Effect Of Farnesyl Pyrophosphate Synthase Inhibitor On Oxidative Stress In Spontaneously Hypertensive Rats

Part One Alteration of enzymes expression in mevalonate pathway: possible role for cardiovascular remodeling in spontaneously hypertensive ratsBackground:Mevalonate pathway is an important metabolic pathway which plays a key role in multiple cellular processes by synthesizing sterol isoprenoids, like cholesterol, and non-sterol isoprenoids, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These non-sterol isoprenoid intermediates of the mevalonate biosynthetic pathway play important roles in the post-translational modification of small GTP-binding proteins (GTPases). Protein prenyltransferases catalyze the addition of the isoprenoids, farnesyl or geranylgeranyl to the C-terminal of GTPases, facilitating their activation. Recent studies have suggested that small GTP-binding proteins Ras and Rho are involved substantially in functional and structural alterations of hypertensive blood vessels and hearts in SHR. Aims:To determine whether the key enzymes expressions of mevalonate pathway in heart, aorta and liver during the process of hypertension in SHR are altered and whether these changes are associated with the cardiovascular remodelling.Methods:At the ages of 3,16 and 25 weeks, systolic blood pressure of SHR and WKY was measured by the tail-cuff method. Serum cholesterol concentrations were determined by +enzymatic methods. Hearts and thoracic aortas were removed for the study of cardiovascular remodelling. The protein expressions of enzymes in hearts, aortas and livers were analysed by western blot, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR)?farnesyl diphosphate synthase (FDPS)?squalene synthetase (SQS)?farnesyltransferase alpha (FNTA)?farnesyltransferase beta (FNTB)?geranylgeranyltransferase type I (GGTase-?).Results:The systolic blood pressure developed gradually over weeks in SHR after 3-week-old, but only slightly in WKY. At 3 weeks of age, blood pressure was not significantly different between SHR and WKY, whereas in both 16-week-old and 25-week-old age groups, blood pressure was significantly higher in SHR than in WKY. SHR had a lower LDL-C level but similar values of STC and HDL-C compared with WKY at 3 weeks of age. At adult stage of 16-week-old and 25-week-old age, STC, LDL-C and HDL-C were lower in SHR than in WKY. The histological measurements showed the mass and myocyte sizes of heart, the media thickness (MT) and media cross-sectional area (MCSA) of thoracic aorta were all increased in SHR since 3 weeks. In heart, there were overexpressions of some enzymes, including HMGR, FDPS. GGTase-?, and a downregulation of SQS in SHR since 3-week-old age. In aorta, besides similar expressions of HMGR, SQS, FDPS and GGTase-?with heart, there were upregulations of FNTA at 16-and 25-week-old age and FNTB at 25-week-old SHR. Western blot demonstrated an overexpression of HMGR and a downregulation of SQS in SHR liver at all ages tested.Conclusions:The cardiovascular remodeling of SHR preceded development of hypertension. The serum cholesterol level was significantly decreased in SHR, and these findings may be associated with lower expression of key enzyme SQS in sterol pathways in liver from SHR at all ages. The up regulation expression of several key enzymes in non-sterol mevalonate pathways, and down regulation expression of SQS in heart and aorta may play a potential pathophysiological role for cardiovascular remodeling in SHR via excessive activation of small GTP-binding proteins. Part Two Effect of farnesyl diphosphate synthase inhibition on endothelial function in spontaneously hypertensive rats vivo—antioxidative stressBackground:Farnesyl diphosphate synthase (FDPS) is a key enzyme in the mevalonate pathway. In previous studies, we found that the expression of key enzymes in the mevalonate pathway, including FDPS, are significantly upregulated in the spontaneously hypertensive rat (SHR). Bisphosphonates inhibit farnesyl pyrophosphate (FPP) synthase, through inhibition of isoprenylation including farnesylation and geranylgeranylation by preventing the synthesis of various isoprenoids such as FPP and