Int J Clin Exp Pathol 2015;8(3):2505-2514 www.ijcep.com /ISSN:1936-2625/IJCEP0004926

Original Article Local renin-angiotensin system regulates hypoxia-induced vascular endothelial growth factor synthesis in mesenchymal stem cells Yue Fan*, Lulu Wang*, Chao Liu, Hongyi Zhu, Lu Zhou, Yu Wang, Xiaowei Wu, Qingping Li Department of Pharmacology, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, P.R.China. *Equal contributors. Received December 16, 2014; Accepted February 20, 2015; Epub March 1, 2015; Published March 15, 2015 Abstract: The use of mesenchymal stem cell (MSC) transplantation for ischemic heart disease has been reported for several years. The main mechanisms responsible for the efficacy of this technique include the differentiation of MSCs into cardiomyocytes and endothelial cells, as well as paracrine effects. However, the differentiation rates of MSCs are very low, and the differentiated cells are not mature. In addition, MSCs undergo massive cell death within a few days after transplantation to the ischemic myocardium. Paracrine effects may thus play a major role in MSCs transplantation. Angiotensin II (Ang II) is known to be produced locally in the ischemic myocardium, but the effects of hypoxia on the local renin-angiotensin system (RAS) in MSCs, and the role of the RAS in hypoxia-induced vascular endothelial growth factor (VEGF) secretion remain unknown. In this study, we demonstrated that hypoxia stimulated the local RAS in MSCs, while pretreatment with the Ang II type 1 (AT1) receptor antagonist losartan reduced hypoxia-induced hypoxia-inducible factor 1α (HIF-1α) and VEGF production. The ERK1/2 inhibitor U0126 and the Akt inhibitor LY294002 also inhibited hypoxia-induced HIF-1α and VEGF production. Overall, these results indicate that the local RAS in MSCs regulates hypoxia-induced VEGF production through ERK1/2, Akt and HIF-1α pathways via the AT1 receptor. Keywords: Mesenchymal stem cell, hypoxia, renin-angiotensin system, vascular endothelial growth factor

Introduction Mesenchymal stem cells (MSCs) are derived from early mesodermal and ectodermal development and exist in tissues such as the bone marrow stroma, adipose tissue, skeletal muscle, and umbilical cord blood [1]. MSCs can differentiate into several cell types including cardiomyocytes, bone, cartilage, and adipose tissue [2-6], both in vitro and in vivo. Ischemic heart disease, such as myocardial infarction, is a leading cause of death and is characterized by cardiomyocyte apoptosis and ventricular remodeling, which eventually lead to heart failure [7]. MSCs have the characteristics of high expansion potential, ease of isolation, and the ability to differentiate into endothelial cells, cardiomyocytes and vascular smooth muscle cells, making them a suitable candidate for regenerative therapy for ischemic heart disease. Several studies have reported that MSCs can significantly attenuate ventricular remodeling and

improve recovery of cardiac function after myocardial infarction [8]. However, the efficacy of stem cell therapy is limited by poor cell survival, and most implanted cells are lost within a few days following transplantation to the heart [9]. Oxygen levels in ischemic regions in the infarcted heart could be as low as 0.2% [10], and apoptosis is thought to be a major factor influencing the effect of MSCs treatment. Hypoxia preconditioning and genetically engineered cells have thus been used to improve the survival rate and therapeutic efficacy of MSCs. The renin-angiotensin system (RAS), which involves the association of renin, angiotensinconverting enzyme (ACE), and angiotensin II (Ang II), has been demonstrated in MSCs [11]. We previously reported that Ang II could induce the synthesis of vascular endothelial growth factor (VEGF) in MSCs via the AT1 receptor [12]. MSCs secrete several growth factors and cytokines, including VEGF, hepatocyte growth fac-

Local RAS regulates hypoxia-induced VEGF synthesis

Figure 1. Characterization and proliferation capacity of MSCs under hypoxia. A. Flow cytometry analysis showed that most of the cultured MSCs expressed CD29 (86.9%) while a small portion of MSCs expressed CD11b/c (5.2%). B. The difference in the proliferation capacity of MSCs after treatment with hypoxia was detected with the CCK-8 assay. Results are mean ± SEM, n = 3/group. *P < 0.01 vs. control.

