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Objectives Oxidative stress induces hypertrophic gene expression and collagen deposition that mediates cardiac hypertrophy and myocardial fibrosis. Vascular peroxidase 1 (VPO1) is a new heme-containing peroxidase, which can utilise hydrogen peroxide (H 2 O 2 ) generated from co-expressed NADPH oxidase (NOX) to produce hypochlorous acid (HOCl) and catalyse peroxidative reactions. Our previous studies had found that NOX/VPO1 pathway-mediated oxidative stress plays an important role in myocardial ischaemia-reperfusion injury. This study was aim to investigate whether VPO1 mediate angiontensin II induced cardiac hypertrophy in H9c2 cells and to explore the underlying mechanism. Methods Cultured H9c2 cells were treated with angiotensin II, cell hypertrophy were measured by cellular surface size, brain natriuretic peptide (BNP) and atrial natriuretic factor (ANF) gene expression. The effects of NADPH oxidase inhibitor, diphenyleneiodonium (DPI), hydrogen peroxide scavenger, catalase, VPO1 inhibitor 4-aminobenzoic acid hydrazide (ABAH) on cell hypertrophy, VPO1 expression, H 2 O 2 and HOCl production were measured. And the effect of ABAH on the mitogen-activated protein kinase pathway including p-38, p-JNK and p-ERK were determined. Results Angiotensin II treatment significantly increased the cellular size and the gene expression of BNP, ANF while up-regulated the VPO1 expression and HOCl production. These effects were inhibited by pretreatment with DPI, catalase and ABAH. Moreover, pretreatment with ABAH abolished the angiotensin II-induced up-regulation of p-ERK activity, not p-38, p-JNK expression. Conclusions These results indicated that VPO1 play a role in anngitotensin II induced cell hyperthrophy via NOX-H 2 O 2 -VPO1-HOCl-ERK1/2 pathway.

Objectives Background and objective Matrix metalloproteinases (MMPs) and Tissue Inhibitor of Matrix Metalloproteinases (TIMPs) associated with atherogenesis and plaque rupture. Sphingomyelinase has a potential regulator role in the activation of MMP via induced ceramide formation. So we evaluated the relationship between sphingomyelinase, ceramide levels and presentation, extent and severity of atherosclerotic coronary artery disease (CAD). Methods Consecutive patients who underwent coronary angiography were randomly included. The serum concentrations of sphingomyelinase, ceramide, MMP-2, MMP-9 and TIMP-1 were analysed with ELISA method in 274 patients. Participants were divided into 5 groups; stable angina pectoris (SAP; n=64), unstable angina pectoris (USAP; n=67), non-ST elevation myocardial infarction (NSTEMI; n=56), acute ST elevation myocardial infarction (STEMI; n=55) and controls (n=30). Coronary angiographic Gensini score was calculated, and the correlation between sphingomyelinase, ceramide, MMP-2, MMP-9 and clinical presentation, extent and severity of atherosclerotic coronary artery disease were analysed. Results sphingomyelinase, ceramide, MMP-2 levels were higher in STEMI and NSTEMI groups compared with USAP, SAP and control groups (STEMI vs USAP p=0.001; STEMI vs SAP p=0.001; STEMI vs control p Conclusions Serum levels of sphingomyelinase, ceramide are elevated in patients with CAD; more so in acute coronary syndromes. sphingomyelinase, ceramide are associated with more extensive and severe CAD (as represented by Gensini score).

Objectives The sphingolipid pathway generates bioactive molecules that are crucial to the regulation of a variety of physiological and pathological processes. Recent studies indicate that plasma secretory acid sphingomyelinase (S-SMase) activity correlates with inflammatory cytokines and the severity of chronic heart failure (CHF). However, the roles of sphingomyelin (SPM) and ceramide (Cer) remain unclear in the heart failure process. Methods To investigate sphingolipids and sphingolipid signalling in patients with left ventricular dysfunction, we measured plasma S-SMase activity, SPM, Cer, N-terminal pro-B-type natriuretic peptide (BNP), high sensitivity C-reactive protein (hsCRP), tumour necrosis factor-α (TNF-α), soluble Fas, soluble Fas ligand and hemodynamic parameters in 423 patients with CHF and in 104 healthy subjects. Results Plasma Cer levels increased stepwise with New York Heart Association (NYHA) functional classification (I, 5.32±1.98; II, 5.81±1.63; III, 6.14±2.14; IV, 6.66±2.61, p Conclusions Elevated plasma Cer levels correlated with the severity of CHF. The accumulation of ceramide was associated with the hemodynamic status, BNP levels, circulating pro-inflammatory cytokines TNF-α and hsCRP, as well as with the soluble apoptosis receptor Fas, suggesting that the sphingolipid signalling pathway is involved in the persistent immune activation and apoptosis associated with CHF, and may contribute to the pathophysiology.

