Guo-Gang Zhang

Involvement of vascular peroxidase 1 in angiotensin II-induced vascular smooth muscle cell proliferation

AIMS Vascular peroxidase 1 (VPO1) is a newly identified haem-containing peroxidase that catalyses the oxidation of a variety of substrates by hydrogen peroxide (H(2)O(2)). Considering the well-defined effects of H(2)O(2) on the vascular remodelling during hypertension, and that VPO1 can utilize H(2)O(2) generated from co-expressed NADPH oxidases to catalyse peroxidative reactions, the aims of this study were to determine the potential role of VPO1 in vascular remodelling during hypertension. METHODS AND RESULTS The vascular morphology and the expression of VPO1 in arterial tissues of spontaneously hypertensive rats and Wistar-Kyoto rats were assessed. The VPO1 expression was significantly increased concomitantly with definite vascular remodelling assessed by evaluating the media thickness, lumen diameter, media thickness-to-lumen diameter ratio and mean nuclear area in artery media in spontaneously hypertensive rats. In addition, in cultured rat aortic smooth muscle cells we found that the angiotensin II-mediated cell proliferation was inhibited by knockdown of VPO1 using small hairpin RNA. Moreover, the NADPH oxidase inhibitor, apocynin, and the hydrogen peroxide scavenger, catalase, but not the ERK1/2 inhibitor, PD98059, attenuated angiotensin II-mediated up-regulation of VPO1 and generation of hypochlorous acid. CONCLUSION VPO1 is a novel regulator of vascular smooth muscle cell proliferation via NADPH oxidase-H(2)O(2)-VPO1-hypochlorous acid-ERK1/2 pathways, which may contribute to vascular remodelling in hypertension.

The ALDH2 Glu504Lys polymorphism is associated with coronary artery disease in Han Chinese: Relation with endothelial ADMA levels

OBJECTIVES We studied the association between mitochondrial aldehyde dehydrogenase (ALDH2) Glu504Lys (rs671 or ALDH2*2) polymorphism and coronary artery disease (CAD), and sought to clarify the mechanisms underlying this association. METHODS The ALDH2 rs671 polymorphism was genotyped in 417 CAD patients and 448 age- and gender-matched controls. All participants were Han Chinese. Human umbilical vein endothelial cells (HUVECs) isolated from 11 human umbilical cords were genotyped, cultured, and exposed to angiotensin II (Ang II, 10(-7)-10(-5)mol/L). Dimethylarginine dimethylaminohydrolase 1 (DDAH1) mRNA expression levels were determined by real-time PCR. Levels of asymmetric dimethylarginine (ADMA) in culture media and cell lysates were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). RESULTS The frequency of carriers of the ALDH2 rs671 A allele (GA+AA) was significantly higher in patients with CAD (47.5%) than in controls (35.0%, p=0.0002). After adjustment for potential confounders, the odds ratio (OR) for CAD for carriers of the rs671 A allele was 1.85 (95% confidence interval [CI]: 1.38-2.49, p=0.00005) in the entire study cohort, and 1.95 (95% CI: 1.40-2.70, p=0.00007) in non-drinkers. In non-drinking controls, the homozygous rs671 AA genotype was associated with significantly lower high-density lipoprotein cholesterol (HDL-C) concentrations compared with rs671 GG homozygotes (p=0.015). HUVEC cells homozygous for the G allele of rs671 showed a significantly higher DDAH1 mRNA expression and lower intracellular ADMA levels compared with heterozygous GA cells (p<0.05, respectively). In homozygous GG cells, high concentrations of Ang II (10(-5)mol/L) decreased DDAH1 mRNA expression and increased intracellular ADMA concentrations. CONCLUSIONS The rs671 polymorphism of ALDH2 is associated with CAD in Han Chinese, possibly by influencing HDL-C levels and endothelial ADMA levels.

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91(phox) subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91(phox) subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91(phox) subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.

Asymmetric dimethylarginine induces TNF-α production via ROS/NF-κB dependent pathway in human monocytic cells and the inhibitory effect of reinioside C

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, has been implicated in vascular inflammation through induction of reactive oxygen species (ROS) and proinflammatory genes in endothelial cells. However, relatively few attentions have been paid to the effect of ADMA on monocytes, one of the important cells throughout all stages of atherosclerosis. In the present study, we found that reinioside C, the main component extracted from Polygala fallax Hemsl., dose-dependently inhibited tumor necrosis factor-alpha (TNF-alpha) production induced by ADMA in monocytes, Furthermore, reinioside C attenuated ADMA-induced generation of reactive oxygen species and activation of nuclear factor-kappaB (NF-kappaB) activity in monocytes in a dose-dependent manner, this effect was inhibited by l-arginine (NOS substrate) and PDTC (inhibitor of NF-kappaB). These data suggest that reinioside C could attenuate the increase of TNF-alpha induced by exogenous ADMA through inhibition ROS/NF-kappaB pathway in monocytes.

