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Valsartan impairs angiogenesis of mesenchymal stem cells through Akt pathway
International Journal of Cardiology 167 (2013) 2765–2774
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Valsartan impairs angiogenesis of mesenchymal stem cells through Akt pathway☆,☆☆ Cheng-I Cheng a, b, Chang-Chun Hsiao a, Shinn-Chih Wu d, Shao-Yu Peng e, Hon-Kan Yip b, Morgan Fu b, Feng-Sheng Wang a, c,⁎ a
Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Kaohsiung, Taiwan Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Department of Medical Research, Chang Gung Memorial Hospital, Kaohsiung, Taiwan d Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan e Institute of Biotechnology, National Taiwan University, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 15 September 2011 Received in revised form 24 June 2012 Accepted 30 June 2012 Available online 16 July 2012 Keywords: Stem cells Angiogenesis Angiotensin II Akt
a b s t r a c t Background: Angiotensin II (AngII) reportedly enhances stem cell proliferation, and type 1 angiotensin II receptor (AT1R) antagonists reduce angiogenesis in a rodent hindlimb ischemic model. Whether AT1R antagonists can alter the angiogenic activity of bone-marrow mesenchymal stem cells (BMSCs) is unknown. The purpose of this study is to investigate whether AT1R antagonists can alter the angiogenic activity of BMSCs and explore the potential mechanism for such an action. Methods: Mouse BMSCs were isolated and treated with AngII, an AT1R antagonist, and a type 2 angiotensin II receptor (AT2R) antagonist. Angiogenic activity of BMSCs was detected by vascular endothelial growth factor (VEGF) secretion and tube formation of human umbilical vein endothelial cells (HUVECs). BMSCs isolated from enhanced green fluorescent protein (eGFP)-transgenic mice were allografted into ischemic hindlimbs in mice. Results: The BMSCs constitutively expressed AT1Rs and AT2Rs. AngII treatment significantly increased VEGF secretion by BMSCs. Valsartan (AT1R antagonist) but not PD123319 (AT2R antagonist) treatment attenuated the AngII-induced promotion of VEGF synthesis by BMSCs. The AngII and AngII receptor antagonist control of angiogenic activity of BMSCs were confirmed by tube formation of HUVECs. AngII treatment promoted phosphorylated Ser473 Akt abundance in BMSCs. RNA interference of an isoform of AT1R, valsartan, and wortmannin treatments attenuated AngII-induced Akt phosphorylation. Allograft of BMSCs significantly increased blood flow and VEGF expression in the gastrocnemius muscles of ischemic hindlimbs, which was attenuated after valsartan treatment. Conclusions: AT1R antagonists, via AT-1R/PI3K/Akt pathways, impair the AngII-induced promotion of angiogenic activity of mouse BMSCs. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cell therapies with bone marrow stem have been employed clinically in recent years as an adjuvant therapy for ischemic cardiomyopathy [1]. Bone marrow mesenchymal stem cells (BMSCs) produce
☆ Sources of funding: This work was supported by the grants from the Taiwan National Science Council (95-2745-B-182A-005) and Kaohsiung Chang Gung Memorial Hospital (CMRPG 860521). ☆☆ Disclosures: No author has any commercial associations or interests, including consultancies, stock ownership, or other competing equity interests or patentlicensing arrangements. ⁎ Corresponding author at: Department of Medical Research, Chang Gung Memorial Hospital, Kaohsiung, 123, Ta-Pei Road, Niao-Song District, Kaohsiung City, 83301, Taiwan. Tel.: +886 7 7317123; fax: +886 7 7322402. E-mail address: [email protected] (F.-S. Wang). 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.06.128
proangiogenic factors, such as vascular endothelial growth factor (VEGF) [2], and chemotaxic factors, such as stromal-derived factor1α (SDF-1α) [3] and stem cell factor (SCF) [4,5] to attract endothelial progenitor cells. Mesenchymal stem cells also differentiate into the endothelial phenotype in vitro [6]. Transplantation of bone marrow stem cells enhances in vivo angiogenesis and results in functional improvement in both the hindlimb ischemic model and myocardial infarction (MI) model in animal studies [7,8]. These findings indicate that BMSCs are potential candidates for the treatment of ischemic disease. Systemic activation of the renin–angiotensin system (RAS) is the hallmark of severe cardiovascular disease, such as heart failure [9] and MI [10]. In patients with hypertension, advanced atherosclerosis, or heart failure, the plasma level of angiotensinogen increases. Angiotensinogen is converted to angiotensin I, which is in turn converted to the effecter peptide of RAS, angiotensin II (AngII), by
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Fig. 2. In vitro angiogenesis assay. (A) Abundance of SCF (left panel), SDF-1α (middle panel), and VEGF (right panel) in culture medium from BMSCs in different treatment groups. Culture media were harvested 7 d after treatment. Data were expressed as mean ± standard error calculated from at least 3 repeated experiments. *: pb 0.05 vs. control. (B) Representative microscopic photographs of capillary tube and vascular ring formation in HUVECs. Cells were cultured in Matrigel (100×) and treated BMSC culture medium for 5 h. Scale bar = 50 μm. (C) Number of capillary tube (left panel) and vascular ring (right panel) in HUVECs. Data were expressed as mean ± standard error calculated from 5 random fields (100×) in each experiment and at least 3 repeated experiments. *: p b 0.05 vs. CM-control.