Objectives To test the role of vascular peroxide 1 (VPO1), a newly identified hame-containing peroxidase in left ventricular remodelling in spontaneously hypertensive rats. Methods Twelve 24-week-old male spontaneously hypertensive rats (SHR) served as SHR group, and 12 age and sex matched Wistar Kyoto rats (WKY) were selected as control group. Systolic blood pressure was measured before experiment. After performing echocardiography analysis, hearts were isolated. Pathological changes of myocardial tissue were measured by HE staining, myocardial collagen was measured by Masson staining, and the expression of VPO1, MMP-2, MMP-9 and TIMP-2 was detected by immunohistochemistry and western blot. Rat heart-derived H9c2 cells were treated with angiotensin II, the cell surface area and the mRNA level of atrial natriuretic peptide, brain natriuretic peptide were measured. Expression of VPO1, MMP-2 and gelatinolytic activity of pro-MMP-2 and the concentration of HOCl was measured. The effect of VPO1 RNA interference on cardiomyocytes hypertrophy, HOCl generation, pro-MMP-2 activity and MMP-2 expression were observed. Furthermore, the direct effects of HOCl on pro-MMP-2 activity and MMP-2 expression were also examined. Results Blood pressure in SHR was significantly higher compared with WKY, increased concentric left ventricular hypertrophy, myocardial cells hypertrophy while the expressions of VPO1, MMP-2, and TIMP-2 protein were significantly up-regulated were found in SHR. In cultured cells, treatment with angiotensin II significantly induced hypertrophy and increased the gelatinolytic activity of pro-MMP-2 and MMP-2 expression while unregulated VPO1 expression and HOCl production. Silencing VPO1 expression significantly suppressed angiotensin II-induced hypertrophy and increased MMP-2 activity concomitantly with decreased HOCl production. Moreover, treatment with HOCl also markedly increased the gelatinolytic activity of pro-MMP-2 and MMP-2 expression. Conclusions VPO1 participating in the extracellular matrix remodelling by activate MMP-2 via HOCl formation, and therefore play an important role in development of left ventricular remodelling.

Objectives Proliferation of vascular smooth muscle cells (VSMCs) induced by angiotensin II (Ang II) plays a vital role in the pathogenesis of hypertension. In the present study, the effect of reinioside C, a main active ingredient of Polygalafallax Hemsl, on proliferation of VSMCs induced by Ang II was investigated. Methods Cell proliferation was measured by two methods; the DNA synthesis and cell cycle were analysed by BrdU marking and flow cytometry. Intracellular ROS level were determined by measuring the oxidative conversion of cell permeable H2DCF to DCF in fluorospectrophotometer. NADPH oxidase subunits (p22phox, gp91phox), AP-1 subunits (c-fos, c-jun) and c-myc were evaluated by real time PCR. ERK1/2 and IkB-a were measured by western-blot. The electrophoretic mobility shift assay for determining the NF-kB DNA-binding activity. Results The results showed that reinioside C attenuated Ang II-induced NADPH oxidase mRNA expression, generation of ROS, ERK1/2 phosphorylation and activation of NF-kB as well as mRNA expression of AP-1 and c-myc in VSMCs in a concentration-dependent manner. These effects of Ang II were also inhibited by diphenyleneiodonium (the NADPHoxidase inhibitor), PD98059 (the ERK1/2 inhibitor) and pyrollidinedithiocarbamate (the NF-kB inhibitor). Conclusions These result suggest reinoside C attenuates AngII-induced proliferation of VSMC via inhibiting NADPH oxidase- ROS- ERK1/2-NF-kB -AP-1 pathway.