Abstract:Aim To explore the predictive value of peripheral blood angiotensinⅡ (AngⅡ), ischemia-modified albumin (IMA) and C-reactive protein (CRP) for venous thromboembolism (VTE). Methods 170 patients with VTE who were treated in Shijiazhuang Peoples Hospital from June 2018 to January 2021 were selected as the study group, and 101 healthy subjects in the same period were selected as the control group. The levels of AngⅡ, IMA and CRP in the two groups were detected in strict accordance with the instructions of ELISA kit. Logistic regression analysis was used to analyze the correlation between AngⅡ, IMA and CRP and VTE. Results The levels of AngⅡ, IMA and CRP in the study group were significantly higher than those in the control group, and the level of D-dimer in the study group was significantly lower than that in the control group (P＜0.05). There was no significant difference in HDL, LDL and platelet count between the two groups (P＞0.05). The levels of triglyceride and fibrinogen in the study group were significantly higher than those in the control group (P＜0.05). Logistic regression analysis showed that AngⅡ, IMA and CRP were independent risk factors for VTE. In the ROC curve, the sensitivity and specificity of AngⅡ, IMA and CRP were 97.6% and 88.9% respectively. Conclusion The three indexes of AngⅡ, IMA and CRP in peripheral blood have good value in the prediction of VTE and are worthy of further promotion in clinic.

Abstract:Aim The purpose of this study was to observe the effect and mechanism of angiotensin(1-7)(Ang(1-7)) on angiotensin Ⅱ(AngⅡ)-induced senescence of human umbilical vein endothelial cell(HUVEC). MethodsHUVEC were cultured in vitro with DMEM high glucose medium containing 10%fetal bovine serum (FBS) for 48 hours and randomly divided into control group, Ang(1-7) group (1 μmol/L), AngⅡ group (1 μmol/L), and AngⅡ＋Ang(1-7) group. Cell senescence β-galactosidase(SA-β-Gal) staining kits were used to detect the number of senescent cell in each group (observed under an optical microscope); reactive oxygen species (ROS) levels were measured using reactive oxygen detection kits, and p53 and dynamin related protein-1(Drp1) expression in each group were detected by Western blot.Results Compared with the control group, both SA-β-Galpositive cell and ROS levels in the AngⅡ group were significantly increased(P＜0.001), and p53 and Drp1 protein expression in the cell were significantly increased (P＜0.01). Compared with AngⅡ group, SA-β-Galpositive cell(P＜0.01), ROS levels(P＜0.001), p53 and Drp1 protein expression(P＜0.05) were decreased in AngⅡ＋Ang(1-7) group. Conclusion Ang(1-7) may inhibit the ROS generation in HUVEC by affecting the p53/Drp1 pathway, and reverse the aging of HUVEC induced by AngⅡ.

Abstract:Aim To study the role and mechanism of dehydroepiandrosterone (DHEA) in the proliferation of vascular smooth muscle cells (VSMCs) induced by angiotensin Ⅱ (AngⅡ). Methods VSMCs were cultured with AngⅡ in vitro, then treated with DHEA. The effect of DHEA on the proliferation of VSMCs induced by AngⅡ was detected by MTS and cell counting. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the effects of DHEA on the expressions of proliferating cell nuclear antigen (PCNA), Krüppel-like factor 5 (KLF5) and inducible nitric oxide synthase (iNOS) in AngⅡ-induced VSMCs. The effect of DHEA on the concentration of peroxynitrite anion (ONOO－) in the medium of AngⅡ-induced VSMCs was examined by enzyme-linked immunosorbent assay (ELISA). Endogenous iNOS was knocked down by transfecting VSMCs with iNOS-specific siRNA (si-iNOS) or nonspecific siRNA (si-Ctrl), and then treated the cells with or without DHEA; Western blot was performed to examine the expression of PCNA. Results The results of MTS and cell counting showed that DHEA could significantly inhibit the proliferation of VSMCs induced by AngⅡ (P＜0.05). The results of qRT-PCR and Western blot showed that DHEA significantly inhibited the expressions of PCNA, KLF5 and iNOS in VSMCs induced by AngⅡ at the level of protein and mRNA (P＜0.05). ELISA results showed that DHEA could reduce the concentration of ONOO－ in culture medium of VSMCs treated with AngⅡ (P＜0.05). Western blot results suggested that DHEA had no effect on AngⅡ-induced PCNA protein expression after knocked down endogenous iNOS (P＞0.05). Conclusion DHEA may inhibit the proliferation of VSMCs induced by AngⅡ probably through down-regulation of KLF5 expression by inhibiting ONOO－ produced by iNOS.