angiotensin converting enzyme (ACE) [11]. By acting on type 1 AngII receptors (AT1Rs) in different organs, AngII promotes vasoconstriction, salt and water retention, secretion of aldosterone, and activation of the sympathetic nervous system [12,13]. Oxidative stress in endothelial cells is also increased due to the additional superoxide production after AT1R stimulation [14]. Various AT1R antagonists have been developed for the treatment of hypertension and heart failure by blocking the activated downstream signaling of AT1R [15]. Although suppression of the over-activated RAS is clinically beneficial for patients with heart failure and hypertension, the application of AT1R antagonists would increase the plasma AngII level in a negative feedback loop [16], leading to activation of type 2 AngII receptors (AT2Rs). Chronic subcutaneous delivery of AngII enhances
angiogenesis after the femoral artery is ligated in the mice hindlimb ischemic model; this phenomenon is blunted when an AT1R antagonist is orally administrated to mice [17]. Although an AT1R antagonist suppresses the angiogenesis effect, an ACE inhibitor has the opposite effect in the hindlimb ischemic model [18]. Stem cell therapy using bone marrow stem cells in animals with myocardial infarction [19] or hindlimb ischemia [20] has resulted in only modest functional improvement, and the improvements in survival and left ventricular systolic function are insignificant in most clinical trials [21,22]. Unlike patients who receive AT1R antagonists for heart failure or hypertension, experimental animals with ischemic disease are seldom administered with pharmaceutical agents when the effects of stem cell transplantation are evaluated. AT1R
Fig. 1. Characteristics and expression of angiotensin receptors in BMSCs harvested from eGFP mice. (A) BMSCs displayed fibroblast morphology (100×) in association with (B) green fluorescence eGFP reaction. Scale bar = 100 µm. (C) Expression of surface markers in BMSCs. Negative and positive immunostaining are expressed in open and red areas as detected by PE-conjugated isotype IgG and antibodies of interest, respectively. Percentage of positively-stained cells is bar scaled in each graph. (D) BMSCs had myocardiac differentiation capacity as evidenced by positive troponin-I immunoflorescence reactions (400×). Scale bar = 25 µm. (E) BMSCs had osteogenic differentiation potency as demonstrated by alizarin red staining (200x). Scale bar = 50 µm. (F) BMSCs had adipogenic differentiation capacity as evidenced by oil-red O staining (100×). Scale bar = 100 µm. (G) BMSCs expressed AT1R and AT2R in the presence or absence of AngII treatment.
