Role Of Vascular Peroxidase1 In Vascular Remodeling In Essential Hypertension

BACKGROUNDStudies have demonstrated that essential hypertension (EH) is a result of imbalance of oxidation and antioxidation. Under hypertensive pathological condition, reactive oxygen species (ROS) are increased and involved in cell damage through a variety of ways. The generation of ROS involves a variety of enzymes such as peroxidase, which catalyzes hydrogen peroxide (H2O2) into hypochlorous acid (HOCL). HOCL has greater toxicity, could directly injuries cells and participates in some signal pathway to affect cell fuction. Vascular peroxidase (VPO) is a newly discovered family member of peroxidases. VPO family including VPO1 and VPO2, both are expressed in the cardiovascular system. The expression of VPO1 play a dominant role in the vascular wall,because of its oxidizing properties and site specificity, we speculate that VPO1 may play an important role in regulating the structure and function of cardiovascular system.Yet there is no method available to measure the level of VPO1 directly. The Escherichia coli (E.coli) are the best common express host in prokaryotic expression. This study used a prokaryotic expression system to prepare VPO1 recombinant protein, establish indirect ELISA detection measurement of VPO1, and then investigated the relationship between VPO1 levels and EH.METHODSInduced with IPTG, the pQE30-VPO1 plasmid was purified in Ni-NTA affinity chromatography and renatured with step dialysis. With the purified protein as antigen, we established indirect ELISA detection measurement of VPO1. Blood was taken from 36 Chinese healthy subjects and 84 EH patients; Plasma was assayed for levels of VPOl.RESULTSThe expression of plasmid pQE30-VPO1 was constructed at 37℃,4 h with 1mol/L IPTG. The level of protein expression was 5%of the E.coli protein, the majority was inclusion body. The purification rate of recombinant protein was 90%, the solubility was 50%.Plasma VPO1 level was significantly higher in EH than healthy subjects (352.1±98.2 ng/ml, P< 0.01), there was no gender difference of VPO1 level.CONCLUSIONThe plasma level of VPO1 has a strong relationship with EH.BACKGROUNDThe development of EH and its complications are closely related to vascular remodeling. The mechanism of vascular remodeling in EH has not yet fully understood. Recent studies suggested that ROS play an important role in the development of vascular remodeling. The first part of our study confirmed that plasma VPO1 level was significantly higher in EH than that of healthy subjects. As the strong oxidizing properties of VPO1 which can catalyze H2O2 to generate HOC1 and other chemical substances, we speculated that VPOl may play a role in vascular remodeling in EH by mediating oxidative stress. In the present study, we combined EH patients and spontaneously hypertensive rats (SHR) to investigate the role of VPO1 in vascular remodeling.METHODS(1) 84 patients with essential hypertension were divided into two groups according to carotid arterial intima-media thickness (IMT) measured by carotid ultrasonography:41 patients with EH and IMT<0.9 mm and 43 patients with EH with carotid arterial IMT≥0.9 mm. Thirty-six healthy volunteers were selected as control group. Blood pressure (BP), height, weight, fasting blood sugar (FBS), triglyceride, cholesterol, high density lipoprotein-cholesterol (HDL-C), low lipoprotein-cholesterol (LDL-C), liver function, renal function were measured in all subjects. The levels of VPO1, H2O2 and catalase (CAT) activity were also measured.(2) 10 12-week-old male spontaneously hypertensive rats (SHR) served as SHR group, and 10 age and sex matched Wistar Kyoto rats (WKY) were selected as control group. All animals were feed for 20 weeks. Systolic blood pressure (SBP) of rats was measured before experiment and week interval. When the experiment was completed, the rats were anesthetized to cut out the thoracic aorta and mesenteric arteries. Slices of these arteries were stained with hematoxylin-eosin staining (HE) and masson staining. Media thickness (MT), lumen diameter (LD), MT/LD, mean nuclear area in artery media were assessed with the computer-assisted image analysis system. The expressions of VPO1 in the thoracic aorta wall and mesenteric arteries were measured with immunohistochemical staining, real-time PCR and western blot, respectively.RESULTS(1) The plasma level of VPO1 and H2O2 increased and the activity of CAT decreased in EH patients.(2) Compared with group EH1, the patients in group EH2 not only had higher Ep and SI, but also had higher VPO1 and H2O2 level, while the catalase activity decreased.(3) The level of SBP and ANGⅡwas significantly higher in SHR than that of WKY.(4) MT, LD, MT/LD and mean nuclear area of thoracic aorta and mesenteric arteries media of SHR group was significantly higher than WKY group.(5) Both VPO1 mRNA and protein expression in thoracic aorta of SHR group were much higher than WKY group; and the percentage of VPO1 staining positive area in thoracic aorta and mesenteric arteries in SHR were strikingly higher than WKY control group.CONCLUSIONRemodeling of carotid artery occurs in EH patients, which is characterized by IMT thickening. Remodeling of thoracic aorta and mesenteric arteries occurs in SHR, which is characterized by arterial media thickening; these phenomeneon of vascular remodeling in hypertension was associated with increased VPO1 level.BACKGROUNDOur previous study found that the increased level of VPO1 associated with the vascular remodeling under pathological state of hypertension. However, the mechanisms responsible for the effect of VPO1 on vascular remodeling remain unknown. NADPH oxidase enzyme is the major sourse of ROS in vascular wall and plays an important role in oxidative stress-mediated vascular smooth muscle cells (VSMC) proliferation, migration in the pathogenesis of vascular remodeling. Recent studies have shown that HOCL could activate the Extracellular signal-regulated kinase1/2 (ERK1/2) signaling pathway to induce cell proliferation. As a member of peroxide family, VPOl could catalyze H2O2 into HOCL. In the present study, therefore, we examined the effects of VPO1 on cell proliferation and its mechanisms.METHODSCultured human aorta vascular smooth muscle cells (HASMCs) were treated with ANGII, the proliferation activity, VPO1 expression, H2O2 and HOCL level were examined. The effect of apocynin, catalase and PD98059 on VPO1 expression and the proliferation activity of cells were observed. The effect of VPO1 RNA interference on ANGⅡ-induced cell proliferation was also observed. Cultured cells were treated with H2O2, VPO1 expression and HOCL level were examined.RESULTS(1) ANGⅡtreatment for 24 h increased the proliferation activity of cells, while the expression of VPO1 in cells and the levels of H2O2 and HOCL in the culture medium were increased.(2) Pretreatment with apocynin or catalase reduced the effects of ANGⅡon cell proliferation and VPO1 expression, while PD98059 only reduced the effects of ANG II on cell proliferation.(3) Interference of VPO1 shRNA significantly inhibited the proliferation activity of cells and the increased level of HOC1 by ANGⅡ.(4) H2O2 treatment for 24 h increased the expression of VPO1 in cells and the levels of HOC1 in the culture medium. Interference of VPO shRNA significantly inhibited the effect of H2O2.CONCLUSIONThe proliferation of vascular smooth muscle cells induced by ANGⅡis related to activation of the Nox/H2O2/VPO1/HOCL/ERK1/2 pathway.