Inflamm. Res. DOI 10.1007/s00011-015-0805-1
Inflammation Research
ORIGINAL RESEARCH PAPER
ACE2 and Ang-(1–7) protect endothelial cell function and prevent early atherosclerosis by inhibiting inflammatory response Yue-Hui Zhang • Yong-huan Zhang • Xue-Fei Dong • Qing-Qing Hao Xiao-Ming Zhou • Qing-Tao Yu • Shu-Ying Li • Xu Chen • Abdulai Fallah Tengbeh • Bo Dong • Yun Zhang
•
Received: 5 March 2014 / Revised: 12 January 2015 / Accepted: 6 February 2015 Ó Springer Basel 2015
Abstract Background Angiotensin-converting enzyme 2 (ACE2) is a counter-regulator against ACE by converting angiotensin II (Ang-II) to Ang-(1–7), but the effect of ACE2 and Ang(1–7) on endothelial cell function and atherosclerotic evolution is unknown. We hypothesized that ACE2 overexpression and Ang-(1–7) may protect endothelial cell function by counterregulation of angiotensin II signaling and inhibition of inflammatory response. Methods We used a recombinant adenovirus vector to locally overexpress ACE2 gene (Ad-ACE2) in human Responsible Editor: Ikuo Morita. Yue-Hui Zhang, Yong-huan Zhang, and Xue-Fei Dong have contributed equally to the work. Y.-H. Zhang Y. Zhang Q.-Q. Hao X.-M. Zhou Q.-T. Yu S.-Y. Li X. Chen A. F. Tengbeh B. Dong Department of Cardiology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China Y. Zhang Q.-Q. Hao X. Chen Department of Pathophysiology, Shandong University School of Medicine, Jinan, Shandong, China Y.-H. Zhang Department of Critical Care Medicine, The Affiliated Baoan Hospital of Nanfang Medical University, Shenzhen, China X.-F. Dong Clinical College of Hebei Medical University, Shijiazhuang, Hebei, China Y.-H. Zhang X.-F. Dong B. Dong (&) Y. Zhang The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong, China e-mail: [email protected]
endothelial cells in vitro and in apoE-deficient mice in vivo. The Ang II-induced MCP-1, VCAM-1 and E-selectin expression, endothelial cell migration and adhesion of human monocytic cells (U-937) to HUVECs by ACE2 gene transfer were evaluated in vitro. Accelerated atherosclerosis was studied in vivo, and atherosclerosis was induced in apoE-deficient mice which were divided randomly into four groups that received respectively a ACE2 gene transfer, Ad-ACE2, Ad-EGFP, Ad-ACE2 ? A779, an Ang-(1–7) receptor antagonist, control group. After a gene transfer for 4 weeks, atherosclerotic pathology was evaluated. Results ACE2 gene transfer not only promoted HUVECs migration, inhibited adhesion of monocyte to HUVECs and decreased Ang II-induced MCP-1, VCAM-1 and E-selectin protein production in vitro, but also decreased the level of MCP-1, VCAM-1 and interleukin 6 and inhibit atherosclerotic plaque evolution in vivo. Further, administration of A779 increased the level of MCP-1, VCAM-1 and interleukin 6 in vivo and led to further advancements in atherosclerotic extent. Conclusions ACE2 and Ang-(1–7) significantly inhibit early atherosclerotic lesion formation via protection of endothelial function and inhibition of inflammatory response. Keywords Atherosclerosis Angiotensin-converting enzyme 2 Ang-(1–7) Gene therapy Endothelial function Inflammation
Introduction A wealth of evidence indicates that the renin-angiotensin system (RAS) is a crucial regulator of cardiovascular
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homeostasis and has an important role in the pathogenesis of endothelial dysfunction and atherosclerosis [1, 2]. Previous studies have shown that adhesion molecular and inflammatory cytokines, for example, MCP-1, VCAM-1 and interleukin 6, are important molecules in the generation of endothelial dysfunction and induction of atherosclerosis. Some study showed that ox-LDL and Ang II produce MCP-1, VCAM-1 and interleukin 6 protein expression. Thus, to inhibit MCP-1, VCAM-1 and other inflammatory cytokines protein expression may helpful to prevent atherosclerotic progression. Most physiological and pathological effects of angiotensin (Ang) II are mediated via Ang II type 1 receptors (AT1R). Ang II binding to the AT1R activates diverse signaling cascades in the vasculature, such as mitogen-activated protein (MAP) kinases, protein tyrosine phosphatases, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase, and Aktmediated regulation of NO synthase, leading to contraction, endothelial dysfunction, and expression of proinflammatory mediators. Ang-(1–7) is mainly formed by the angiotensin-converting enzyme 2, which opposes many Ang II–stimulated actions. Ang-(1–7) causes vascular vasodilation, inhibits cell growth, protects endothelial cell activity and inhibits vascular pathology. Our study and other reports suggested that ACE2 and Ang-(1–7) may play an important role in the pathogenesis of atherosclerosis, diabetic cardiovascular complication and coronary heart disease [3, 4]. Sampaio et al. reported that Ang II-induced phosphorylation of the MAP kinases was counterregulated by Ang-(1–7) in endothelial cells. Su et al. [5] found that Ang-(1–7) reversibly inhibited Ang II-induced activation of ERK1/2, p38MAP kinase and attenuated Ang II–stimulated production of transforming growth factor beta 1 in proximal tubular cells. These studies indicated that Ang-(1–7) may play an important protective mechanism in the endothelium by counterregulation of potentially deleterious effects of Ang II [6]. An important member of the RAS, angiotensinconverting enzyme 2 (ACE2), which competes with ACE by converting vasoconstrictive Ang-II into vasodilative Ang-(1–7),was found to be expressed in cardiovascular disease and brain disease [7, 8]. Our recent study in a rabbit model of atherosclerosis demonstrated that ACE2 stabilized aortic plaques at a late stage [9]. However, little is known about whether ACE2 and Ang-(1–7) protect endothelial function In vitro and In vivo in early atherosclerosis. Thus, the current study was undertaken to test the hypothesis that ACE2 overexpression and Ang(1–7) may protect endothelial cell function by counterregulation of angiotensin II signaling and inhibition of inflammatory response. We used an ACE2 gene transfer in human endothelial cells in vitro and in a mouse model of hypercholesterolemia in vivo and the endothelial function,
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atherosclerosis extent and inflammatory response were evaluated.