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expression occurs in human bone marrow hematopoietic and stromal cells [23]. Local activation of RAS also occurs in the left ventricle after acute MI [10], which is speculated to be crucial for homing of hematopoietic stem cells into the infarcted area to repair damaged myocardium [24]. Additionally, AngII is now known to promote proliferation of HSCs [25]. Therefore, antagonism of AT1R may potentially cause biologic changes in stem cells, and we hypothesize that AT1R antagonists may alter the AngII-mediated angiogenic activity of BMSCs. In this study, we designed in vivo and in vitro experiments to elucidate the potential effects of AngII and AT1R on angiogenesis promoted by BMSCs. 2. Materials and methods
pluripotency of BMSCs, in vitro differentiation into osteogenic, cardiomyogenic, and adipogenic phenotypes was performed. A total of 5 × 104 mice BMSCs were seeded onto 24 well-plates and cultured in respective condition medium. Alizarin red staining, Oil-Red-O staining, and immunofluorescent (IF) staining of anti-mouse cardiac troponin-I (Tn-I) were performed to confirm osteogenesis, adipogenesis, and cardiomyogenesis, respectively. The details of primary culture, flow cytometry, and in vitro differentiation are provided in the Supplemental data. 2.3. Flow cytometry BMSCs were trypsinized and concentrated to 1 × 106 cells/100 μL PBS. Then they were incubated at 4 °C with 5 μL phycoerythrin-conjugated antibodies against mice for 30 min in the dark, rinsed twice with 2 mL PBS and fixed with freshly prepared, cold 1% paraformaldehyde (Sigma-Aldrich). Detection of phycoerythrin labeling was accomplished on a Cytomics 500 equipped with 570 nm argon laser (Beckman Coulter, Fullerton, CA) using CXP software with a minimum of 10,000 events counted.
2.1. Experimental animals 2.4. Reagents and in vitro cell treatment The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. All experimental protocols for animal studies were approved by the Animal Care Committee at our institute. Transgenic enhanced green fluorescent protein (eGFP) mice were kindly provided by Dr. Shinn-Chih Wu (Department of Animal Science and Technology, National Taiwan University). eGFP mice were generated by homologous recombination in embryonic stem cells and backcrossed 10 times into the ICR background, and eGFP is tagged on β-tubulin. Wild type ICR mice were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan), as control. 2.2. Identification of BMSCs Eight-week-old eGFP mice were euthanized by carbon dioxide gas inhalation followed by cervical dislocation, and BMSCs were isolated as described previously [26]. The cell cultures within 7 passages were used for the experiment. Flow cytometry with anti-mouse CD11b (BioLegend, Santa Fe, CA), CD29 (BioLegend), CD31 (eBioscience, San Diego, CA), CD34 (eBioscience), CD44 (BioLegend), CD45 (eBioscience), CD73 (BD Biosciences, San Diego, CA), CD86 (eBioscience), CD105 (eBioscience), CD117 (BD Biosciences), CD133 (BD Biosciences), CD166 (BD Biosciences), MHC-I (BD Biosciences), MHC-II (BD Biosciences), and Sca-1 (eBioscience) antibodies conjugated with phycoerythrin were used to identify BMSCs. To evaluate the
BMSCs were seeded at the density of 1 × 105/cm2 and treated with 10−7 M AngII (Sigma-Aldrich, St. Louis, MO), 10−5 M AT1R antagonist valsartan (Novartis, Basle, Switzerland), 10−5 M AT2R antagonist PD123319 (Sigma-Aldrich), or 10−8 M wortmannin (PI3K inhibitor) (Sigma-Aldrich) as indicated. For the experiments investigating AngII effects on Akt phosphorylation and VEGF transcription, undifferentiated BMSCs were cultured in serum-free for 3 h and subjected to the treatment of AngII and various pharmacological inhibitors as indicated. The expression of AT1aR (an isoform of AT1R) was knocked down by delivering small interfering RNA (siRNA) (Ambion, Austin, TX) into BMSCs using electroporation protocols. Scramble RNA was used as scrambled controls. As previously described [27], BMSCs were harvested at 24, 48, 72, and 96 h after electroporation to examine the expression of AT1aR by real time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The details of electroporation and RNA interfering are described in the Supplementary data. 2.5. Western blot Protein extracts were obtained from cell lysates of BMSCs and homogenized gastrocnemius muscles. The samples were probed with goat anti-AT1R (1:1000 dilution) (Abcam), goat anti-AT2R (1:2000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA),
Fig. 3. Effects of AngII and valsartan on phosphorylated Ser473-Akt and VEGF expression in BMSCs. (A) Representative immunoblotting and density plot of phosphorylated Ser473Akt in BMSCs under AngII with or without valsartan and wortmannin as indicated. *: pb 0.05 vs. baseline. (B) Expression of VEGF after treatment with AngII, valsartan or wortmannin for 6 h as indicated. Transcription of VEGF mRNA was determined by quantitative RT-PCR. *: p b 0.05 vs. control. (C) Representative immunoblotting and density plot of phosphorylated Ser473-Akt in BMSCs under AngII with or without PD123319 and wortmannin as indicated. *: pb 0.05 vs. baseline. . #: p b 0.05 vs. AngII. (D) Expression of VEGF after treatment with AngII, PD123319, or wortmannin for 6 h as indicated. Transcription of VEGF mRNA was determined by quantitative RT-PCR. *: p b 0.05 vs. control. #: p b 0.05 vs. AngII.