Inhibition of NOX/VPO1 pathway and inflammatory reaction by trimethoxystilbene in prevention of cardiovascular remodeling in hypoxia-induced pulmonary hypertensive rats.

Abstract: Recent studies show that resveratrol exerts beneficial effects on prevention of pulmonary hypertension. This study is performed to explore the effects of trimethoxystilbene, a novel resveratrol analog, on rat pulmonary vascular remodeling and right ventricular hypertrophy in hypoxia-induced pulmonary arterial hypertension (PAH) and the underlying mechanisms. Sprague–Dawley rats were placed in a chamber and exposed to 10% O2 continuously for 4 weeks to induce PAH. The effects of trimethoxystilbene (5 or 10 mg/kg per day, intragastric [i.g.]) and resveratrol (as a positive control, 25 mg/kg per day, i.g.) on hypoxia-induced PAH vascular remodeling and right ventricle hypertrophy were evaluated. At the end of experiments, the index for pulmonary vascular remodeling and right ventricle hypertrophy, inflammatory cell infiltration in lung tissue, the plasma levels and lung tissue contents of hydrogen peroxide (H2O2), the mRNA and protein levels for NADPH oxidases (NOX2, NOX4) and vascular peroxidase 1 (VPO1) in pulmonary artery or right ventricle were measured. The results showed that trimethoxystilbene treatment significantly attenuated hypoxia-induced pulmonary vascular remodeling (such as decrease in the ratio of wall thickness to vessel external diameter) and right ventricle hypertrophy (such as decrease in the ratio of right ventricle weight to the length of the tibia), accompanied by downregulation of NOX2, NOX4, and VPO1 expression in pulmonary artery or right ventricle, decrease in H2O2 production and inflammatory cell infiltration in lung tissue. Trimethoxystilbene is able to prevent pulmonary vascular remodeling and right ventricle hypertrophy in hypoxia-induced rat model of PAH, which is related to inhibition of the NOX/VPO1 pathway-mediated oxidative stress and the inflammatory reaction.

Vascular peroxidase 1 catalyzes the formation of hypohalous acids: characterization of its substrate specificity and enzymatic properties.

The heme-containing peroxidase family comprises eight members in humans. The physiological and pathophysiological roles of heme-containing peroxidases are not well understood. Phagocyte-derived myeloperoxidase (MPO) utilizes chloride and bromide, in the presence of hydrogen peroxide (H(2)O(2)), to generate hypochlorous acid and hypobromous acid, potent oxidizing species that are known to kill invading pathogens. Vascular peroxidase 1 (VPO1) is a new member of the heme-containing peroxidase family; VPO1 is highly expressed in the cardiovascular system, lung, liver, pancreas, and spleen. However, functional roles of VPO1 have not been defined. In this report, we demonstrate the capacity for VPO1 to catalyze the formation of hypohalous acids, and characterize its enzymatic properties. VPO1, like MPO but unlike lactoperoxidase, is able to generate hypochlorous acid, hypobromous acid, and hypothiocyanous acid in the presence of H(2)O(2). Under physiological pH and concentrations of halides (100μM KBr, 100μM KSCN, and 100mM NaCl), VPO1 utilizes approximately 45% of H(2)O(2) for the generation of hypobromous acid, 35% for hypothiocyanous acid, and 18% for hypochlorous acid. The specific activity of VPO1 is ∼10- to 70-fold lower than that of MPO, depending on the specific substrate. These studies demonstrate that the enzymatic properties and substrate specificity of VPO1 are similar to MPO; however, significantly lower catalytic rate constants of VPO1 relative to MPO suggest the possibility of other physiologic roles for this novel heme-containing peroxidase.

The role of vascular peroxidase 1 in ox-LDL-induced vascular smooth muscle cell calcification.

Reactive oxygen species (ROS)-induced osteogenic differentiation of vascular smooth muscle cells (VSMCs) is associated with the pathogenesis of vascular calcification. Vascular peroxidase 1 (VPO1), a peroxidase in the cardiovascular system, utilizes the hydrogen peroxide (H2O2) produced by co-expressed NADPH oxidases to produce hypochlorous acid (HOCl) and catalyze peroxidative reactions. The aim of this study was to determine whether VPO1 plays a role in the osteogenic differentiation of VSMCs in the setting of the vascular calcification induced by oxidized low-density lipoprotein (ox-LDL). In cultured primary rat VSMCs, we observed that the expression of VPO1 was significantly increased in combination with increases in calcification, as demonstrated via increased mineralization, as well as increased alkaline phosphatase (ALP) activity and up-regulated runt-related transcription factor 2 (Runx2) expression in ox-LDL-treated cells. Ox-LDL-induced VSMC calcification and Runx2 expression were both inhibited by knockdown of VPO1 using a small interfering RNA or by an NADPH oxidase inhibitor. Moreover, the knockdown of VPO1 in VSMCs suppressed the production of HOCl and the phosphorylation of AKT, ERK and P38 MAPK. Furthermore, HOCl treatment facilitated the phosphorylation of AKT, ERK1/2 and P38 MAPK and the expression of Runx2, whereas LY294002 (a specific inhibitor of PI3K), U0126 (a specific inhibitor of ERK1/2) and SB203580 (a specific inhibitor of P38 MAPK) significantly attenuated the HOCl-induced up-regulation of Runx2. Collectively, these results demonstrated that VPO1 promotes ox-LDL-induced VSMC calcification via the VPO1/HOCl/PI3K/AKT, ERK1/2, and P38 MAPK/Runx2 signaling pathways.