Materials and methods Preparation of ACE2 Ad vector The murine ACE2 cDNA was amplified by RT-PCR from RNA of mouse kidney. The amplified product was first cloned into a pMD18-T vector (Invitrogen, Carlsbad, CA), then subcloned into pDC316 with the EcoRI and SalIsites. The ACE2 cDNA sequence in the pDC316-ACE2 plasmid was confirmed by sequencing. A recombinant Ad carrying the murine ACE2 (Ad-ACE2) was prepared by use of the AdMax system (Microbix Biosystems, Toronto, ON, Canada). HUVECs culture and gene transfer Human umbilical vein endothelial cells (HUVECs) were obtained from American type culture collection. HUVECs were cultured in M199 medium supplemented with 20 % fetal calf serum. To determine the efficacy of Ad-ACE2 gene transfer, HUVECs were cultured and plated at a density of 5 9 105 cells per well in 24-well tissue culture dishes and allowed to grow to confluence in M199 containing 10 % FCS under standard culture conditions. HUVECs were transfected In vitro with Ad-ACE2 or Ad-EGFP of 100 pfu/cell or PBS, ACE2 mRNA and protein expressions were detected on day 3 after transfection. HUVECs incubated with Ang-II for 24 h were divided into four groups that received Ad-ACE2, Ad-EGFP, AdACE2 ? A779 (1 lmol l-1), and control group, respectively. Ad-ACE2 (1 9 106 pfu) or Ad-EGFP (1 9 106 pfu) was transfected into cells which were harvested at 24 h after gene transfection for western blot analysis. HUVECs migration assay HUVECs migration was analyzed by a modified Boyden’s chamber method. HUVECs (2.5 9 104 per well) were transfected in vitro with Ad-ACE2 or Ad-EGFP at 100 pfu/cell. HUVECs were plated at a density of 2.5 9 104 cells per well in 16-well tissue culture dishes and transfected in vitro with Ad-ACE2 or Ad-EGFP at 100 pfu/cell. AngII was added to the culture medium at a final concentration of 10-6mol l-1 for 24 h. The HUVECs were added onto the upper surface and Medium 199 containing 5 % serum (HUVECs) was added into the lower chamber of the plate. The experiment lasted for 24 h at 37 °C. The migrated cells on the lower surface were fixed and stained with crystal violet. The number of migration cells was counted.
ACE2 and Ang-(1–7) protect endothelial cell function
Human monocytic cells (U-937) adhering to endothelial cells HCAECs were activated with AngII for 16 h and the HCAECs were divided into four groups, Ad-ACE2, AdEGFP, Ad-ACE2 ? A779 (1 lmol l-1) and control group. Ad-ACE2 (1 9 106 pfu) or Ad-EGFP (1 9 106 pfu) was transfected into the HCAECs for 24 h at 100 multiplicities of infection. The PKH26-labeled U-937 was added to each group for 45 min. The fluorescence intension was correlated with the percentage of the PKH26-labeled U-937 adhering to HUVECs and the adhesion occurrence of the PKH26-labeled U-937 to HUVECs was calculated by use of a 544-/590-nm filter set.
intraperitoneal injection of ketamine (150 mg kg-1) and xylazine (10 mg kg-1). Mini-osmotic pumps (Alzet Model 2004, Du rect Corp, Cupertino, CA, USA.) were implanted subcutaneously in mice to deliver A779 (200ng kg-1 min-1) for 28 days. Mice were maintained on the high-cholesterol diet for an additional 4 weeks. At the end of experiment, the mice were killed by CO2 asphyxiation. The aortas were removed after in situ perfusion with PBS followed by 4 % paraformaldehyde. The paraffin-embedded cross-sections of aortas were prepared for the histology and immunohistochemical analyses. All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document no. 55, 2001) and the Animal Care Committee of Shandong University.
ELISA Pathology and immunohistochemical analysis The level of MCP-1, VCAM-1 and E-selectin in HUVECs in vitro and the level of MCP-1, VCAM-1 and interleukin 6 (IL-6) in the serum of apoE-deficient mice were measured by a commercial ELISA kit (eBioscience, Hatfield, UK) according to the manufacturer’s instruction. Western blot
Paraffin-embedded arteries were dewaxed and rehydrated. Serial sections were stained with oil red O stain and macrophage expressions were identified using appropriate primary antibodies (Abcam Cambridge, USA). After incubation with biotinylated secondary antibody, the avidin– biotin complex immunoperoxidase technique was used.
ACE2 protein expression from membrane protein was detected in HUVECs. The samples (100 lg protein)were subjected to 14 % SDS-PAGE and then transferred to nitrocellulose membranes. After incubation in blocking solution (4 % nonfat milk), membranes were incubatedwith goat polyclonal antibody against ACE2 (Santa Cruz Biotechnology, USA) overnight at 4 °C. Membranes were washed and then incubated with 1:1000 dilution second antibodies for 1 h. Then the membrane was incubated with enhanced chemiluminescence reagent, and relative intensities of protein bands were analyzed by a MSF-300G Scanner (Microtek Lab, Nikon, Japan).
Measurement of total antioxidant capacity (TAC) activity and superoxide dismutase (SOD) activity
Animal model and gene transfer
Data were expressed as mean ± standard deviation (SD). SASStat View-J 5.0 (SAS Institute, Gary, IN, USA) was used for analysis. One-way ANOVA and Student’s t test were applied to analyze the difference among different animal and cell groups. A p value \ 0.05 was considered statistically significant.