tor, insulin-like growth factor-1, basic fibroblast growth factor, and hypoxia inducible factor-1α (HIF-1α) and so on [13, 14]. VEGF acts as a critical growth factor in MSCs-mediated protective effects in infarcted hearts by enhancing cell survival and angiogenic capacity [15, 16]. VEGF expression induction is mediated by the transcription factor HIF-1α [17, 18], and HIF-1α expression in turn is regulated by phosphorylation via the PI3K/Akt and MAPK pathways [19]. However, little is known about the effects of hypoxia on the local RAS in MSCs and on VEGF production by MSCs. The purposes of this study were therefore to investigate the effects of the local RAS on VEGF production by MSCs under hypoxic conditions, and the roles of the ERK, Akt and HIF-1α pathways on these effects. Materials and methods Animals Healthy male Sprague-Dawley rats (weighing 70 to 80 g for isolation of MSCs) were obtained 2506

from the Vital River Laboratory Animal Co., Ltd., Beijing Laboratory Animal Research Center (Beijing, China). All procedures involving animals were approved by the Ethics Committee for Animal Experiments of Nanjing Medical University. Efforts were made to minimize suffering of animals. Reagents Losartan were purchased from Merck (Darmstadt, Germany). PD123319 were from Tocris (Bristol, GB). LY294002, U0126 were obtained from Sigma (MO, USA). Isolation, culture and phenotype characterization of MSCs MSCs were obtained as reported previously [12]. Briefly, bone marrow was flushed from rat femurs and tibiae. The marrow mononuclear cells were then resuspended, centrifuged and seeded into 25-cm2 flasks and cultured at a density of 1×106 cells/ml at 37°C in a mixture Int J Clin Exp Pathol 2015;8(3):2505-2514

Local RAS regulates hypoxia-induced VEGF synthesis

Figure 2. Effects of hypoxia on local RAS expression in MSCs. A. AT1, AT2, and ACE mRNA levels in MSCs after exposure to hypoxia for 24 h. B. AngII levels in MSCs supernatants after exposure to hypoxia for 24 h. Results are mean ± SEM, n = 3/group (each experiment was repeated on three separate occasions). *P < 0.05 vs. control; **P < 0.01 vs. control.

of 95% air and 5% CO2 in DMEM (Gibco, California, USA) containing 10% fetal bovine serum (Gibco), penicillin (100 U/ml) and streptomycin (100 mg/ml). Nonadherent hematopoietic cells were removed after 48 h, and the medium was replaced every 3 days for adherent cells. Each primary culture was replated to new flasks when the MSCs reached approximately 80% confluence. Cells were used from passage 2-6. MSCs were characterized by flow cytometry. Exposure of cells to hypoxia MSCs were exposed to hypoxic conditions in a hypoxia cell culture chamber (Don Whitley Scientific, Shipley, GB) using a gas mix of 5% CO2, and 95% N2 and maintained at 37°C for 24 h. The chamber was purged with the hypoxic gas mix according to the manufacturer’s instructions to generate hypoxia. Cells were aseptically transferred into the chamber upon reaching about 80% confluency. Cell counting kit-8 (CCK-8) assay for cell proliferation Cell proliferation was determined using CCK-8 solution (Beyotime, Beijing, China) according to the manufacture’s instruction. Cells in 96-well plate were added with 10 μl CCK-8 solution, and incubated for another 1 hours at 37°C. Absorbances of each well were quantified at 450 nm using a microplate reader. 2507

Quantitative real-time polymerase chain reaction (PCR) analysis Total RNA was extracted from cells using Trizol (Invitrogen Life Technologies, CA, USA) and reverse-transcribed according to standard protocols (Promega, WI, USA). Quantitative PCR was performed on an ABI 7300 PCR instrument (Applied Biosystems, Foster City, CA, USA) using a SYBR Green kit. The PCR conditions provided by the manufacturer were as follows: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The primer sequences were: AT1 receptor: sense 5-CCTCTACGCCAGTGTGTTCC-3, antisense 5-GCCAAGCCAGCCATCAGC-3; AT2 receptor: sense 5-CCCCTTGTTTGGTGTATGGC-3, antisense 5-AGGCAATCCCAGCAGACC-3; ACE: sense 5-ACGGAAGCATCACCAAGGAG-3, antisense 5-TGGCACATTCGCAGGAACG-3; VEGF: sense 5-GCGGGCTGCTGCAATG-3, antisense 5-TGCAACGCGAGTCTGTGTTT-3; β-actin: sense 5-GCACCGCAAATGCTTCTA-3, antisense 5-GGTCTTTACGGATGTCAACG-3. Each sample was tested in triplicate, and relative gene expression data were analyzed using the 2-ΔΔCT method. mRNA levels were normalized using the β-actin housekeeping gene and compared with mRNA levels in the control group. Radioimmunoassay (RIA) The amounts of Ang II were analyzed in supernatants secreted under all experimental conditions. Supernatants were collected, centrifuged Int J Clin Exp Pathol 2015;8(3):2505-2514

