Study Of Mevalonate Pathway In Cardiovascular System Of Spontaneously Hypertensive Rats

Part One Effect of farnesyl diphosphate synthase inhibition on endothelial function in spontaneously hypertensive rats and its mechanismsBackground:Famesyl diphosphate synthase(FDS),an essential enzyme in the mevalonate pathway,catalyzes the synthesis of isoprenoid intermediates.The latter is needed for protein isoprenylation of RhoA for its function to negatively regulate expression and phosphorylation of endothelial nitric oxide synthase(eNOS),thereby negatively regulating the production of nitric oxide(NO).Up to date endothelial dysfunction characterized by an impairment of the production and release of the endothelial-derived NO is a strong predictor of cardiovascular disease.We previously reported that spontaneously hypertensive rat(SHR) in which endothelial dysfunction occurred,had higher FDS when compared with Wistar-Kyoto rat(WKY).Aims:To determine whether chronic inhibition of FDS can improve the endothelial function in SHR and explore its mechanisms.Methods:After 12-week administration of alendronate(low dose of 1 mg/kg/day;high dose of 10 mg/kg/day),endothelium-dependent and -independent vasorelaxation were measured in isolated aortic rings.Activation of RhoA in aorta was determined by pull-down assay.Aortic expression of eNOS was determined by Western blot.Serum nitric oxide(NO) end products were determined using Griess method.Results:High-dose of alendronate was able to improve the impaired endothelium-dependent vasodilation in SHR.Biochemically,alendronate was able to suppress the RhoA activation,but upregulate the eNOS expression in a dose-dependent manner.Conclusions:Our data suggested that chronic FDS inhibition could improve the endothelial function in SHR,and the upregulation of eNOS expression as a result of inhibition of RhoA activation might be an important mechanism. Part Two Studies of tissue cholesterol content and HMG-CoA reducatse expression/activity in spontaneously hypertensive rats and effects of atorvastatin on themBackground and Aim:Cardiovascular remodeling is closely associated with cholesterol and is attenuated by statins.The spontaneously hypertensive rat(SHR) has a low serum cholesterol level and evident cardiovascular remodeling.3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) reductase is the rate limiting enzyme of cholesterol synthesis pathway, which is also called mevalonate pathway.The aims of the present study were to characterize the effects of HMG-CoA reductase inhibitor,atorvastatin,on tissue cholesterol content and HMG-CoA reductase expression and activity in four tissues from SHR:liver and extrahepatic tissues(heart,aorta and kidney).Methods:Eight-week old SHR and normotensive Wistar-Kyoto rats(WKY) were treated daily with atorvastatin(50 mg/kg) for 10 weeks.Cholesterol levels of serum and tissues (liver,heart,aorta and kidney) were determined by commercial enzymatic methods. Western blot analysis and high performance liquid chromatogram(HPLC) were used to assay the expression and activity of enzyme respectively.Results:The levels of total tissue cholesterol in SHR livers were significantly higer,but in SHR extrahepatic tissues were similar when compared with WKY rats.The expression and activity of HMG-CoA reductase in all these tissues were stringly higher in SHR than in WKY rats.Treatment with atorvastatin decreased cholesterol content and HMG-CoA reductase expression and activity in all four tissues of SHR.However,in WKY,atorvastatin only altered HMG-CoA reductase in liver,where the protein expression was upregulated but the enzyme activity was decreased.Conclusions:The present study demonstrates that the effects of atorvastatin on tissue cholesterol content and HMG-CoA reductase are strain-and tissue-specific. Part Three Effect of sodium ferulate on rat thoracic aorta and its mechanismsBackground:Sodium ferulate(SF) is a sodium salt of ferulic acid(FA).FA is the active ingredient of the traditional Chinese herbal medicine such as Radix Angelicae Sinensis, Rhizoma Chuanxiong.In vitro and in vivo studies revealed that SF or FA possessed beneficial pharmacological effects including its strong antioxidant,antiinflammatory, platelet aggregation inhibitory,and free radical-scavenging activities.Aims:This study was designed to investigate the effects of SF on rat isolated aortas and the possible mechanisms.Methods:Isometric tension was recorded in response to drugs in organ bath.Cytosolic free Ca²⁺ concentration([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells(RASMC).Results:SF relaxed the isolated aortic rings precontracted with phenylephrine(PE) or high-K⁺ in a concentration- dependent manner,and mechanical removal of endothelium did not significantly modify the SF-induced relaxation.In Ca²⁺-free solution,SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ or PE pre-challenged rings,and suppressed the transient contraction induced by PE or caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium,glibenclamide,4-aminopyridine,and barium chloride.In addition, SF concentration- dependently reduced the contraction induced by phorbol-12- myristate-13-acetate,an activator of protein kinase C(PKC),in the absence of extracellular Ca²⁺.In RASMC,SF had no effect on PE- or KCl-induced[Ca²⁺]i increase either in the presence or in the absence of external Ca²⁺.Conclusions:These results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle.SF had no effect on[Ca²⁺]i,the direct inhibition of the common pathway after[Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction,while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.