Male apoE-deficient mice on a C57BL/6 background (10–12 weeks of age) were obtained from Beijing University Animal Research Center. The mice were kept in separate cages in a 12:12 h light:dark cycle, allowed to acclimatize for 1 week before the study started, and then fed with a high-fat diet (0.25 % cholesterol and 15 % cocoa butter) for another 8 weeks. The thirty-six mice were randomly divided into Ad-ACE2, Ad-EGFP, AdACE2 ? A779 and control groups (n = 9 in each group) that received a suspension of Ad-ACE2 (2.5 9 109pfu), Ad-EGFP (2.5 9 109 pfu), Ad-ACE2 ? A779 and no injection, respectively. The Ad-ACE2 and Ad-EGFP were injected via the tail vein. The mice were anaesthetised by
HUVECs were divided into four groups, Ad-ACE2, AdACE2 ?A779 or Ad-EGFP and control group at the dose of 100 pfu/cell. The HUVECs were stimulated with Ang II (10-6 mol l-1) for 24 h. Cell lysate samples were collected by fractionation. The total antioxidant capacity (TAC) activity and superoxide dismutase (SOD) activity were measured by assay kit (NJBC, Nanjing, China) as previously reported [10]. Statistical analysis
Results ACE2 gene and protein expression in HUVECs We first established the efficacy of the Ad-ACE2 infection in vitro. The ACE2 gene and ACE2 activity were evaluated by RT-PCR and a fluorogenic substrate method in HUVECs
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Y.-H. Zhang et al. Fig. 1 Expression of ACE2 mRNA, protein in HUVECs. a ACE2 mRNA expression in HUVECs detected by real-time RT-PCR, *p \ 0.01 versus control, Ad-EGFP. b ACE2 protein expression in HUVECs by western blot. c Quantitative analysis of results in b. *p \ 0.01 versus control, AdEGFP
transfected by Ad-ACE2. The expression of ACE2 mRNA was observed in Ad-ACE2, Ad-EGFP and Control groups. The ACE2 mRNA expression was significantly higher in Ad-ACE2 group than those in Ad-EGFP and control groups (Fig. 1a). ACE2 protein expression was also statistically higher in Ad-ACE2 group than those in Ad-EGFP and control groups (Fig. 1b, c). These data suggest that transfection with Ad-ACE2 was successful. Effects of ACE2 and Ang-(1–7) on HUVECs function We determined the effects of ACE2 on HUVECs migration. Our results showed that Ad-ACE2 promoted HUVECs migration in comparison with the Ad-EGFP or control group (Fig. 2a, b), and A779 partially reversed the effect of HUVECs migration by ACE2 overexpression; the results indicated that ACE2 may improve endothelial cell activity and Ang-(1–7) plays an important role in the protection of endothelial cell activity. The effects of ACE2 overexpression on the adhesion of U-937 to HUVECs were also evaluated, and the result showed that the adhesion of U-937 to HUVECs was significantly decreased in Ad-ACE2 group than those in AdEGFP or control group. Conversely, A779 partially reversed the effect of the adhesion of U-937 to HUVEC in Ad-ACE2 ? A779 group (Fig. 2c). Effects of ACE2 on MCP-1, VCAM-1 and E-selectin expression in HUVEC in vitro Overexpression of ACE2 inhibited the Ang II-induced expression of MCP-1, VCAM-1 and E-selectin expression
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in HUVECs. A779 treatment reversed the inhibitory effect of ACE2 overexpression (Fig. 3c–e, p \ 0.01, p \ 0.01, p \ 0.05). Effects of ACE2 on Ang II–stimulated TAC and SOD activity in HUVECs The results showed that the TAC and SOD activity were statistically higher in Ad-ACE2 group than those in AdEGFP (p \ 0.01) and control (p \ 0.01) groups (Fig. 3a, b). Conversely, with the administration of A779 in the AdACE2 ? A779 group, the TAC and SOD activity were decreased compared with those in the Ad-ACE2 group (p \ 0.05) (Fig. 3a, b). Effects of ACE2 and Ang-(1–7) on atherosclerotic lesions The oil red O-positive areas were remarkably reduced in Ad-ACE2 group in comparison with that in AdEGFP (p \ 0.01) and control (p \ 0.01) groups (Fig. 4a, b). It is noteworthy that after administration of A779, the oil red O-positive areas in Ad-ACE2 ? A779 group were significantly increased compared with that in the Ad-ACE2 group (p \ 0.01). The macrophage infiltration was statistically lower in the Ad-ACE2 group than that in the Ad-EGFP, and control groups (all p \ 0.01). In contrast, the macrophage infiltration was higher in Ad-ACE2 ? A779 group than that in AdACE2 group (p \ 0.05), indicating that administration of A779 increases the extent of macrophage infiltration (Fig. 4c).
ACE2 and Ang-(1–7) protect endothelial cell function Fig. 2 HUVECs migration assay and adhesion of U-937 cells to HUVECs. a HUVECs migration assay. b Quantitative evaluate of HUVECs migration. *p \ 0.01 versus Ad-EGFP or control group. §p \ 0.05 versus Ad-ACE2. c Effects of ACE2 on the adhesion of U-937 cells to HUVECs. *p \ 0.01 versus Ad-EGFP or control group. § p \ 0.05 versus Ad-ACE2
ACE2 gene transfer attenuated the level of MCP-1, VCAM-1 and interleukin 6 in serum in ApoE-deficient mice The result suggested that the levels of MCP-1, VCAM-1 and interleukin 6 were significantly lower in the Ad-ACE2 group than those in the Ad-EGFP and control (p \ 0.01, in all) groups (Fig. 5a–c). However, administration of A779 resulted in a significant increase in MCP-1, VCAM-1 and interleukin 6 in Ad-ACE2 ? A779 group compared with that in Ad-ACE2 group (p \ 0.01, p \ 0.01, p \ 0.05).
Discussion Our study founded that Ang II significantly induced endothelial dysfunction, including production of MCP-1, VCAM-1 and E-selectin and inhibition migration of endothelial cells and decreased antioxidant capacity. However, ACE2 gene transfer inhibited Ang II-induced production of MCP-1, VCAM-1 and E-selectin, increased antioxidant capacity and decreased the endothelial dysfunction in vitro. ACE2 gene significantly inhibited early atherosclerotic lesions in apoE-deficient mice and this effect was associated with decreased levels of MCP-1,
VCAM-1 and interleukin 6. A779 partially reversed the effect of HUVECs migration and increased the extent of atherosclerosis and inflammatory response by ACE2 overexpression. This study suggested that ACE2 gene transfer and Ang-(1–7) protect endothelial function, inhibit inflammatory response and have an important role in the pathogenesis of early stage of atherosclerosis. Endothelial cell function plays an important role in prevention of early atherosclerosis. In certain diseases, the production of ROS increased aberrantly and may exceed the capacity of cellular enzymatic and nonenzymatic antioxidants, and then contribute to trigger sustained endothelial activation. Endothelial cell activation and dysfunction is a prerequisite for vascular pathogenesis. It can lead to vascular vasoconstriction, inflammatory response, and promote atherosclerotic evolution [11]. Study demonstrated that RAS plays a crucial role in the pathogenesis of cardiovascular disease. Recent study found that intracellular Ang-II played an important role in diabetesinduced organ damage [12]. Singh et al. [13] demonstrated that intracellular Ang-II was produced in cardiac myocytes and fibroblasts in high glucose conditions, which up regulates TGF-b and collagen synthesis and contributes to extracellular matrix accumulation. Intracellular angiotensin II production is correlated with cardiomyocyte apoptosis,