geranylgeranyl pyrophosphate (GGPP). Bisphosphonate alendronate was able to improve the endothelial function in SHR and the upregulation of eNOS expression by suppression of small GTPase RhoA activation, which needs geranylgeranylation by GGPP. Excess of ROS generation synthesized by NADPH oxidase is critically involved in the breakdown of endothelial function in SHR. The activated Racl by geranylgeranylation is a prerequisite of NADPH oxidase activation.Aims:To determine whether FDPS inhibitor ibandronate improves the endothelial function in SHR, at least in part, through inhibition of excessive oxidative response via the Rac1/NADPH oxidase pathway. Methods:After 4-week administration of ibandronate (1mg/kg/day), endothelium-dependent and independent vasorelaxation were measured in isolated aortic rings. The level of ROS in aorta was then quantitatively measured fluorometrically. and the enzymatic activities of NADPH oxidase was evaluated spectrophotometrically with commercial assay kits. Activation of Rac1 in aorta was determined by pull-down assay. Aortic expression of p47phox in both cytoplasmic and membrane fractions and Rac1 was determined by western blot.Conclusions:FPP synthase inhibitor ibandronate exerted antioxidant effects in the vasculature of SHR mediated by Racl/NADPH oxidase pathway. These effects of ibandronate may contribute to the vasoprotective effects for impaired endothelium in SHR. Part Three Role of farnesylpyrophosphate synthase in angiotensin?-mediated oxidative stressBackground:In vascular cells, ROS production can be induced by humoral factors such as angiotensin?(Ang?). An abnormal elevation of circulating Ang?is critically involved in the excess oxidative stress in spontaneously hypertensive rat (SHR). Many kinds of pathways are involved in Ang?-induced excess oxidative stress response of vascular smooth muscle cells including the Rac1/NADPH oxidase signaling.Farnesyl pyrophosphate synthase (FDPS) synthesize of various isoprenoids such as FPP and geranylgeranyl pyrophosphate (GGPP). The latter is needed for protein isoprenylation and activation of small GTP-binding protein, including Racl. Activation and translocation of Rac1 from the cytosolic compartment to the cell membrane induce activation and upregulation of NADPH oxidase expression, which contributes to the excessive generation of ROS.Aims:To explore whether inhibition of FDPS synthase by ibandronate (IBAN) could interfere with the oxidative stress responses induced by Ang?in cultured vascular smooth muscle cells (VSMCs). and whether it involves Racl/NADPH oxidase pathway.Methods:VSMCs from SHR and Wistar-Kyoto rats (WKY) were stimulated with Ang?for 3 h. In some experiments, cells were pre-exposed for 24 h to IBAN (10-5 mol/L), IBAN plus Geranylgeraniol (GGOH,3×10-5 mol/L), IBAN plus Farnesol (FOH,3×10-5 mol/L), IBAN plus Mevalonate (MEV?10"4 mol/L)?GGTI-286 (selective inhibitor of GGTase I, 10-5 mol/L) or Racl Inhibitor (10-4 mol/L). The generation of ROS for the VSMCs was evaluated by a fluorometry assay using intracellular oxidation of DCFH-DA, and the enzymatic activities of NADPH oxidase was evaluated spectrophotometrically with commercial assay kits. Activation of Rac1 was determined by pull-down assay. The expression of p47phox in both cytoplasmic and membrane fractions and Racl in VSMCs were determined by western blot.Results:Angiotensin?(Ang?) concentration dependently increased ROS production in cultured VSMCs from WKY and SHR. The Ang?-induced responses were greater in SHR VSMCs, but significantly reduced by ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, expression of NADPH oxidase subunit p47phox, NADPH oxidase and Racl GTP-binding activity in VSMCs. Addition of GGOH, but not FOH or MEV reversed the inhibitory effects of ibandronate. Moreover, inhibitor of geranylgeranyl-transferase GGTI-286 and Racl mimicked the effect of ibandronate on this excessive oxidative response.Conclusions:FDPS inhibitor ibandronate exerted cellular antioxidant effects induced on Ang?stimulation in cultured SHR VSMCs, and this effect may be mediated at least partly by reduction the activated form of Rac1 via blocking of geranylgeranylation.