Abstract:Aim To study the effect of A1166C polymorphism of angiotensin Ⅱ type 1 receptor gene (AGTR1) on arteriosclerosis. Methods Pulse wave velocity (PWV) was chosen as an index for evaluating arteriosclerosis. Topic-related English literatures were searched in PubMed, EMBASE, Cochrane Library and Web of Science. The retrieval time was from the establishment of the database to October 2018. Two authors independently selected appropriate observational studies based on the corresponding inclusion and exclusion criteria. Revman 5.2 software and STATA 11.0 software were used for Meta-analysis. Results A total of seven literatures (n＝1 687) was included in the Meta-analysis. The results showed that there was no significant correlation between AGTR1 A1166C polymorphism and PWV in general population and white population. In Asian population, the PWV of allele C carriers was significantly higher than that of AA genotype (AC＋CC vs AA:SMD＝0.3,5%CI 0.06-0.60, P＝0.02). Conclusions AGTR1 A1166C polymorphism may be associated with arteriosclerosis in Asians. Allele C may be a risk factor for PWV elevation in Asian population.

Abstract:Aim RNA interference was used to downregulate angiotensin converting enzyme (ACE) and angiotensinⅡtype 1 receptor (AT1R), and to observe its effects on blood pressure and brain tissue injury in spontaneously hypertensive rats (SHR). Methods SHR was randomly divided into 5 groups:blank control group, virus control group, Ad5-ACE-shRNA treatment group, Ad5-AT1R-shRNA treatment group, Ad5-ACE-AT1R-shRNA treatment group, and WKY normal blood pressure control group was set up. All rats in each group were injected 1 time in the first and seventeenth day of the experiment. On the third day after the first injection, enzyme-linked immunosorbent assay (ELISA) was used to detect the contents of ACE and Ang Ⅱ in rat brain, real-time fluorescent quantitative PCR was used to detect the expression of ACE mRNA and AT1R mRNA in brain tissue, Western blot was used to detect the expression of ACE and AT1R protein in brain tissue. At the end of the experiment, brain tissue was examined by light microscopy to observe the brain structure. Results On the third day after the first injection, the tail arterial pressure of Ad5-ACE-shRNA treatment group, Ad5-AT1R-shRNA treatment group and Ad5-ACE-AT1R-shRNA treatment group decreased by 25.2±5.3 mmHg, 23.5±4.8 mmHg and 27.1±6.4 mmHg, 24.4±5.6 mmHg, 23.6±4.9 mmHg and 26.3±6.9 mmHg respectively on the 14th day, and the blood pressure rose on the 17th day after Ad5-ACE-shRNA treatment and Ad5-AT1R-shRNA treatment after the second injection The tail arterial pressure of Ad5-ACE-AT1R-shRNA treatment group decreased again. On the 23rd day, the arterial pressure decreased by 40.2±4.5 mmHg, 37.8±3.9 mmHg and 43.7±7.3 mmHg respectively before the first injection. On the 37th day, The blood pressure rose to the level before the second injection on the 40th day. The blood pressure of the blank control group and the virus control group continued to increase by 26 mmHg, there was no significant difference between the two groups; the tail arterial pressure of the normal blood pressure control group had no obvious change. The expression of ACE mRNA in brain tissue of Ad5-ACE-shRNA treatment group and Ad5-ACE-AT1R-shRNA treatment group and the expression of AT1R mRNA in the brain tissue of Ad5-AT1R-shRNA treatment group and Ad5-ACE-AT1R-shRNA treatment group were significantly lower than those of blank control group and virus control group (P＜0.05), but there was no significant difference compared with normal blood pressure control group. The expression of AT1R protein in brain tissue of Ad5-ACE-shRNA treatment group, Ad5-AT1R-shRNA treatment group and Ad5-ACE-AT1R-shRNA treatment group was significantly lower than that of blank control group and virus control group (P＜0.05). The light microscope showed that the brain tissue structure of Ad5-ACE-shRNA treatment group, Ad5-AT1R-shRNA treatment group and Ad5-ACE-AT1R-shRNA treatment group was significantly improved.Conclusion RNA interference downregulated ACE and AT1R expression, reduced blood pressure in spontaneously hypertensive rats and improved brain injury, especially Ad5-ACE-AT1R-shRNA treatment had obvious effect and less side effect. RNA interference has a good application prospect for gene therapy of hypertension.