C.-I. Cheng et al. / International Journal of Cardiology 167 (2013) 2765–2774 rabbit anti-Akt (1:1000 dilution) (Cell Signaling Technology, Danvers, MA), rabbit antiphospho-Akt (Ser473) (Cell Signaling Technology), and mouse anti-VEGF (1:1000 dilution) (Abcam) antibodies, respectively, as described in the Supplementary data. Mouse anti-β-actin antibody (1:2000) (Chemicon International) was used as an internal control.
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dishes overnight in triplicate, and then different treatment conditions (i.e., control, AngII, AngII with valsartan) were applied for 7 days. The supernatant at days 0, 1, 4, and 7 was collected for ELISA using mouse VEGF, SDF-1α, and SCF Immunoassay Kit (R&D System Inc., Minneapolis, MN) according to the manufacturer's instructions. Optical density at 405 nm was determined by a microplate reader, and the concentrations of different samples were determined from the standard curves.
2.6. Quantitative real time RT-PCR Total RNA was extracted from cell lysates of BMSCs using the RNeasy Mini Kit (Qiagen, Qiagen AB, Solona, Sweden) according to the manufacturer's instructions. Reverse transcription was then performed with Roche Transcription First Structure cDNA Synthesis Kit (Roche Diagnostics) by using 1 μg of total RNA according to the manufacturer's instructions. Quantitative real time PCR was performed as described in the Supplemental data. The sequences of primers are listed in the Supplementary table. 2.7. Enzyme-linked immunosorbent assay (ELISA) The concentration of VEGF, SDF-1α, and SCF in the supernatant was measured by ELISA at indicated days. A total of 1 × 105 eGFP mice BMSCs were seeded to 6 well-
2.8. Capillary tube and vascular ring formation assay Matrigel-induced capillary tube and vascular ring formation using human umbilical vein endothelial cells (HUVECs) were performed to assess the angiogenic activity of BMSCs in triplicate. Briefly, Matrigel (BD Biosciences) was diluted to 4 mg/mL with cold PBS and added to 24-well plates in a total volume of 200 μL per well. Plates stood at 37 °C for 30 min to form a gel layer. After gel formation, 2 × 105 cells of human umbilical vein endothelial cell line were applied to each well, along with the addition of 1 mL supernatant from different treatment conditions described above for ELISA (day 7). The plates were then incubated at 37 °C for 24 h with 5% CO2. Following incubation, HUVECs were observed, and the formation of capillary
Fig. 4. Effect of AT1aR RNA interference on Akt phosphorylation and VEGF expression in BMSCs. (A) AT1aR RNA interference decreased AT1aR mRNA expression. mRNA expression in cell cultures was detected by quantitative RT-PCR. *: p b 0.05 vs. scramble control. Representative immunoblotting of (B) AT1R expression and (C) phosphorylated Ser473-Akt expression in AT1aR RNA interference- (upper panel) and scramble control-transfected (lower panel) BMSCs. (D) AT1aR RNA interference decreased VEGF mRNA expression in BMSCs. mRNA expression in cell cultures was detected by quantitative RT-PCR 8 h after treatment. Data was expressed as mean ± standard error calculated from at least 3 repeated experiments. *: p b 0.05 vs. vehicle group.
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tubes and vascular rings was quantified in 3 randomly chosen fields in triplicate at 100× magnification.
2.9. Hindlimb ischemic model and transplantation of eGFP BMSCs Six of eight-week-old male ICR mice in each group were pretreated for 2 weeks with 20 mg/kg/day of valsartan (provided by Novartis) or vehicle dissolved in drinking water. A total of 3 × 105 BMSCs from eGFP mice suspended in 50 μL phosphate-buffered saline (PBS) (separated into 2 injections) (n = 6 with valsartan; n = 6 without valsartan) or PBS alone (n = 6 with valsartan; n = 6 without valsartan) were injected into the left gastrocnemius muscle 24 h after ligation of the femoral artery under the anesthesia with intraperitoneal 200 mg/kg ketamine and 10 mg/kg xylazine. An analgesic of 0.1 mg/kg buprenorphine was administered subcutaneously to relieve pain when observed. Laser Doppler blood flow (LDBF) imaging was then performed as described in the Supplementary data.