Elevation of ceramide and activation of secretory acid sphingomyelinase in patients with acute coronary syndromes.

BackgroundAlthough there are several reported evidences for a pathogenic role of sphingolipid signaling in atherosclerosis, peripheral blood levels of ceramide and secretory acid sphingomyelinase (S-SMase) activity in patients with acute coronary syndromes (ACS) have not been evaluated. Methods and resultsA total of 304 CAD patients and 52 healthy individuals were divided into four groups: control group (n=52), stable angina pectoris (SAP) group (n=98), unstable angina pectoris (UAP) group (n=92), and acute myocardial infarction (AMI) group (n=114). Plasma levels of sphingomyelin (SPM) were elevated in patients with UAP and AMI compared with those in the control and SAP participants. Plasma ceramide levels and S-SMase activity in patients with ACS (including UAP and AMI) on day 0 were significantly higher than those in the control and SAP participants. Elevation in plasma ceramide levels in patients with UAP and AMI was sustained until a day after percutaneous coronary intervention or day 7, respectively. Moreover, in patients with UAP, S-SMase activity elevation on day 0 was followed by a gradual decrease toward the SAP range up to a day after percutaneous coronary intervention. In patients with AMI, elevation in S-SMase activity showed a peak on day 3. ConclusionSerial changes in plasma ceramide and S-SMase activity were documented in patients with ACS. These findings provide an insight into the molecular mechanism of plaque destabilization.

Vascular peroxidase 1: A novel enzyme in promoting oxidative stress in cardiovascular system

Vascular peroxidase 1 (VPO1) is a recently identified novel family member of peroxidases in cardiovascular system. As an enzyme that is downstream of NADPH oxidases (NOX), VPO1 functions to utilize NOX - derived hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl), a strong oxidant which is believed to greatly promote oxidative stress. Under multiple conditions, NOX is activated concomitantly with an increase in superoxide anion (O2(.-)) and H2O2 production. The latter is converted to HOCl by VPO1. In this process (O2(.-) → H2O2 → HOCl), the oxidant reactivities of reactive oxygen species (ROS) are significantly increased and therefore the oxidative stress is dramatically amplified. Several lines of evidence suggest that the NOX/VPO1 pathway - mediated oxidative stress plays an important role in myocardial ischemia-reperfusion injury, endothelial cell apoptosis and/or smooth muscle cell proliferation. In addition, VPO1 can be secreted into the extracellular space to participate in extracellular matrix formation, suggesting that VPO1 may also play a role in cardiovascular remodeling (such as fibrosis). This function is independent of the peroxidase activity of VPO1.

Vascular VPO1 expression is related to the endothelial dysfunction in spontaneously hypertensive rats

Reactive oxygen species (ROS) contributes to endothelial dysfunction that is involved in the pathogeneses of hypertension. Vascular peroxidase 1 (VPO1) can utilize ROS to catalyze peroxidative reactions, possibly enhancing endothelial dysfunction. This study is to identify VPO1s involvement in endothelial dysfunction and hypertension. Sixty-four spontaneously hypertensive rats (SHRs) and 64 age-matched, bodyweight controlled normotensive Wistar-Kyoto rats (WKYs) were randomly grouped and studied at the age of 5, 8, 13 and 20 weeks (16 animals, each). Blood pressure and vasodilator responses to acetylcholine in aortic rings were observed. The expressions of VPO1 and endothelial NO synthase (eNOS) in aortas were assessed by quantitative reverse transcription-PCR and western blotting analysis. Plasma concentrations of hydrogen peroxide (H2O2) and NO, NOX activity, hypochlorous acid (HOCl) production, and 3-nitrotyrosine content in aortic homogenates were also determined in this study. Along with the development of hypertension in SHR rats, VPO1 expression was up-regulated together with a significant increase in NOX activity, HOCl production, 3-nitrotyrosine content, and plasma H2O2 level compared with WKYs at 8, 13 and 20 weeks of age. In contrast, blood NO levels were decreased and aortic relaxation to acetylcholine was deteriorated in SHRs. The over-expression of VPO1 during the development of hypertension, accompanied by the endothelial dysfunction, the decreased NO levels, the elevated NOX and ROS activities, indicates a clear connection between VPO1 gene and hypertension. VPO1 may pathogenetically contribute to hypertension via signal pathways involving NOX-H2O2-VPO1-HOCl or JNK/p38 MAPK although further studies are needed to determine the precise mechanisms.