitation after sodium pentobarbital anesthesia. Blood from each animal was collected, and the serum was isolated for biochemical analysis including TC, the activities of the antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) and nitric oxide (NO). The heart was quickly excised, the heart weight: body weight ratio (mg?g) was calculated for each rat. Then, hearts were sectioned into four slices along a plane parallel to the atrioventricular ring. The middle slice was immediately prepared for histological examination. The remaining samples were stored at -80℃prior to biochemical assays (including CAT, SOD, Gpx and NO) or western blot analysis of phosphorylated myosin phosphatase target subunit (p-MYPT1) and endothelial NO synthase (eNOS). The liver was also quickly excised, the liver weight: body weight ratio (g?g) was calculated for each rat, then, sections of liver tissues were set aside for histological examination.Results: In hypercholesterolemic rats, we found the serum levels of TC, the liver weight: body weight ratio (g?g), and the p-MYPT1 expression significantly elevated, while the activities of CAT, SOD, Gpx and NO production in serum and cardiac tissue, as well as cardiac eNOS expression decreased. Remarkably, administrating fasudil (10, 30 mg/kg per day) to hypercholesterolemic rats for 2 weeks, ROCK activity and serum TC level were siginificantly suppressed, the activities of antioxidant enzymes and the concentration of NO in serum and cardiac tissue, as well as cardiac eNOS expression were potently elevated. In addition, fasudil notably suppressed the extent of lipid peroxidation and attenuated the histopathological changes in the heart and liver of hypercholesterolemic rats.Conclusions: These results suggeste that ROCK pathway is involved in hypercholesterolemia-induced oxidative stress and endothelial dysfunction. ROCK inhibitor, fasudil, may improve lipid metabolism and has anti-oxidant effect, which supports the hypothesis that ROCK is a potential therapeutic target for hypercholesterolemia.Part 3 Roles of Rho kinase signaling pathway in proliferation of vascular smooth muscle cell induced by oxidized LDLObjective: To study the role of ROCK pathway in cell proliferation of cultured rat VSMC.Methods: Cell cultures of rat aortic VSMCs were prepared using an explant method and those VSMCs at passages 4-6 were used in all experiments. Cells were serum deprived for 24h and treated with 0.1, 1.0 and 10μM fasudil for 1 h, then exposed to ox-LDL (50mg/L) for 24 h. The following indices were determined. (1) The proliferative activity of VSMCs was estimated by MTT assay. (2) Expressions of matrix metalloproteinases (MMP)-2 and MMP-9 of VSMCs were determined by western blot. (3) The content of MDA and the activity of SOD in supernatant were tested by colorimetric method. Results: Stimulation of VSMCs with ox-LDL significantly increased cell proliferation. Incubation of VSMCs with ox-LDL (50mg/L) induced a significant increase in MMP-2 and MMP-9 expression. The content of MDA in supernatant increased while the activity of SOD decreased. Pretreating with 0.1μM fasudil notably decreased MMP-2 expression, while had no effects on ox-LDL-induced cell proliferation, MMP-9 expression, content of MDA and activity of SOD. Pretreating with 1.0 or 10μM fasudil, ox-LDL-induced cell proliferation was significantly suppressed as well as MMP-2 and MMP-9 expressions were markedly inhibited. In addition, 1.0 or 10μM fasudil decreased the MDA content and increased the SOD activity in supernatant.Conclusions: These results suggeste that ROCK is involved in VSMC proliferation induced by ox-LDL. Inhibition of ROCK could suppress VSMC proliferation through alleviating oxidative stress and down-regulating MMP-2 and MMP-9 expressions.CONCLUSIONS1 Activated ROCK pathway is involved in inflammatory response, oxidative stress and endothelial dysfunction in hypercholesterolemic rats.2 Fasudil, a specific ROCK inbitor could exert anti-inflammatory, anti-oxidant effects and improve endothelial function through inhibiting ROCK pathway in hypercholesterolemic rats.3 ROCK is involved in VSMC proliferation induced by ox-LDL. Inhibition of ROCK could suppress VSMC proliferation through alleviating oxidative stress and down-regulating MMP-2 and MMP-9 expressions.