The Expression Of VPO1in Cardiovascular Remodeling And The Intervention Effect Of Losartan In SHR Rats

Chapter1Involvement of VPO1in the pathogenesis of vascular remodeling in different week-old spontaneously hypertensive ratsBackgroundHypertension is a kind of systemic arterial systolic and (or) diastolic blood pressure which continues to rise as the main features of systemic disease. The main pathological changes in hypertension are vascular remodeling and left ventricular hypertrophy, involving changes of parenchymal cells (such as myocardial cells, smooth muscle cells and endothelial cells) and stromal cells (such as cardiac fibroblasts and extracellular matrix), causing volume increase of myocardial cells and hyperplasia of stromal. Additionally, along with blood pressure development, vascular resistance increases and causes corresponding increase of left ventricular afterload, resulting in left ventricular hypertrophy. Furtherrmore, it can lead to left ventricular failure. The performances in blood vessels include vascular endothelial dysfunction, smooth muscle cell hypertrophy and extracellular matrix deposition. Vascular remodeling and left ventricular remodeling are the central link during the development of of hypertension. Numerous studies show that oxidative stress is one of the mechanisms of vascular remodeling and left ventricular remodeling in hypertension.Oxidative stress can cause damages by reactive oxygen species (ROS) which are excessive accumulated. NADPH oxidase (NOX) and peroxidase are the important source of reactive oxygen species in blood pressure regulation.Vascular peroxidase1(VPO1), a recently reported member of heme-containing peroxidase family, are mainly expressed in the cardiovascular system. Studies have shown that VPOl, similar to myeloperoxidase (MPO), is able to catalysis H2O2(the weak oxidant) to HOC1(the strong oxidant), which promotes the damage of body cell. It can be speculated that VPO1may play an important role in oxidative stress.In this chapter spontaneously hypertensive rats (SHRs) of different week-old and controlled normotensive Wistar-Kyoto rats (WKYs) were selected to investigate the correlation between VPO1expression and the vascular remodeling in thoracic and mesenteric aortas, as well as the relationship between VPO1expression and the level of oxidative stress. Furthermore, the effects of losartan on vascular remodeling in hypertensive rats and mechanisms involved were approached. MethodsA total of48male SHRs aged at4weeks with a bodyweight of65-85g/rat, and another32male and body-weight matched WKYs, were purchased. Adapted fed a week later, the animals were randomly divided into following groups:①Groups WKY5and SHR5(n=8, each), the rats at5week-old;②Groups WKY8and SHR8(n=8, each), the rats at8week-old;③Group WKYB and SHR13(n=8, each), the rats at13week-old;④Group WKY20and SHR20(n=8, each), the rats at20week-old;⑤low-dose losartan Group (15mg/kg/day, Los(L));⑥high-dose losartan Group (30mg/kg/day, Los (H)). Additionally, the13week-old SHRs had been administrated with the different dose of losartan mentioned above daily for7weeks, meanwhile SHR and WKY groups were given an equal volume of saline. Animals were weighed weekly to adjust the dose, and conventional fed until experiments terminated at20weeks of age. The left carotid artery was catheterized for monitoring blood pressure and collecting blood sampling for measuring concentrations of angiotensin Ⅱ (Ang Ⅱ) and hydrogen peroxide (H2O2). It has also been done to determinate aortic NADPH oxidase(NOX) activity. Both of the RNA and protein of thoracic aorta, as well as the RNA of mesenteric artery were extracted to evaluate the VPO1expression with Real-time PCR or Western Blot methods. Thoracic aorta and mesenteric artery were embedded in4% paraformaldehyde and then moved to the paraffin for morphological experiments of hematoxylin-eosin (HE), masson staining and immunohistochemistry.Results1. Compared with the same week-old WKY rats, blood pressure in SHR8, SHR13and SHR20increased significantly, and the vascular remodel of thoracic aorta and mesenteric artery in SHR13and SHR20were obvious;2. Compared with the same week-old WKY rats, levels of plasma Ang Ⅱ and H2O2in SHR8, SHR13and SHR20were significantly increased, and NOX activity of aortic tissue was significantly increased, which reached the highest magnitude in SHR20;3. Compared with the same week-old WKY rats, VPO1mRNA expression of mesenteric