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Fig. 3 Antioxidant ability, MCP-1, VCAM-1 and E-selectin protein expression in HUVECs. a T-AOC activity in HUVECs. *p \ 0.01, versus control, Ad-EGFP groups. §p \ 0.05 versus Ad-ACE2 group. b SOD activity in HUVECs. *p \ 0.01, versus control, Ad-EGFP groups, §p \ 0.05 versus Ad-ACE2 group. c E-selectin protein level in HUVECs. *p \ 0.01, versus control, Ad-EGFP and AdACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP
groups. d VCAM-1 protein level in HUVECs. *p \ 0.01, versus control, Ad-EGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. e MCP-1 protein level in HUVECs. *p \ 0.01, versus control, Ad-EGFP. §p \ 0.05 versus AdACE2 ? A779 group. p \ 0.01, versus control and Ad-EGFP groups
Fig. 4 Pathological staining of the atherosclerotic lesions. a Oil red O-positive atherosclerotic lesion (upper) and macrophage expression (lower) in four groups (original magnification 9 400). b Quantification of the oil red O-positive staining area. *p \ 0.01, versus control, AdEGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. c Quantification of the macrophage expression staining area. *p \ 0.01, versus control, Ad-EGFP. §p \ 0.05 versus Ad-ACE2 ? A779 group. p \ 0.01, versus control and Ad-EGFP groups
oxidative stress, and cardiac fibrosis. Study also found that Ang-II enhances the uptake and oxidation of LDL through LOX-1 by endothelial cells, macrophages and SMCs and
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also induces endothelial cell injury and foam cell formation. Ang-II also produces MCP-1, VCAM-1, E-selectin and interleukin 6 expression. Study showed that adhesion
ACE2 and Ang-(1–7) protect endothelial cell function
Fig. 5 Level of MCP-1, VCAM-1 and Interleukin 6 in serum in male apoE-deficient mice. a MCP-1 level in four groups. *p \ 0.01, versus control, Ad-EGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. b VCAM-1 level in four groups. *p \ 0.01, versus control, Ad-EGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. c Interleukin 6 level in four groups. *p \ 0.01, versus control, Ad-EGFP groups. § p \ 0.05, versus Ad-ACE2 ? A779 group. p \ 0.01, versus control and Ad-EGFP groups
molecular and inflammatory cytokines take part in various steps involved in the pathogenesis of atherosclerosis, and the inhibition of adhesion molecular and inflammatory cytokines expression has been shown to protect endothelial cell activity and prevent progression of atherosclerosis [14, 15]. Kobat et al. [16] found that the levels of soluble LOX1 were statistically higher in patients with proximal/middle segment of the LAD lesions than those with distal segments of the LAD lesions in stable coronary artery disease, the result suggested that soluble LOX-1 was a novel biochemical marker and may provide important clue in the treatment of coronary artery disease. Some study showed that Ang II induces expression of the VCAM-1 and ICAM1 in HUVECs and takes part in the pathogenesis of atherosclerosis [17]. Studies also demonstrated that Ang II is an important stimulator of p38MAP kinase, ERK1/2 kinase. These kinases play important role in Ang II signaling and vascular function, including cell growth, migration, inflammation. Recent study showed that Ang(1–7) has protective effects in the cardiovascular and kidney diseases through counterbalancing the physiological actions of Ang II [18–21]. Lovren et al. [21] found that
ACE2 confers endothelial function and limits aberrant vascular responses. Our previous study demonstrated that ACE2 increases the stability of atherosclerotic plaque via decreased Ang-II and increased Ang-(1–7) level [9]. Previous study reported that Ang-(1–7) reduced smooth muscle growth and the neointima/media thickness ratio in balloon-injured carotid artery after arterial stenting in rats. Tesanovic et al. found that Ang-(1–7) has both vasoprotection and anti-atherosclerotic effects. The mechanisms include the restoration of nitric oxide bioavailability and the involution in the Mas receptor signaling pathway [22]. Study showed that Ang-(1–7) protects endothelial cell function directly and indirectly. Ang-(1–7) stimulates NO release and causes vasodilatation via Akt-dependent pathways [23]. Another studies found that Ang-(1–7) inhibits Ang II–mediated phosphorylation of p38, ERK MAP kinase, thereby resulting in negative modulation on Ang II effect. Ang-(1–7) antagonizes Ang II-induced activation of protein kinase C, p38MAP and ERK1/2 kinase in vascular smooth muscle cells and human endothelial cells. In the present study, ACE2 gene transfer promoted HUVECs migration and inhibited adhesion of monocyte to HUVECs. In the meanwhile, ACE2 overexpression not only inhibited Ang II-induced MCP-1, VCAM-1 and E-selectin protein production in vitro, but also decreased the level of MCP-1, VCAM-1 and interleukin 6 and inhibited atherosclerotic plaque evolution in vivo. Furthermore, administration of A779 increased the level of MCP-1,VCAM-1 and interleukin 6 in vivo and led to further advancements in atherosclerotic extent. These results indicated that ACE2 effectively improves endothelial cell function, inhibits inflammatory response and Ang-(1–7) may play an important role during this process. The cellular responses in the vasculature including contraction, growth, and inflammation by Ang-II are mediated via generation of reactive oxygen species (ROS). Much evidence demonstrated that vascular endothelial cell and smooth muscle cell NAD(P)H oxidase is a major source of vascular ROS. Both decreased ability of antioxidants and increased production of oxidants play an important roles in the pathogenesis of atherosclerosis. Touyz et al. and Sampaio et al. demonstrated that Ang II promoted ROS production in VSMC and endothelial cell. Our in vitro studies showed that Ang II decreased the TAC and SOD activity. Conversely, ACE2 gene transfer increased the antioxidant ability as manifested with the increased TAC and SOD activity, whereas administration of A779 resulted in decreased antioxidant ability. Theses results indicated that ACE2 gene transfer increased the ability of antioxidant, indicating that ACE2 represents a protective mechanism in the endothelium by increasing ability of antioxidant. Ant-(1–7) plays a crucial role in counterbalance deleterious effects induced by Ang II.
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In summary, our study demonstrated that ACE2 overexpression and Ang-(1–7) protect endothelial cell function and inhibit atherosclerotic evolution. The mechanism of protecting endothelial cell function and anti-atherosclerotic effect of ACE2 and Ang-(1–7) resulted from counter regulation of Ang II signaling, inhibition of inflammatory response and increasing the ability of antioxidant. Acknowledgments We would like to express our thanks to the teachers at the Key Laboratory of Cardiovascular Remodeling and Function Research, Shandong University. This study was supported by the National 973 Basic Research Program of China (No. 2013CB530700), the National Natural Science Foundation of China (No. 81170207) and the Program of State Chinese Medicine Administration Bureau (No. JDZX2012113).
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15. Conflict of interest work.