Abstract:Aim To investigate the effects of Ezrin gene knockout on calpain-1 activity and adenosine triphosphate binding cassette transporter A1(ABCA1) protein levels in RAW264.7 cells. Methods RAW264.7 cells transfected with small interfering RNA (siRNA) targeting Ezrin were subject to real time PCR and Western blot to detect the inhibition ratio. Added with angiotension Ⅱ(AngⅡ) or AngⅡ+ Ezrin siRNA, calpain-1 protein expression and activity were determined by using Western blot. Then, the expression of ABCA1 protein was examined by Western blot after the treatment with AngⅡ,Ezrin siRNA, calpain inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucine (ALLN). Results Ezrin mRNA and protein expression in Ezrin siRNA groups were inhibited(P<0.05). The calpain-1 expression were upregulated by AngⅡ treatment (4.92±0.23 vs 1.00±0.17, P<0.05), which were reversed by Ezrin siRNA (0.23±0.21 vs 4.92±0.23, P<0.05). Therefore, the ABCA1 protein expression was inhibited by addition of AngⅡ(0.167±0.055 vs 0.732±0.072), which were reversed by Ezrin siRNA (0.611±0.048 vs 0.167±0.055, P<0.05). There were no statistically significant differences at ABCA1 protein levels between AngⅡ+Ezrin siRNA group and AngⅡ+Ezrin siRNA+ALLN group. Conclusion Ezrin might play an important role in AngⅡ-induced ABCA1 protein attenuation via calpain-1 pathway.

Abstract:Aim To investigate the role of endogenous sulfur dioxide (SO2) in inhibiting cardiomyocyte autophagy in angiotensin Ⅱ (AngⅡ)-induced myocardial hypertrophic mice. Methods 16 healthy 9-week-old C57BL mice were randomly divided into wild type control group (WT Con group) and wild type＋AngⅡ group (WT AngⅡ group); 16 cardiac-specific aspartate aminotransferase 2 (AAT2) transgenic mice were randomly divided into AAT2 control group (AAT2 Con group) and AAT2＋AngⅡ group (AAT2 AngⅡ group); 8 mice in each group. The mouse was subcutaneously implanted with a capsule osmotic pressure pump pre-filled with normal saline or AngⅡ in the back for continuous administration for 4 weeks. Total heart weight/body weight (HW/BW) ratio was detected in 4 groups of mice. HE staining was used to observe the changes of myocardial cell structure. The expression of α-myosin heavy chain (α-MHC) was detected by immunohistochemistry staining. The content of SO2 in myocardial tissue was detected by high performance liquid chromatography. Western blot was used to detect the expressions of endogenous SO2 producing enzymes AAT1 and AAT2, cardiomyocyte phenotypic markers α-MHC and β-MHC as well as myocardial autophagy indicators Beclin-1, LC3, Atg4B and p62. Results Compared with WT Con group, the SO2 content was significantly reduced (P＜0.01) but the expression of AAT1 protein had no obvious changes (P＞0.05), the expression of AAT2 protein was significantly decreased (P＜0.05), in the myocardial tissue of the mice in WT AngⅡ group; HW/BW was increased significantly (P＜0.01), and myocardial fibers were thickened; Immunohistochemistry showed that the expression of α-MHC protein was decreased in cardiomyocyte cytoplasm; Western blot results showed that the expression of α-MHC protein in cardiomyocytes was significantly decreased (P＜0.01), the expression of β-MHC protein was significantly increased (P＜0.01), and cardiomyocyte autophagy was significantly increased which was demonstrated by the fact that the ratio of LC3Ⅱ/LC3Ⅰ was significantly increased, the expressions of Beclin-1 and Atg4B protein were significantly increased, and the expression of p62 protein was significantly reduced (all P＜0.01). However, compared with WT Con group, the SO2 content and AAT2 protein expression in AAT2 AngⅡ group were significantly increased (P＜0.01), AAT1 protein expression did not significantly change (P＞0.05), and the HW/BW ratio was significantly decreased (P＜0.05); The thickness of myocardial fibers was significantly reversed; The conversion of α-MHC protein to β-MHC protein was significantly reduced (P＜0.01), and the level of autophagy was significantly reduced in cardiomyocytes. Conclusion Endogenous SO2/AAT2 system can inhibit the cardiomyocyte autophagy and myocardial hypertrophy in the AngⅡ-treated mice.