2.10. Immunohistochemistry (IHC) staining and IF staining IHC staining was performed using gastrocnemius muscles in the legs with hindlimb ischemia, and IHC staining was performed with rabbit anti-von Willebrand factor (vWF) antibody (1:250 dilution) (Chemicon International, Temecula, CA) as previously described [28]. Capillary density was then calculated as capillaries per field and per muscle fiber in 3 randomly chosen fields of each specimen (6 mice in each group) at 200× magnification. The details of immunocytochemistry and IHC staining are described in the Supplemental data. To elucidate the in vivo endothelial differentiation and paracrine function of eGFP mice BMSCs after transplantation, the tissue from gastrocnemius muscles was processed as described in the IHC procedure, and tissue sections were subjected to IF staining with mouse anti-VEGF antibody (1:500 dilution) (Abcam, Cambridge, MA), and rabbit anti-vWF antibody (1:250 dilution). Images were generated by Zeiss LMS5 Live confocal microscope (Zeiss, Oberkochen, Germany). The details of the IF are shown in the Supplementary data.
2.11. In vivo fluorescence tracking In order to confirm whether transplanted eGFP BMSCs were retained in the gastrocnemius muscles, mice receiving eGFP BMSCs transplantation after hindlimb ischemia were examined by Lumazone In Vivo Imaging System (MAG Biosystems, Pleasanton, CA) at day 15 after anesthesia with intramuscular ketamine 50 mg/kg and xylocaine 50 mg/kg. 2.12. Statistical analysis Data were presented as mean ± SEM and analyzed using a two-tailed t test or one-way ANOVA processed by the operating SPSS 11.0 computer program (SPSS Inc., Chicago, IL). A probability value b0.05 is considered statistically significant.
3. Results 3.1. Constitutive expression of AT1R and AT2R in BMSCs We verified whether bone marrow stromal cells (BMSCs) in eGFP mice expressed AT1R or AR2R. Microscopic observation showed that BMSCs displayed spindle-shape, fibroblast-like morphology (Fig. 1A) and green fluorescence (Fig. 1B). Flow cytometric analyses revealed that BMSCs were positive for CD29 (86.2%), CD44 (98.0%), CD73 (75.2%), CD105 (90.7%), Sca-1 (93.8%) and MHC-I (93.3%), but negative for CD11b (0.5%), CD31 (0.3%), CD34 (0.5%), CD45 (0.4%), CD86 (0.3%), CD117 (0.5%), CD133 (0.7%), CD166 (0.9%), and MHC-II (0.5%) (Fig. 1C). Additionally, BMSCs differentiated toward myogenic, osteogenic, and adipogenic lineages, as demonstrated by positive stain
Fig. 5. Angiogenesis in ischemic limbs with or without BMSCs or valsartan treatment. (A) Representative laser Doppler images of ischemic hindlimb at 15 days after BMSCs transplantation into ischemic mice with or without valsartan administration. (B). Treatment with valsartan reduced the promoting effect of BMSCs graft on blood flow in ischemic limbs. *: p b 0.05 vs. PBS group. (C) Representative images of capillary vessels as demonstrated by positive vWF immunostaining in gastricnemius muscle of ischemic limb at 14 days after BMSCs or valsartan treatment (200×). Scale bar = 50 µm. (D) Administration of valsartan attenuated the promotional effects of BMSCs on capillary vessel distribution in ischemic limbs. Data was calculated from 3 randomly-selected fields (left panel) and 30 random muscle fibers (right panel) from 6 animals in each group. *: P b 0.05; **: P b 0.001; ****: P b 0.0001 vs. PBS group.