artery, as well as VPO1mRNA and protein expression of thoracic aortia were significantly increased in SHR8, SHR13and SHR20;4. Losartan decreased blood pressure and reversed vascular remodeling in dose-dependent manner in SHRs, meanwhile reducing plasma H2O2concentrations, aortic NOX activity and VPO1protein expression in SHRs. Conclusion1. The mechanisms of vascular remodeling in SHRs were related to the enhanced oxidative stress, and VPOl’s upregulation were related to vascular remodeling, which may involve the NOX/VPO1pathway;2. Losartan’s capacity of anti-remodeling in hypertensive rats may be related to the inhibition NOX/VPO1pathway, in which oxidative stress was reduced. Chapter2Involvement of VPO1in the pathogenesis of myocardial remodeling in different week-old spontaneously hypertensive ratsBackgroundMyocardial remodeling is defined that myocardial tissue makes adaptive response to a variety of pathological conditions (pressure overload, ischemia, high renin levels, etc.), including cardiac hypertrophy, interstitial fibrosis, myocardial cells apoptosis and embryonic genes re-expression. Reconstruction during hypertension early-stage maintains normal heart function and has a certain positive role in short term, but the long-lasting reconstruction will gradually lead cardiac function deteriorating to heart failure, the decompensated emergence. Recent studies have shown that oxidative stress plays an important role in hypertensive left ventricular remodeling. A large accumulation of reactive oxygen species and/or reduced antioxidant capacity cause oxidative stress. The enzymes producing ROS include NADPH oxidase(NOX), peroxidase, xanthine oxidase, monoamine oxidase and etc. VPO1, a newly discovered member of the heme-containing peroxidase family, expresses mainly in the cardiovascular system. Based on the VPO1’s peculiarity to catalyze H2O2(weak oxidant) to HOC1(strong oxidant), with the same SHR rats model we continued to investigate whether VPO1was Involved in the pathogenesis of myocardial remodeling. Furthermore, the effects of losartan on ventricular remodeling in hypertensive rats and mechanisms involved were approached.MethodsBased on animal groups in Chapter1, there were six groups of animals:①Group SHR5and WKY5;②Group SHR8and WKY8;③Group SHR13and WKY13;④Group SHR20and WKY20;⑤Group Los (L);⑥Group Los (H), n=8each. Echocardiography was given to evaluate heart structure and cardiac function changes at the end of the drugs treatment. The right tibias were dissociated to measure the length (TL), while hearts harvested, and the heart weight (HW) and left ventrucular weight (LVW) were recorded normalized by tibia length. Left ventricle were embedded in4%paraformaldehyde and then move to the paraffin for hematoxylin-eosin (HE), masson staining and immunohistochemistry. Both of the RNA and protein of ventricular tissue were extracted. Myocardial hypertrophy markers (BNP, a-SA, β-MHC), fibrosis markers (collagen type Ⅰ, Ⅲ and CTGF), and apoptosis makers (Bax and Bcl-2) were determined by Real-time PCR. Left ventriculars were pulverized to measure HOC1and H2O2concentrations, and NADPH oxidase (NOX) activity. Meanwhile, NOX2and VPO1expression in left ventricular were detected by Western blot.Results1. Compared with the same week-old WKY rats, blood pressure in SHR8, SHR13and SHR2o increased significantly; ventricular end-diastolic left ventricular posterior wall and interventricular septum were markedly thickened in SHR13and SHR2o; in contrast to left ventricular diastolic dysfunction, systolic function was not significantly changed;2. Compared with the same week-old WKY rats, collagen deposition significantly increased, while mRNA expressions of BNP, α-SA, β-MHC, Ⅰ, Ⅲcollagen, CTGF and Bax were significantly upregulated, and on the opposite, Bcl-2was significantly down-regulated in SHR13and SHR2o;3. Compared with the same week-old WKY rats, left ventricular HOC1and H2O2concentrations, NOX activity and the mRNA and protein expressions of NOX2and VPO1were significantly increased in SHRg, SHR13and SHR20;4. Compared with control group, Losartan reversed left ventricular remodeling in dose-dependent manner, meanwhile reducing left ventricular HOC1and H2O2concentration, NOX activity, NOX2and VPO1protein expression in SHRs.Conclusion1. The mechanisms of left ventricular remodeling in SHRs were related to the enhanced oxidative stress, and VPO1’s upregulation were related to ventricular remodeling, which may