Abstract:Aim To observe the relationship between the angiotensin Ⅱ Type 1 receptor autoantibody (AT1-AA) and the local inflammation of plaques in atherosclerotic animal model. Methods AT1-AA positive and negative sera were collected and purified from patients with hypertension complicated with acute coronary syndrome. Atherosclerosis models were established in 30 rabbits with hyperlipidemia induced by balloon injury. The animals were randomly divided into 6 groups:(1)control group; (2)low concentration AT1-AA [20 μg/(kg·d)] injection group (LA group); (3)high concentration AT1-AA [40 μg/(kg·d)] injection group (HA group); (4)losartan [20 mg/(kg·d)] lavage＋high concentration AT1-AA injection group (L＋HA group); (5)simvastatin [4 mg/(kg·d)] lavage＋high concentration AT1-AA injection group (S＋HA group); (6)7aa [1.5 mg/(kg·d)] lavage＋high concentration AT1-AA injection group (7aa＋HA group). Each group was treated differently. The abdominal aortas of rabbits were taken out and stained with HE.Image-Pro Express system was used to measure the percentage of atheromatous plaques in the area of lumen. The expressions of MMP-2 in aortic tissue were detected by western blot. Results The values of total cholesterol and low density lipoprotein cholesterol were obviously increased after four weeks. Excluding the control group, the levels of serum AT1-AA in the 8th and 10th week were significantly higher than those at the beginning in the other groups. In LA group and HA group, the relative plaque areas were 46.99%±13.06% and 66.11%±19.67%, respectively, which were significantly higher than those in control group (27.71%±7.46%), L＋HA group (34.27%±12.38%), S＋HA group (24.03%±8.56%) and 7aa＋HA group (28.54%±12.50%) (all P＜0.05). The expressions of MMP-2 in LA group and HA group were significantly higher than those in other groups (all P＜0.05). Conclusion AT1-AA can accelerate the atheromatous plaque formation in rabbit atherosclerosis model and promote the inflammatory reaction and cells proliferation in local plaque.

Abstract:Aim To investigated the role and mechanism of macrophage in endothelial dysfunction and cardiac hypertrophy in AngⅡ-induced hypertensive mice. Methods C57BL/6 mice were randomly divided into four groups:normal+PBS group, normal+clodronate liposome (CL) group, Ang Ⅱ+PBS group, Ang Ⅱ+CL group. PBS or CL was injected via tail vein,and Ang Ⅱ was delivered by implantation of osmotic mini-pump. The systolic blood pressure (SBP) was measured by tail-cuff method, SBP was measured at 3 time points:at baseline, 7 days and 14 days after AngⅡ infusion. HE staining was used to measure myocardial hypertrophy, endothelium dependent relaxation to acetylcholine in aortic rings was determined by organ chamber bath, the protein expression of p-eNOS, p-ERK1/2 , TNF-α, IL-1β, TGF-β1, fibronectin was determined by Western blot. Results Compared with the normal+PBS mice, AngⅡ+PBS significantly increased systolic blood pressure (44%,P<0.05), macrophage infiltration in myocardial tissue (54%, P<0.05), heart weight (29%, P<0.05) as well as single myocardial cell area (48%, P<0.05), impaired acetylcholine-induced endothelium dependent relaxation (Emax-35%, P<0.05). The treatment with CL significantly reduced SBP (-25.28%, P<0.05), the area of single myocardial cell (-38.83%, P<0.05), and improve acetylcholine-induced endothelium dependent relaxation (Emax 12.63%, P<0.05) in AngⅡ hypertensive mice. CL treatment also restored the expression of p-eNOS, p-ERK1/2, TNF-α, IL-1β, TGF-β1, fibronectin induced by AngⅡ (P<0.05). Conclusions The results demonstrate that CL protects against AngⅡ-induced endothelial dysfunction and myocardial damage and remodeling, the underlying mechanisms may involve reduction in myocardial macrophage infiltration and macrophage-derived cytokines.

Abstract:Aim To explore a disintergrin and metalloproteinase 17 (ADAM17) regulating the protective effect of calcitonin gene-related peptide (CGRP) against proliferation of vascular smooth muscle cell (VSMC) and its mechanism.Methods The rat thoracic aortic smooth muscle cell line (A10VSMC) was used as the object of study. RNA interference was adopted to reduce ADAM17 expression. Western blot and real-time quantitative PCR were used to detect the expression levels of protein and mRNA. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to evaluate cell viability of VSMC.Results CGRP could markedly attenuate angiotensin Ⅱ-induced VSMC proliferation. There were significant increases in expression levels of ADAM17 protein and mRNA in VSMC stimulated with angiotensin Ⅱ for 24 hours. Pretreatment with CGRP for 30 minutes significantly reduced the expression levels of ADAM17 protein and mRNA, whereas the effects could be reversed by CGRP receptor antagonist CGRP8-37. The results of RNA interference showed that ADAM17 siRNA significantly inhibitted angiotensin Ⅱ-mediated VSMC proliferation. Conclusion The reduction of ADAM17 expression may be the mechanism by which CGRP inhibits angiotensin Ⅱ-mediated VSMC proliferation.