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for troponin-I expression (Fig. 1D), Alizarin red staining (Fig. 1E), and Oil-Red-O (Fig. 1F). Experiments were conducted to verify whether BMSCs expressed AT1R or AT2R. Immunoblotting showed that BMSCs constitutively expressed AT1R and AT2R (Fig. 1G). 3.2. AT1R antagonists attenuate the AngII-mediated angiogenic activity of BMSCs We tested whether AT1R or AT2R signaling affected angiogenic activities of BMSCs. ELISA results showed that AngII treatment significantly increased VEGF levels in culture medium harvested from BMSCs. The AT1R antagonist valsartan significantly attenuated the AngII-induced promotion of VEGF secretion by BMSCs (Fig. 2A). The AT2R antagonist PD123319 did not change the AngII-induced increase in VEGF. These findings indicated that activation of AT1R enhances the secretion of VEGF. Exposure to AngII, valsartan, or PD123319 did not significantly affect SCF or SDF-1α secretion by BMSCs. Experiments were also conducted to determine whether culture medium harvested from AngII or valsartan-treated BMSCs could affect the angiogenesis of HUVECs. Microscopic observation showed that cells in the Ang II group had increased tube and vessel ringlike morphology. These effects were reduced after valsartan treatment (Fig. 2B). Valsartan treatment significantly attenuated the promotional effects of AngII on the number of tubes and vascular rings in HUVEC. PD123319 did not markedly affect the angiogenic activity of BMSCs (Fig. 2C). 3.3. AT1R antagonists attenuate the AngII-mediated Akt signaling and VEGF expression in BMSCs We investigated whether AngII or an AT1R antagonist could affect Akt phosphorylation or VEGF expression. BMSCs were incubated in the serum-free conditions. Immunoblotting showed that AngII treatment promoted phosphorylated Ser473-Akt expression in 45 min and 60 min, respectively. Valsartan or PI3K inhibitor wortmannin treatment attenuated the AngII-induced enhancement of phosphorylated Ser473-Akt levels (Fig. 3A). Treatment with valsartan or wortmannin alone or combined treatment with valsartan and wortmannin did not significantly affect the baseline levels of phosphorylated Akt (Fig. 3A). Quantitative RT-PCR analyses revealed that treatment with valsartan or wortmannin alone or combined treatment with valsartan and wortmannin significantly reduced the promotional effects of AngII on the VEGF expression in cell cultures (Fig. 3B). Notably, treatment with valsartan alone did not significantly reduce the baseline VEGF expression whereas valsartan and wortmannin synergistically block the AngII-induced VEGF transcription. Treatment with PD123319 (an AT2R antagonist) did not significantly change the promotional effects of AngII on the concentrations of phosphorylated Akt (Fig. 3C) or the expression of VEGF in BMSCs (Fig. 3D). Analytic results indicate that AngII increased VEGF expression through AT1R-dependent Akt signaling in BMSCs. Moreover, compared with the scrambled control, AT1aR RNA interference significantly decreased the expression of AT1R mRNA (Fig. 4A), AT1R1 protein (Fig. 4B), phosphorylated Ser473-Akt (Fig. 4C), and VEGF (Fig. 4D) in the AngII-treated BMSCs.
Fig. 6. In vivo VEGF abundance in the ischemic hindlimb tissues. (A) Representative immunoblot images of VEGF gastrocnemius muscle in ischemic limb at 15 days after BMSCs or valsartan treatment. (B) Intensity of VEGF immunoblots. Treatment with valsartan reduced the BMSC-induced enhancement of VEGF concentrations in ischemic limbs. Data was calculated from 6 animals in each group. *: Pb 0.05 vs. PBS treatment alone group.
showed that gastrocnemius muscles in the ischemic legs after BMSC transplantation displayed increased capillary density, as evidenced by vWF expression (Fig. 5C). Histomorphometric analyses showed that treatment with valsartan significantly attenuated the BMSCinduced increase in capillary vessel number (Fig. 5D). Gastrocnemius muscle strongly displayed VEGF expression following BMSC transplantation. The injured tissue weakly expressed VEGF immunoreactivity after valsartan treatment (Fig. 6A and B). 3.5. eGFP BMSCs did not differentiate into endothelial phenotype after transplantation We determine whether BMSCs could differentiate early in the hindlimb ischemic model. In vivo fluorescence observation showed that injured sites strongly displayed green fluorescence at 2 weeks after transplantation of eGFP BMSCs, which suggested the allografted eGFP BMSCs were still viable in injured sites (Fig. 7A). Confocal microscopic images showed that green fluorescent eGFP BMSCs codisplayed red fluorescence VEGF immunoreactivity (Fig. 7B, left panel) but not vWF immunostaining (Fig. 7B, right panel). These effects were attenuated after valsartan treatment. Analytic results suggest that graft with BMSCs enhanced angiogenesis in the ischemic hindlimbs was through induction of VEGF but not endothelial differentiation of BMSCs. 4. Discussion