involve the NOX/VPO1pathway;2. Losartan’s capacity of anti-remodeling in hypertensive rats may be related to the inhibition NOX/VPO1pathway, in which oxidative stress was reduced. Chapter3Involvement of VPO1in the pathogenesis of endothelial dysfunction in different week-old spontaneously hypertensive ratsBackgroundHypertension is a major risk factor for cardiovascular disease, and more and more evidence show oxidative stress caused by a large accumulation of reactive oxygen species (ROS) is main pathogenesis of hypertension. The enzymes producing ROS in Hypertensive heart, kidneys and central nervous system include xanthine oxidase enzyme, nitric oxide synthase uncoupled (NOS), mitochondrial respiratory enzymes and NADPH oxidase (NOX). NOX, the major source of ROS in cardiovascular system, can catalyze the production of superoxide and other downstream reactive oxygen species. There are7kinds of NOX family members catalytic subunit, namely NOX1-5and Duoxl and Duox2(dual oxidase). So far, NOX catalytic subunits confirmed in mammalian cardiovascular systems is mainly NOX2and NOX4.Peroxidase is widely distributed in the body, and highly cell/tissue-specific distribution. It is involved in host defense reactions, the biosynthesis of thyroid hormones and extracellular matrix, and so on. In pathological conditions it can catalyze hydrogen peroxide (H2O2) and chloride ions to produce hypochlorous acid, thereby oxidizing proteins, lipids and DNA, and leading to cell and organization. Nitric oxide (NO), a vasodilation factor, is produced by endothelial nitric oxide synthase (eNOS) and maintains vascular diastolic function. The decrease of eNOS synthesis and/or bioavailability can lead to endothelial dysfunction, and endothelial dysfunction is the important reason why high blood pressure causes damage to target organs and related complications.Vascular peroxidasel (VPO1), a newly discovered peroxidase family member, is mainly expressed in vascular endothelial cell, smooth muscle cell and cardiac cell in the cardiovascular system. The specificity of VPO1and substrate is similar to MPO, i.e. weak oxidant H2O2is catalyzed strong oxidant hypochlorite, further promoting oxidative stress to vascular tissues.It had been concluded that VPO1was involved in the development of hypertension in the section above, and in this section with the same SHR rats model we continued to investigate NOX activity status, ROS production,and oxidative stress, furthermore weather VPO1was involved in endothelial dysfunction along with hypertension’progress.MethodsBased on animal groups in Chapter1, there were six groups of animals:①Group SHR5and WKY5;②Group SHR8and WKY8;③Group SHR13and WKY13;④Group SHR20and WKY20;⑤Group Los (L);⑥Group Los (H), n=8each. The left carotid artery was catheterized for monitoring blood pressure and collecting blood sampling to measure concentrations of NO and H2O2concentration. Thoracic aortas were freed to determinate endothelium-dependent relaxation response, and pulverized to measure HOC1and3-nitrotyrosine’s concentrations, and NADPH oxidase (NOX) activity. The RNA and protein of thoracic aorta were extracted to determine NOX4, eNOS and VPO1expression with the Real-time PCR or Western Blot test.Results1. Compared with the same week-old WKY rats, blood pressure in SHR8, SHR13and SHR20increased significantly, while SHR13and SHR20with endothelium-dependent diastolic dysfunction;2. Compared with the same week-old WKY rats, plasma H2O2level in SHR8, SHR13and SHR20was significantly increased, while HOCl,3-nitrotyrosine, NOX activity and NOX4mRNA and protein expression of aorta were significantly increased;3. Compared with the same week-old WKY rats, plasma concentration of NO, eNOS mRNA and protein expression of aorta were significantly reduced in SHR8, SHR13and SHR20;4. Losartan decreased HOCl,3-nitrotyrosine, NOX activity and NOX4and VPO1mRNA and protein expression of aorta, in contrast to increase plasma NO concentration and aorta eNOS mRNA and protein expression in dose-dependent manner,which finally improved vascular endothelial function. Conclusion1. Hypertension can lead to vascular endothelial dysfunction, and the anhanced oxidative stress may be one of the mechanisms;2. VPO1was related to endothelial dysfunction in hypertensive rats, which may involve the NOX/VPO1pathway;3. Losartan reduced blood pressure and the extent of oxidative stress, reducing VPO1expression as well, which improved endothelial function.