Local RAS regulates hypoxia-induced VEGF synthesis

Figure 3. Local RAS regulated hypoxia-induced VEGF secretion. A. Effect of losartan on hypoxiainduced HIF-1α protein levels. B. Effect of losartan on hypoxia-induced VEGF protein levels. C. Effect of losartan on hypoxia-induced VEGF mRNA levels. Results are mean ± SEM, n = 3/group (each experiment was repeated on three separate occasions). *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. hypoxia.

to remove cell debris, and stored at -80°C for analysis. Ang II levels were analyzed by RIA using a commercially available RIA kit (Beifang, China), according to the manufacturer’s instructions. Western blotting Cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and then lysed with ice-cold lysis buffer (1% Triton X-100, 20 mmol/l HEPES (pH 7.5), 5 mmol/l MgCl2, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulphonyl fluoride, and 1 mg/ml each of leupeptin, aprotinin, and pepstatin) for 30 min. The cell lysates were centrifuged at 12,000 g for 10 min at 4°C and

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the supernatant was collected and stored at -80°C until use. Equal amounts of protein (30 μg) were mixed with sodium dodecyl sulfate (SDS) sample buffer (0.125 M Tris-HCl, pH 6.8, 10% glycerol, 2% β-mercaptoethanol, 2% SDS and 0.1% bromophenol blue) and boiled for 5 min. The samples were then electrophoresed on 10% and 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a standard protocol [12]. The membrane was blocked in 5% non-fat dried milk powder in TBST solution (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature, and then incubated with primary antibody at 4°C overnight. Primary antibodies against Akt (1:1000), phosphorylated

Int J Clin Exp Pathol 2015;8(3):2505-2514

Local RAS regulates hypoxia-induced VEGF synthesis

Figure 4. Phosphorylation level of ERK1/2 and Akt. Inhibition of AngII type 1 (AT1) receptor, AngII type 2 (AT2) receptor, ERK1/2 and Akt respectively in MSCs by pretreatment with inhibitors for 1 h followed by exposure to hypoxia for 24 h. A and C. Phospho-ERK1/2 levels in MSCs, B and D. Phospho-Akt levels in MSCs. Results are mean ± SEM, n = 3/group (each experiment was repeated on three separate occasions). *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. hypoxia.

(p) Akt (1:1000), and pERK1/2 (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). HIF-1α antibody was purchased from Novus Biologicals (CO, USA), total ERK antibody was purchased from Bioworld Technology (MN, USA), and VEGF antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The bound antibodies were detected using horseradish peroxidaseconjugated secondary antibodies for 2 h. After 2509

washing in PBST, bands were visualized using an enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Buckingham- shire, UK) system. Statistical analysis All data are presented as mean ± SEM. Data were analyzed using Student’s t-tests and values of P < 0.05 were considered statistically significant. Int J Clin Exp Pathol 2015;8(3):2505-2514

Local RAS regulates hypoxia-induced VEGF synthesis

Figure 5. Effects of inhibitors of ERK1/2 and Akt on the expression of HIF-1α and VEGF in MSCs. Pretreatment with different inhibitors for 1 h followed by exposure to hypoxia for 24 h. (A) HIF-1α protein levels in MSCs; (B) VEGF protein levels in MSCs; and (C) VEGF mRNA levels in MSCs after exposure to hypoxia for 24 h. Results are mean ± SEM, n = 3/group (each experiment was repeated on three separate occasions). *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. hypoxia.