3.4. AT1R antagonists attenuate the in vivo angiogenesis in the hindlimb ischemic model with intramuscular transplantation of BMSCs
4.1. Mechanism of angiogenesis
We tested whether BMSCs or valsartan treatment changed angiogenesis in mice with ischemic hindlimbs. BMSCs from eGFPtransgenic mice were allografted into the injured site. BMSCs transplantation significantly increased blood flow in injured tissues compared to control group. Administration of valsartan significantly attenuated the BMSC graft-induced enhancement of blood flow in ischemic hindlimbs (Fig. 5A and B). Immunohistochemical observation
The present study demonstrates that activation of AT1R/PI3K/Akt is crucial for the angiogenic effect of BMSCs, which is attenuated by pharmacological inhibition or AT1aR RNA interference. Unlike a previous study in a mice MI model [29] which showed eGFP BMSCs incorporated into vessel after bone marrow transplantation, our study hardly recognized the co-localization of endothelial markers with eGFP expressed in BMSCs in the early phase of in vivo angiogenesis
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Fig. 7. Representative fluorescence photographs of grafted BMSCs in ischemic hindlimb. (A) Localization of exogenous eGFP-BMSC (green fluorescence) in ischemic hindlimb by using an in vivo fluorescence tracing system 15 d after various treatments as indicated. Left panel: white light. Middle panel: green fluorescence. Right panel: merged image. (B) Representative confocal images of VEGF expression (red fluorescence; let panel) and vWF (red fluorescence; right panel) in eGFP-BMSC (green fluorescence) in gastrocnemius muscle. Specimens were probed by using antibodies against VEGF and vWF conjugated with Alexa Fluor 568, respectively. Nuclei in cells were recognized by DAPI (blue fluorescence). Scale bar = 100 μm.
with confocal imaging. Even though BMSCs may potentially express endothelial markers in vivo, formation of functional capillary networks is not feasible during the early stage of angiogenesis, suggesting that trans-differentiation is less likely to be the mechanism of angiogenesis. Increased production of VEGF in muscle proportional to capillary density and increased in vitro secretion of VEGF detected by ELISA in our study suggested that enhanced angiogenesis by BMSCs was mainly due to the paracrine effect and not the direct formation of capillaries by BMSCs. Several pro-angiogenic factors, including VEGF and FGF [30], and chemo-attractants, such as SDF-1α and SCF, are involved in bone marrow stem cell-related angiogenesis [31]. In this study, we demonstrated that the AT1R antagonist attenuates the AngII-mediated VEGF production; the effect of AngII on the secretion of SDF-1a and SCF is neutral. Furthermore, a previous study [25]
suggested that the anti-proliferative effect of the AT1R antagonist could be another mechanism attenuating the angiogenesis of BMSCs. Further investigation is warranted to study the effect of AT1R antagonism on the in vivo survival of BMSCs. 4.2. Angiogenesis and renin–angiotensin system (RAS) In the rodent hindlimb ischemic model, ACEI and the AT1R antagonist demonstrate opposite effects on angiogenesis, although both exert similar effects in the treatment of hypertension and heart failure. The pro-angiogenic effect of ACEI is mediated by the bradykinin B2 receptor-dependent up-regulation of endothelial nitric oxide synthetase (eNOS) and is independent of VEGF expression [32]. On the contrary, the action of AngII on angiogenesis is mediated by the AT1R-related expression of VEGF [33] and does not exist in eNOS-
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deficient mice [34]. This finding suggests that AngII enhanced ischemia-induced angiogenesis through AT1R activation involves the VEGF/eNOS-dependent pathway. Furthermore, impaired angiogenesis in hindlimb ischemia in AT1aR-deficient mice was rescued successfully by peripheral blood mononuclear cells derived from wild type mice rather than those derived from AT1aR-deficient mice [35]. These findings also indicate that the AngII/AT1R/VEGF pathway positively modulates ischemia-induced angiogenesis, and our study confirmed that RAS axis is important for the pro-angiogenic effects of BMSCs. 