Involvement Of VPO1in Hypertensive Cardiac Remodeling

Background Cardiac remodeling in response to stress and injury, such as pressure or volume overload, neurohormones and cytokines, is characterized by cardiac hypertrophy and myocardial inflammation and fibrosis, which is a major risk factor for cardiovascular morbidity and mortality and a leading cause of chronic heart failure. A large body of evidence shows that oxidative stress plays an important role in the pathological process of cardiac remodeling. Stimulus mentioned above can activate the NADPH oxidases (NOX), a major resource of ROS, and result in large production of supreoxide (O2-) and peroxide (H2O2) with subsequent downstream of redox-sensitive signaling pathways, which is involved in the development of pathologic process of hypertrophy response and collagen deposition.Vascμlar peroxidase1(VPO1) is a newly identified novel family member of peroxidases in cardiovascular system. VPO1as an enzyme that is downstream of NOX, functions to utilize NOX-derived hydrogen peroxide (H2O2) to produce a strong oxidant-hypochlorous acid (HOCl), and thus the oxidative stress is dramatically amplified. Several lines of evidence suggest that the NOX/VPO1pathway-mediated oxidative stress plays an important role in myocardial ischemia-reperfusion injury, endothelial cell apoptosis and smooth muscle cell proliferation. In addition, VPO1can be secreted into the extracellular space to participate in extracellular matrix formation, suggesting that VPO1may also play a role in cardiac remodeling, The present study was first to determine whether VPO1is implicated in hypertensive cardiac remodeling in vivo, and then to investigate whether VPO1is involved in Ang II induced cardiac remodeling in vitro and to explore the underlying mechanism.Chapter I Relationship between VPO1and hypertensive cardiac remodelingObjectives:To investigate the relationship between VPO1and hypertensive cardiac remodeling.Methods:Twelve male spontaneously hypertensive rats (20-week-old) were served as hypertension group (SHR group), and Twelve male Wistar-Kyoto rats with the same age served as the normotensive control group (WKY group). Blood pressure was measured and after performing echocardiography analysis, the heart was isolated. Pathological changes of myocardial tissue was measured by HE staining, myocardial collagen deposition was measured by Masson staining, and the expression of VPO1was detected by immunohistochemistry。The expression of Nox2, Nox4and VPO1was also determined by western blot and the mRNA expression of type I Ⅲ collagen, Nox2、Nox4and VPO1was measured by real time-PCR. Results:Compared with WKY group, blood pressure, thickness of ventricle, collagen deposition, mRNA and protein expression of Nox2、Nox4and VPO1, mRNA expression of type Ⅰ、Ⅲ collagen were all significantly increased in SHR group, whereas cardiac diastolic function is impaired in SHR group.Conclusion:The present reserch indicates that VPO1may be involved in the process of Hypertensive rats cardiac remodeling via NOX/VP01-signaling pathways. Chapter Ⅱ VPO1mediates Ang Ⅱ-induced myocardial remodeling in H9c2cellsObjective:To investigate whether VPO1mediates Ang Ⅱ-induced cardiac remodeling in H9c2cells and to explore the underlying mechanism.Methods:(1) The effect of Ang Ⅱ on the VPO1expression in H9c2cells was observed. H9c2cells were treated with different concentrations of Ang Ⅱ (10-7M,10-6M,10-5M Ang Ⅱ) for24h, and H9c2cell were treated with Ang II for different time (12h,24h,48h). The expressions of VPO1were determined by Western-Blot and Real-Time PCR.(2) To study the effect of NADPH inhibitor diphenyleneio-donium (DPI), catalase, VPO1inhibitor-ABAH on Ang Ⅱ induced cardiac remodeling. Cultured H9c2cells were randomly divided into five groups:control group:cells were incubated in high glucose DMEM containing1%fetal bovine serum for24hours; Ang Ⅱ treatment group:cells were incubated in high glucose DMEM containing10-6M Ang II for24hours; Ang Ⅱ+DPI group:cells were pretreated with100μM DPI (NADPH oxidase inhibitor) for2hours prior to Ang II exposure; Ang Ⅱ+Catalase group: cells were pretreated with300μM Catalase for2hours prior to Ang II exposure; Ang II+ABAH group:cells were pretreated with400μM ABAH for2hours prior to Ang Ⅱ exposure; The mRNA and proteins expression of Nox2、Nox4and VPO1were determined by western blot and real time-PCR. Hypertrophy assay:H9c2cell were stained with crystal violet and the cellular size were calculated by Image software. Gene expression of BNP、ANF was tested by RT-PCR. Fibrosis assay: collagen type Ⅰ、Ⅲ gene expression was measured by RT-PCR, and collagen content was valued by Sirus Red staining. To explore the underlying mechanism:the protein expression of p-38、p-ERK and p-JNK were evaluated by western blot. The level of H2O2and HOC1were detected respectively through ELISA and3-chlorotyrosine protein immuno assay. Results:(1) In vitro, these results suggest that VPO1is unregulated by Ang II in a dose-depended manner, which can be abrogated both in gene and protein level by different inhibitors, such as NADPH inhibitors-DPI, catalase, and the VPO1inhibitors (aminobenzoic acid hydrazide, ABAH).(2) Compared with the control group, Ang Ⅱ (10’6M) significantly increased the expression of Nox2, VPO1, the collagen content, the cellular size, and also up-regulated the gene expression of BNP, ANF, type In Ⅲ collagen. In addition, Ang II also increased the production of H2O2and the protein expression of3-chlorotyrosine protein. These effects of Ang II on H9c2cells were inhibited in a different degree by the pretreatment of DPI, catalase and the VPO1inhibitor (ABAH).(3)Ang II induced myocardial remodeling, followed by up-regulating the MAPK (p-ERK, p-38, p-JNK) protein expression, which were attenuated by the NADPH oxidase inhibitor (DPI) and catalase. However, the VPOl inhibitor (ABAH) can only inhibit the up-regulation of p-ERK induced by Ang II.Conclusion:The present study has demonstrated that VPO1is at least part involved in the process of the Ang Ⅱ induced cardiac remodeling, both in myocardial hypertrophy and fibrosis. The results indicate that VPO1play a role via Nox2/H2O2/VPO1/HOCl/ERK1/2signaling pathway in the pathology of Ang Ⅱ induced cardiac remodeling.