The authors report no conflicts of interest in this
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Inflammation Research
ORIGINAL RESEARCH PAPER
ACE2 and Ang-(1–7) protect endothelial cell function and prevent early atherosclerosis by inhibiting inflammatory response Yue-Hui Zhang • Yong-huan Zhang • Xue-Fei Dong • Qing-Qing Hao Xiao-Ming Zhou • Qing-Tao Yu • Shu-Ying Li • Xu Chen • Abdulai Fallah Tengbeh • Bo Dong • Yun Zhang
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Received: 5 March 2014 / Revised: 12 January 2015 / Accepted: 6 February 2015 Ó Springer Basel 2015
Abstract Background Angiotensin-converting enzyme 2 (ACE2) is a counter-regulator against ACE by converting angiotensin II (Ang-II) to Ang-(1–7), but the effect of ACE2 and Ang(1–7) on endothelial cell function and atherosclerotic evolution is unknown. We hypothesized that ACE2 overexpression and Ang-(1–7) may protect endothelial cell function by counterregulation of angiotensin II signaling and inhibition of inflammatory response. Methods We used a recombinant adenovirus vector to locally overexpress ACE2 gene (Ad-ACE2) in human Responsible Editor: Ikuo Morita. Yue-Hui Zhang, Yong-huan Zhang, and Xue-Fei Dong have contributed equally to the work. Y.-H. Zhang Y. Zhang Q.-Q. Hao X.-M. Zhou Q.-T. Yu S.-Y. Li X. Chen A. F. Tengbeh B. Dong Department of Cardiology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China Y. Zhang Q.-Q. Hao X. Chen Department of Pathophysiology, Shandong University School of Medicine, Jinan, Shandong, China Y.-H. Zhang Department of Critical Care Medicine, The Affiliated Baoan Hospital of Nanfang Medical University, Shenzhen, China X.-F. Dong Clinical College of Hebei Medical University, Shijiazhuang, Hebei, China Y.-H. Zhang X.-F. Dong B. Dong (&) Y. Zhang The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong, China e-mail: [email protected]
endothelial cells in vitro and in apoE-deficient mice in vivo. The Ang II-induced MCP-1, VCAM-1 and E-selectin expression, endothelial cell migration and adhesion of human monocytic cells (U-937) to HUVECs by ACE2 gene transfer were evaluated in vitro. Accelerated atherosclerosis was studied in vivo, and atherosclerosis was induced in apoE-deficient mice which were divided randomly into four groups that received respectively a ACE2 gene transfer, Ad-ACE2, Ad-EGFP, Ad-ACE2 ? A779, an Ang-(1–7) receptor antagonist, control group. After a gene transfer for 4 weeks, atherosclerotic pathology was evaluated. Results ACE2 gene transfer not only promoted HUVECs migration, inhibited adhesion of monocyte to HUVECs and decreased Ang II-induced MCP-1, VCAM-1 and E-selectin protein production in vitro, but also decreased the level of MCP-1, VCAM-1 and interleukin 6 and inhibit atherosclerotic plaque evolution in vivo. Further, administration of A779 increased the level of MCP-1, VCAM-1 and interleukin 6 in vivo and led to further advancements in atherosclerotic extent. Conclusions ACE2 and Ang-(1–7) significantly inhibit early atherosclerotic lesion formation via protection of endothelial function and inhibition of inflammatory response. Keywords Atherosclerosis Angiotensin-converting enzyme 2 Ang-(1–7) Gene therapy Endothelial function Inflammation
Introduction A wealth of evidence indicates that the renin-angiotensin system (RAS) is a crucial regulator of cardiovascular
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homeostasis and has an important role in the pathogenesis of endothelial dysfunction and atherosclerosis [1, 2]. Previous studies have shown that adhesion molecular and inflammatory cytokines, for example, MCP-1, VCAM-1 and interleukin 6, are important molecules in the generation of endothelial dysfunction and induction of atherosclerosis. Some study showed that ox-LDL and Ang II produce MCP-1, VCAM-1 and interleukin 6 protein expression. Thus, to inhibit MCP-1, VCAM-1 and other inflammatory cytokines protein expression may helpful to prevent atherosclerotic progression. Most physiological and pathological effects of angiotensin (Ang) II are mediated via Ang II type 1 receptors (AT1R). Ang II binding to the AT1R activates diverse signaling cascades in the vasculature, such as mitogen-activated protein (MAP) kinases, protein tyrosine phosphatases, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase, and Aktmediated regulation of NO synthase, leading to contraction, endothelial dysfunction, and expression of proinflammatory mediators. Ang-(1–7) is mainly formed by the angiotensin-converting enzyme 2, which opposes many Ang II–stimulated actions. Ang-(1–7) causes vascular vasodilation, inhibits cell growth, protects endothelial cell activity and inhibits vascular pathology. Our study and other reports suggested that ACE2 and Ang-(1–7) may play an important role in the pathogenesis of atherosclerosis, diabetic cardiovascular complication and coronary heart disease [3, 4]. Sampaio et al. reported that Ang II-induced phosphorylation of the MAP kinases was counterregulated by Ang-(1–7) in endothelial cells. Su et al. [5] found that Ang-(1–7) reversibly inhibited Ang II-induced activation of ERK1/2, p38MAP kinase and attenuated Ang II–stimulated production of transforming growth factor beta 1 in proximal tubular cells. These studies indicated that Ang-(1–7) may play an important protective mechanism in the endothelium by counterregulation of potentially deleterious effects of Ang II [6]. An important member of the RAS, angiotensinconverting enzyme 2 (ACE2), which competes with ACE by converting vasoconstrictive Ang-II into vasodilative Ang-(1–7),was found to be expressed in cardiovascular disease and brain disease [7, 8]. Our recent study in a rabbit model of atherosclerosis demonstrated that ACE2 stabilized aortic plaques at a late stage [9]. However, little is known about whether ACE2 and Ang-(1–7) protect endothelial function In vitro and In vivo in early atherosclerosis. Thus, the current study was undertaken to test the hypothesis that ACE2 overexpression and Ang(1–7) may protect endothelial cell function by counterregulation of angiotensin II signaling and inhibition of inflammatory response. We used an ACE2 gene transfer in human endothelial cells in vitro and in a mouse model of hypercholesterolemia in vivo and the endothelial function,
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atherosclerosis extent and inflammatory response were evaluated.