Results Characterization and proliferation capacity of MSCs under hypoxia To characterize the phenotype of the isolated MSCs, expression of MSCs surface marker was analyzed. The cultured MSCs were positive for CD29 antigens and negative for CD11b/c antigen (Figure 1A). CCK-8 assay demonstrated that hypoxia significantly inhibited cell proliferation compared with untreated cells (Figure 1B). Effect of hypoxia on local RAS in MSCs The local RAS including the AT1 receptor, AT2 receptor, and ACE was significantly induced in 2510

cultured MSCs by hypoxia and serum deprivation for 24 h compared with controls. mRNA levels of each component were significantly increased (Figure 2A). The concentration of Ang II in the supernatant was also significantly increased compared with the control (Figure 2B). Local RAS regulated hypoxia-induced VEGF secretion VEGF mRNA expression was significantly increased after exposure of serum-deprived MSCs to hypoxia for 24 h. Because of the increasing expression levels of AT1 and AT2 receptors induced by hypoxia, we further examined the effects of the AT1 receptor antagonist losartan (1 μM) and the AT2 receptor antagonist PD123319 (1 μM) on hypoxia-induced Int J Clin Exp Pathol 2015;8(3):2505-2514

Local RAS regulates hypoxia-induced VEGF synthesis VEGF secretion. As shown in Figure 3, losartan significantly inhibited the hypoxia-induced increase in VEGF mRNA expression, but PD123319 had no effect on VEGF mRNA levels. Similarly, hypoxia-induced VEGF protein secretion was also inhibited by the AT1 receptor antagonist losartan. PI3K-Akt and MEK-ERK pathway were involved in hypoxia-induced VEGF expression in MSCs To investigate the effects of hypoxia on the PI3K-Akt and MEK-ERK pathways in MSCs, serum-deprived MSCs were incubated under hypoxic conditions for 24 h. Akt and ERK1/2 phosphorylation levels were significantly increased in MSCs underwent hypoxia compared with the cells that incubated under normoxic conditions. This hypoxia-stimulated activation of Akt and ERK1/2 phosphorylation was reversed by pretreatment with the Akt inhibitor LY294002 and the ERK inhibitor U0126, respectively (Figure 4C, 4D). To determine which receptor was involved, we pretreated the AT1 receptor with losartan and the AT2 receptor with PD123319 on Akt and ERK1/2 phosphorylation levels. And we found that, as shown in Figure 4A, 4B, losartan significantly inhibited hypoxia-induced ERK1/2 and Akt phosphorylation, but PD123319 had little effect. The Akt inhibitor LY294002 (10 μM) and the ERK inhibitor U0126 (10 μM) were used than to determine if hypoxia increased VEGF production through Akt- and ERK-dependent mechanisms. As shown in Figure 5, both LY294002 and U0126 decreased hypoxia-induced VEGF expression and secretion. These results suggest that the local RAS regulated hypoxiainduced VEGF secretion via PI3K-Akt and MEKERK pathways. HIF-1α was involved in local RAS-Akt and RASERK pathways To investigate if the local RAS regulated hypoxia-induced VEGF secretion in MSCs via HIF-1α, we investigated the effect of losartan and PD123319 respectively on HIF-1α expression. As show in Figure 3, losartan significantly reduced HIF-1α expression, while PD123319 had no effect. To further investigate the relationship between HIF-1α and the Akt and ERK1/2 signaling pathways, we examined the effects of LY294002 and U0126 on HIF-1α