Materials and methods Preparation of ACE2 Ad vector The murine ACE2 cDNA was amplified by RT-PCR from RNA of mouse kidney. The amplified product was first cloned into a pMD18-T vector (Invitrogen, Carlsbad, CA), then subcloned into pDC316 with the EcoRI and SalIsites. The ACE2 cDNA sequence in the pDC316-ACE2 plasmid was confirmed by sequencing. A recombinant Ad carrying the murine ACE2 (Ad-ACE2) was prepared by use of the AdMax system (Microbix Biosystems, Toronto, ON, Canada). HUVECs culture and gene transfer Human umbilical vein endothelial cells (HUVECs) were obtained from American type culture collection. HUVECs were cultured in M199 medium supplemented with 20 % fetal calf serum. To determine the efficacy of Ad-ACE2 gene transfer, HUVECs were cultured and plated at a density of 5 9 105 cells per well in 24-well tissue culture dishes and allowed to grow to confluence in M199 containing 10 % FCS under standard culture conditions. HUVECs were transfected In vitro with Ad-ACE2 or Ad-EGFP of 100 pfu/cell or PBS, ACE2 mRNA and protein expressions were detected on day 3 after transfection. HUVECs incubated with Ang-II for 24 h were divided into four groups that received Ad-ACE2, Ad-EGFP, AdACE2 ? A779 (1 lmol l-1), and control group, respectively. Ad-ACE2 (1 9 106 pfu) or Ad-EGFP (1 9 106 pfu) was transfected into cells which were harvested at 24 h after gene transfection for western blot analysis. HUVECs migration assay HUVECs migration was analyzed by a modified Boyden’s chamber method. HUVECs (2.5 9 104 per well) were transfected in vitro with Ad-ACE2 or Ad-EGFP at 100 pfu/cell. HUVECs were plated at a density of 2.5 9 104 cells per well in 16-well tissue culture dishes and transfected in vitro with Ad-ACE2 or Ad-EGFP at 100 pfu/cell. AngII was added to the culture medium at a final concentration of 10-6mol l-1 for 24 h. The HUVECs were added onto the upper surface and Medium 199 containing 5 % serum (HUVECs) was added into the lower chamber of the plate. The experiment lasted for 24 h at 37 °C. The migrated cells on the lower surface were fixed and stained with crystal violet. The number of migration cells was counted.
ACE2 and Ang-(1–7) protect endothelial cell function
Human monocytic cells (U-937) adhering to endothelial cells HCAECs were activated with AngII for 16 h and the HCAECs were divided into four groups, Ad-ACE2, AdEGFP, Ad-ACE2 ? A779 (1 lmol l-1) and control group. Ad-ACE2 (1 9 106 pfu) or Ad-EGFP (1 9 106 pfu) was transfected into the HCAECs for 24 h at 100 multiplicities of infection. The PKH26-labeled U-937 was added to each group for 45 min. The fluorescence intension was correlated with the percentage of the PKH26-labeled U-937 adhering to HUVECs and the adhesion occurrence of the PKH26-labeled U-937 to HUVECs was calculated by use of a 544-/590-nm filter set.
intraperitoneal injection of ketamine (150 mg kg-1) and xylazine (10 mg kg-1). Mini-osmotic pumps (Alzet Model 2004, Du rect Corp, Cupertino, CA, USA.) were implanted subcutaneously in mice to deliver A779 (200ng kg-1 min-1) for 28 days. Mice were maintained on the high-cholesterol diet for an additional 4 weeks. At the end of experiment, the mice were killed by CO2 asphyxiation. The aortas were removed after in situ perfusion with PBS followed by 4 % paraformaldehyde. The paraffin-embedded cross-sections of aortas were prepared for the histology and immunohistochemical analyses. All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document no. 55, 2001) and the Animal Care Committee of Shandong University.
ELISA Pathology and immunohistochemical analysis The level of MCP-1, VCAM-1 and E-selectin in HUVECs in vitro and the level of MCP-1, VCAM-1 and interleukin 6 (IL-6) in the serum of apoE-deficient mice were measured by a commercial ELISA kit (eBioscience, Hatfield, UK) according to the manufacturer’s instruction. Western blot
Paraffin-embedded arteries were dewaxed and rehydrated. Serial sections were stained with oil red O stain and macrophage expressions were identified using appropriate primary antibodies (Abcam Cambridge, USA). After incubation with biotinylated secondary antibody, the avidin– biotin complex immunoperoxidase technique was used.
ACE2 protein expression from membrane protein was detected in HUVECs. The samples (100 lg protein)were subjected to 14 % SDS-PAGE and then transferred to nitrocellulose membranes. After incubation in blocking solution (4 % nonfat milk), membranes were incubatedwith goat polyclonal antibody against ACE2 (Santa Cruz Biotechnology, USA) overnight at 4 °C. Membranes were washed and then incubated with 1:1000 dilution second antibodies for 1 h. Then the membrane was incubated with enhanced chemiluminescence reagent, and relative intensities of protein bands were analyzed by a MSF-300G Scanner (Microtek Lab, Nikon, Japan).
Measurement of total antioxidant capacity (TAC) activity and superoxide dismutase (SOD) activity
Animal model and gene transfer
Data were expressed as mean ± standard deviation (SD). SASStat View-J 5.0 (SAS Institute, Gary, IN, USA) was used for analysis. One-way ANOVA and Student’s t test were applied to analyze the difference among different animal and cell groups. A p value \ 0.05 was considered statistically significant.
Male apoE-deficient mice on a C57BL/6 background (10–12 weeks of age) were obtained from Beijing University Animal Research Center. The mice were kept in separate cages in a 12:12 h light:dark cycle, allowed to acclimatize for 1 week before the study started, and then fed with a high-fat diet (0.25 % cholesterol and 15 % cocoa butter) for another 8 weeks. The thirty-six mice were randomly divided into Ad-ACE2, Ad-EGFP, AdACE2 ? A779 and control groups (n = 9 in each group) that received a suspension of Ad-ACE2 (2.5 9 109pfu), Ad-EGFP (2.5 9 109 pfu), Ad-ACE2 ? A779 and no injection, respectively. The Ad-ACE2 and Ad-EGFP were injected via the tail vein. The mice were anaesthetised by
HUVECs were divided into four groups, Ad-ACE2, AdACE2 ?A779 or Ad-EGFP and control group at the dose of 100 pfu/cell. The HUVECs were stimulated with Ang II (10-6 mol l-1) for 24 h. Cell lysate samples were collected by fractionation. The total antioxidant capacity (TAC) activity and superoxide dismutase (SOD) activity were measured by assay kit (NJBC, Nanjing, China) as previously reported [10]. Statistical analysis
Results ACE2 gene and protein expression in HUVECs We first established the efficacy of the Ad-ACE2 infection in vitro. The ACE2 gene and ACE2 activity were evaluated by RT-PCR and a fluorogenic substrate method in HUVECs
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Y.-H. Zhang et al. Fig. 1 Expression of ACE2 mRNA, protein in HUVECs. a ACE2 mRNA expression in HUVECs detected by real-time RT-PCR, *p \ 0.01 versus control, Ad-EGFP. b ACE2 protein expression in HUVECs by western blot. c Quantitative analysis of results in b. *p \ 0.01 versus control, AdEGFP
transfected by Ad-ACE2. The expression of ACE2 mRNA was observed in Ad-ACE2, Ad-EGFP and Control groups. The ACE2 mRNA expression was significantly higher in Ad-ACE2 group than those in Ad-EGFP and control groups (Fig. 1a). ACE2 protein expression was also statistically higher in Ad-ACE2 group than those in Ad-EGFP and control groups (Fig. 1b, c). These data suggest that transfection with Ad-ACE2 was successful. Effects of ACE2 and Ang-(1–7) on HUVECs function We determined the effects of ACE2 on HUVECs migration. Our results showed that Ad-ACE2 promoted HUVECs migration in comparison with the Ad-EGFP or control group (Fig. 2a, b), and A779 partially reversed the effect of HUVECs migration by ACE2 overexpression; the results indicated that ACE2 may improve endothelial cell activity and Ang-(1–7) plays an important role in the protection of endothelial cell activity. The effects of ACE2 overexpression on the adhesion of U-937 to HUVECs were also evaluated, and the result showed that the adhesion of U-937 to HUVECs was significantly decreased in Ad-ACE2 group than those in AdEGFP or control group. Conversely, A779 partially reversed the effect of the adhesion of U-937 to HUVEC in Ad-ACE2 ? A779 group (Fig. 2c). Effects of ACE2 on MCP-1, VCAM-1 and E-selectin expression in HUVEC in vitro Overexpression of ACE2 inhibited the Ang II-induced expression of MCP-1, VCAM-1 and E-selectin expression
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in HUVECs. A779 treatment reversed the inhibitory effect of ACE2 overexpression (Fig. 3c–e, p \ 0.01, p \ 0.01, p \ 0.05). Effects of ACE2 on Ang II–stimulated TAC and SOD activity in HUVECs The results showed that the TAC and SOD activity were statistically higher in Ad-ACE2 group than those in AdEGFP (p \ 0.01) and control (p \ 0.01) groups (Fig. 3a, b). Conversely, with the administration of A779 in the AdACE2 ? A779 group, the TAC and SOD activity were decreased compared with those in the Ad-ACE2 group (p \ 0.05) (Fig. 3a, b). Effects of ACE2 and Ang-(1–7) on atherosclerotic lesions The oil red O-positive areas were remarkably reduced in Ad-ACE2 group in comparison with that in AdEGFP (p \ 0.01) and control (p \ 0.01) groups (Fig. 4a, b). It is noteworthy that after administration of A779, the oil red O-positive areas in Ad-ACE2 ? A779 group were significantly increased compared with that in the Ad-ACE2 group (p \ 0.01). The macrophage infiltration was statistically lower in the Ad-ACE2 group than that in the Ad-EGFP, and control groups (all p \ 0.01). In contrast, the macrophage infiltration was higher in Ad-ACE2 ? A779 group than that in AdACE2 group (p \ 0.05), indicating that administration of A779 increases the extent of macrophage infiltration (Fig. 4c).
ACE2 and Ang-(1–7) protect endothelial cell function Fig. 2 HUVECs migration assay and adhesion of U-937 cells to HUVECs. a HUVECs migration assay. b Quantitative evaluate of HUVECs migration. *p \ 0.01 versus Ad-EGFP or control group. §p \ 0.05 versus Ad-ACE2. c Effects of ACE2 on the adhesion of U-937 cells to HUVECs. *p \ 0.01 versus Ad-EGFP or control group. § p \ 0.05 versus Ad-ACE2
ACE2 gene transfer attenuated the level of MCP-1, VCAM-1 and interleukin 6 in serum in ApoE-deficient mice The result suggested that the levels of MCP-1, VCAM-1 and interleukin 6 were significantly lower in the Ad-ACE2 group than those in the Ad-EGFP and control (p \ 0.01, in all) groups (Fig. 5a–c). However, administration of A779 resulted in a significant increase in MCP-1, VCAM-1 and interleukin 6 in Ad-ACE2 ? A779 group compared with that in Ad-ACE2 group (p \ 0.01, p \ 0.01, p \ 0.05).
Discussion Our study founded that Ang II significantly induced endothelial dysfunction, including production of MCP-1, VCAM-1 and E-selectin and inhibition migration of endothelial cells and decreased antioxidant capacity. However, ACE2 gene transfer inhibited Ang II-induced production of MCP-1, VCAM-1 and E-selectin, increased antioxidant capacity and decreased the endothelial dysfunction in vitro. ACE2 gene significantly inhibited early atherosclerotic lesions in apoE-deficient mice and this effect was associated with decreased levels of MCP-1,
VCAM-1 and interleukin 6. A779 partially reversed the effect of HUVECs migration and increased the extent of atherosclerosis and inflammatory response by ACE2 overexpression. This study suggested that ACE2 gene transfer and Ang-(1–7) protect endothelial function, inhibit inflammatory response and have an important role in the pathogenesis of early stage of atherosclerosis. Endothelial cell function plays an important role in prevention of early atherosclerosis. In certain diseases, the production of ROS increased aberrantly and may exceed the capacity of cellular enzymatic and nonenzymatic antioxidants, and then contribute to trigger sustained endothelial activation. Endothelial cell activation and dysfunction is a prerequisite for vascular pathogenesis. It can lead to vascular vasoconstriction, inflammatory response, and promote atherosclerotic evolution [11]. Study demonstrated that RAS plays a crucial role in the pathogenesis of cardiovascular disease. Recent study found that intracellular Ang-II played an important role in diabetesinduced organ damage [12]. Singh et al. [13] demonstrated that intracellular Ang-II was produced in cardiac myocytes and fibroblasts in high glucose conditions, which up regulates TGF-b and collagen synthesis and contributes to extracellular matrix accumulation. Intracellular angiotensin II production is correlated with cardiomyocyte apoptosis,