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expression and found that HIF-1α expression was strongly inhibited by both LY294002 and U0126 (Figure 5). These results suggest that HIF-1α acted as a downstream effector of the local RAS, Akt and ERK1/2 signaling pathways in MSCs. Discussion The present study examined the response of bone marrow-derived MSCs to hypoxic conditions in vitro. The results confirmed previous findings that hypoxia increased HIF-1α and HIF1α-mediated VEGF expression levels [20, 21], and those effect could be inhibited by a PI3K/ Akt inhibitor. We also demonstrated for the first time that the components of the local RAS in MSCs, including the AT1 receptor, AT2 receptor, ACE, and Ang II, were all increased after exposure to hypoxia, and that the AT1 receptor antagonist losartan reduced VEGF secretion via the Akt, ERK and HIF-1α pathways. MSCs have been used as cell-based therapy for ischemic heart disease for several years. Transplantation of MSCs has been shown to attenuate ventricular remodeling and improve cardiac performance after myocardial infarction [10, 22-24]. The main mechanisms responsible for these actions are thought to be via cell differentiation and paracrine effects. Cardiomyocyte differentiation has attracted the attention of many researchers, and 5-azacytidine, suberoylanilide hydroxamic acid, and co-culture with neonatal cardiomyocytes have been used to promote MSCs differentiation into cardiomyocytes [25-27]. Other studies found that VEGF and simvastatin could induce MSCs differentiation into endothelial cells [28, 29]. However, the differentiation rate was low, and it has been reported that MSCs differentiation towards cardiomyocytes produced cardiac progenitor cells [30], and that their endothelial differentiation failed to produce mature endothelial cells [31]. Paracrine effects may therefore be considered to play the major role in MSCs transplantation and cardiac protection. VEGF is critical for angiogenesis and has been shown to be important in MSCs transplantation for ischemic heart disease [32-34]. VEGF also exerts anti-apoptotic effects [35]. Hypoxia is known to induce VEGF secretion in MSCs [20]. HIF-1α is the primary hypoxia mediator and plays an important role in VEGF secretion. However, little is known

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Local RAS regulates hypoxia-induced VEGF synthesis about the changes in the local RAS in MSCs exposed to hypoxia, or about the signaling mechanisms associated with hypoxia-induced VEGF production in MSCs. The RAS in the circulatory system, heart, and kidney participates in the regulation of cardiovascular function in the steady state, and in controlling electrolytes equilibrium and blood pressure. Accumulating evidence suggests that Ang II plays a key role in cardiovascular pathology and remodeling [36, 37], and Ang II levels are increased in cardiac tissues after myocardial infarction [38]. The RAS also exists in MSCs [11], and the results of the present study demonstrated that hypoxia increased the level of RAS in MSCs, and blockade of the AT1 receptor reduced hypoxia-induced VEGF production. We propose that hypoxia induces VEGF synthesis and secretion through upregulating the level of RAS in MSCs after transplantation, which thus play a role in myocardial protection. In addition, our previously unpublished observation showed that transplantation of Ang II-pretreated MSCs resulted in better cardiac function than untreated MSCs. The PI3K/Akt and ERK pathways are implicated in a variety of cellular functions, including cell survival, apoptosis and proliferation, and are activated in response to hypoxia and cytokines. We previously reported that the Akt pathway and ERK pathways were involved in transforming growth factor-β and Ang II-induced VEGF synthesis [12, 39]. Secretion of paracrine factors has been shown to be increased in MSCs overexpressing Akt. ERK also participates in the differentiation of MSCs into endothelial cells and cardiomyocytes [25, 40]. HIF-1α is a key mediator of cellular responses to hypoxia and can directly influence the activation of angiogenic factors. Our results demonstrated that the local RAS regulated hypoxia-induced VEGF mRNA and protein expression levels via the Akt, ERK and HIF-1α pathways. The increase in VEGF production in MSCs exposed to hypoxia was associated with increased Akt and ERK1/2 phosphorylation. Pretreatment with losartan reduced both HIF-1α-activated VEGF production and Akt and ERK1/2 phosphorylation, while pretreatment with LY294002 and U0126 attenuated the HIF-1α-activated VEGF production. These findings demonstrate that Akt and ERK1/2 signaling may mediate MSCs production of VEGF through regulation of HIF-1α, and

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that the local RAS may regulate the levels of Akt and ERK1/2 phosphorylation. In conclusion, the results of this study demonstrate that the local RAS in MSCs is increased in response to hypoxia, and that stimulation of the local RAS mediates MSCs production of VEGF. Acknowledgements This study was supported by the National Science Foundation of China (No. 30973534, 81173052) and the Jiangsu College Graduate Research and Innovation Program (No. CXZZ13_0604). Disclosure of conflict of interest None. Address correspondence to: Dr. Qingping Li, Department of Pharmacology, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, P.R. China. Fax: 86-25-86868469; E-mail: pharm_qpli@ njmu.edu.cn

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Int J Clin Exp Pathol 2015;8(3):2505-2514

Local renin-angiotensin system regulates hypoxia-induced vascular endothelial growth factor synthesis in mesenchymal stem cells.

The use of mesenchymal stem cell (MSC) transplantation for ischemic heart disease has been reported for several years. The main mechanisms responsible...
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