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Fig. 3 Antioxidant ability, MCP-1, VCAM-1 and E-selectin protein expression in HUVECs. a T-AOC activity in HUVECs. *p \ 0.01, versus control, Ad-EGFP groups. §p \ 0.05 versus Ad-ACE2 group. b SOD activity in HUVECs. *p \ 0.01, versus control, Ad-EGFP groups, §p \ 0.05 versus Ad-ACE2 group. c E-selectin protein level in HUVECs. *p \ 0.01, versus control, Ad-EGFP and AdACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP
groups. d VCAM-1 protein level in HUVECs. *p \ 0.01, versus control, Ad-EGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. e MCP-1 protein level in HUVECs. *p \ 0.01, versus control, Ad-EGFP. §p \ 0.05 versus AdACE2 ? A779 group. p \ 0.01, versus control and Ad-EGFP groups
Fig. 4 Pathological staining of the atherosclerotic lesions. a Oil red O-positive atherosclerotic lesion (upper) and macrophage expression (lower) in four groups (original magnification 9 400). b Quantification of the oil red O-positive staining area. *p \ 0.01, versus control, AdEGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. c Quantification of the macrophage expression staining area. *p \ 0.01, versus control, Ad-EGFP. §p \ 0.05 versus Ad-ACE2 ? A779 group. p \ 0.01, versus control and Ad-EGFP groups
oxidative stress, and cardiac fibrosis. Study also found that Ang-II enhances the uptake and oxidation of LDL through LOX-1 by endothelial cells, macrophages and SMCs and
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also induces endothelial cell injury and foam cell formation. Ang-II also produces MCP-1, VCAM-1, E-selectin and interleukin 6 expression. Study showed that adhesion
ACE2 and Ang-(1–7) protect endothelial cell function
Fig. 5 Level of MCP-1, VCAM-1 and Interleukin 6 in serum in male apoE-deficient mice. a MCP-1 level in four groups. *p \ 0.01, versus control, Ad-EGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. b VCAM-1 level in four groups. *p \ 0.01, versus control, Ad-EGFP and Ad-ACE2 ? A779 groups. p \ 0.01, versus control and Ad-EGFP groups. c Interleukin 6 level in four groups. *p \ 0.01, versus control, Ad-EGFP groups. § p \ 0.05, versus Ad-ACE2 ? A779 group. p \ 0.01, versus control and Ad-EGFP groups
molecular and inflammatory cytokines take part in various steps involved in the pathogenesis of atherosclerosis, and the inhibition of adhesion molecular and inflammatory cytokines expression has been shown to protect endothelial cell activity and prevent progression of atherosclerosis [14, 15]. Kobat et al. [16] found that the levels of soluble LOX1 were statistically higher in patients with proximal/middle segment of the LAD lesions than those with distal segments of the LAD lesions in stable coronary artery disease, the result suggested that soluble LOX-1 was a novel biochemical marker and may provide important clue in the treatment of coronary artery disease. Some study showed that Ang II induces expression of the VCAM-1 and ICAM1 in HUVECs and takes part in the pathogenesis of atherosclerosis [17]. Studies also demonstrated that Ang II is an important stimulator of p38MAP kinase, ERK1/2 kinase. These kinases play important role in Ang II signaling and vascular function, including cell growth, migration, inflammation. Recent study showed that Ang(1–7) has protective effects in the cardiovascular and kidney diseases through counterbalancing the physiological actions of Ang II [18–21]. Lovren et al. [21] found that
ACE2 confers endothelial function and limits aberrant vascular responses. Our previous study demonstrated that ACE2 increases the stability of atherosclerotic plaque via decreased Ang-II and increased Ang-(1–7) level [9]. Previous study reported that Ang-(1–7) reduced smooth muscle growth and the neointima/media thickness ratio in balloon-injured carotid artery after arterial stenting in rats. Tesanovic et al. found that Ang-(1–7) has both vasoprotection and anti-atherosclerotic effects. The mechanisms include the restoration of nitric oxide bioavailability and the involution in the Mas receptor signaling pathway [22]. Study showed that Ang-(1–7) protects endothelial cell function directly and indirectly. Ang-(1–7) stimulates NO release and causes vasodilatation via Akt-dependent pathways [23]. Another studies found that Ang-(1–7) inhibits Ang II–mediated phosphorylation of p38, ERK MAP kinase, thereby resulting in negative modulation on Ang II effect. Ang-(1–7) antagonizes Ang II-induced activation of protein kinase C, p38MAP and ERK1/2 kinase in vascular smooth muscle cells and human endothelial cells. In the present study, ACE2 gene transfer promoted HUVECs migration and inhibited adhesion of monocyte to HUVECs. In the meanwhile, ACE2 overexpression not only inhibited Ang II-induced MCP-1, VCAM-1 and E-selectin protein production in vitro, but also decreased the level of MCP-1, VCAM-1 and interleukin 6 and inhibited atherosclerotic plaque evolution in vivo. Furthermore, administration of A779 increased the level of MCP-1,VCAM-1 and interleukin 6 in vivo and led to further advancements in atherosclerotic extent. These results indicated that ACE2 effectively improves endothelial cell function, inhibits inflammatory response and Ang-(1–7) may play an important role during this process. The cellular responses in the vasculature including contraction, growth, and inflammation by Ang-II are mediated via generation of reactive oxygen species (ROS). Much evidence demonstrated that vascular endothelial cell and smooth muscle cell NAD(P)H oxidase is a major source of vascular ROS. Both decreased ability of antioxidants and increased production of oxidants play an important roles in the pathogenesis of atherosclerosis. Touyz et al. and Sampaio et al. demonstrated that Ang II promoted ROS production in VSMC and endothelial cell. Our in vitro studies showed that Ang II decreased the TAC and SOD activity. Conversely, ACE2 gene transfer increased the antioxidant ability as manifested with the increased TAC and SOD activity, whereas administration of A779 resulted in decreased antioxidant ability. Theses results indicated that ACE2 gene transfer increased the ability of antioxidant, indicating that ACE2 represents a protective mechanism in the endothelium by increasing ability of antioxidant. Ant-(1–7) plays a crucial role in counterbalance deleterious effects induced by Ang II.
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In summary, our study demonstrated that ACE2 overexpression and Ang-(1–7) protect endothelial cell function and inhibit atherosclerotic evolution. The mechanism of protecting endothelial cell function and anti-atherosclerotic effect of ACE2 and Ang-(1–7) resulted from counter regulation of Ang II signaling, inhibition of inflammatory response and increasing the ability of antioxidant. Acknowledgments We would like to express our thanks to the teachers at the Key Laboratory of Cardiovascular Remodeling and Function Research, Shandong University. This study was supported by the National 973 Basic Research Program of China (No. 2013CB530700), the National Natural Science Foundation of China (No. 81170207) and the Program of State Chinese Medicine Administration Bureau (No. JDZX2012113).
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15. Conflict of interest work.
The authors report no conflicts of interest in this
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