4.3. AT2R vs. AT1R in angiogenesis of BMSCs Conflicting results have been observed regarding the roles of AT1R and AT2R in AngII-related angiogenesis. AT2R activation by AngII may increase hypoxia-induced endothelial sprouting in mice hearts through the bradykinin B2 receptor/nitric oxide/cGMP pathway [36]. Blockage of AT1R during AngII stimulation may counterregulate the activation of AT2R, subsequently leading to the apoptosis of smooth muscle cells and the pro-angiogenic effect of endothelial cells. On the other hand, AngII was found to increase eNOS expression and cGMP release in bovine aortic endothelial cells overexpressing AT1R, and this effect was further accentuated by the AT1R antagonist [37]. Another study using the hindlimb ischemic model in Agtr2 −/Y mice showed that AT2R may negatively modulate ischemia-induced angiogenesis through an activation process, and the up-regulation of Bcl-2 and eNOS was detected independent of VEGF expression in the ischemic legs of Agtr2 −/Y mice [17]. In our present study, the secretion of VEGF caused by AngII was not affected by PD123319, suggesting that AT2R-mediated anti-angiogenesis was not observed in our model using BMSCs. Similarly, angiogenesis was not altered in AT1R-deficient mice when mice were treated simultaneously with 30 mg/kg/day of PD123319 and implanted with peripheral blood mononuclear cells [35]. Although AT1R and AT2R are both expressed on BMSCs, the effects of AngII on angiogenesis are mainly mediated through AT1R. The self-renewal ability dominantly drives BMSCs away from the AT2R-related anti-proliferative and antiangiogenic effect. 4.4. Akt signaling in BMSCs Signaling of AngII-mediated angiogenesis and proliferation in stem cells occurs via several different mechanisms. PI3K/Akt signaling is important for the maintenance of pluripotency and viability in human embryonic stem cells [38]. One study has shown that AngII stimulates DNA synthesis by increasing cyclin and cyclin-dependent kinase, which is mediated through the AT1R-dependent Ca ++/phosphokinase C pathway and the epidermal growth factor receptor/ PI3K/Akt/mTOR-dependent pathway [39]. Another recent publication also indicates AngII stimulated the synthesis of VEGF in MSCs through the activation of ERK1/2 and the Akt pathway via AT1R activation [40]. Furthermore, Akt overexpression in MSCs promoted cardiac repair more significantly by enhancing paracrine function, including the secretion of VEGF [41]. These findings, along with the results of our present study, indicate that AT1R/PI3K/Akt signaling is crucial for the angiogenesis of BMSCs. Nevertheless, how AngII activates PI3K through AT1R and how Akt induces the transcription of VEGF in BMSCs remain unclear. Future study should focus on exploring these issues. 4.5. Clinical implication AT1R antagonists are widely used to treat a variety of cardiovascular diseases. Additional benefits include halting the progression of atherosclerosis [42], reducing restenosis of intracoronary stents [43], and preventing post-MI remodeling of the left ventricle [44].
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Although direct antagonism of AT1R reduces the bradykinin-related side effects induced by ACEI, these agents do not result in better clinical outcomes (i.e., prevention of MI and cardiovascular death) [45]. As previously mentioned, AT1R antagonists slow ischemiainduced angiogenesis in the hindlimb ischemic model and in in vitro cell survival. Our study also confirms that this agent may impair ischemia-induced angiogenesis of mice BMSCs. Despite the over-activation of RAS in cardiovascular disease, further evaluation is warranted, especially for the patients receiving stem cell therapy for severe ischemic conditions.
5. Conclusion To our knowledge, this is the first time that an AT1R antagonist is demonstrated to attenuate the in vivo angiogenesis of BMSCs. Based on the results of this study, we conclude that AT1R antagonists impair the AngII-related angiogenesis of mice BMSCs by inactivation of the AT-1aR/PI3K/Akt-related pathway. Application of AT1R antagonists in patients receiving stem cell therapy for ischemic conditions needs further evaluation. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijcard.2012.06.128.
Acknowledgments This work was supported in part by the grants from the Taiwan National Science Council (95-2745-B-182A-005) and Kaohsiung Chang Gung Memorial Hospital (CMRPG 860521). The authors also thank the Center for Laboratory Animals for the use of the facilities.
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Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Valsartan impairs angiogenesis of mesenchymal stem cells through Akt pathway☆,☆☆ Cheng-I Cheng a, b, Chang-Chun Hsiao a, Shinn-Chih Wu d, Shao-Yu Peng e, Hon-Kan Yip b, Morgan Fu b, Feng-Sheng Wang a, c,⁎ a
Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Kaohsiung, Taiwan Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Department of Medical Research, Chang Gung Memorial Hospital, Kaohsiung, Taiwan d Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan e Institute of Biotechnology, National Taiwan University, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 15 September 2011 Received in revised form 24 June 2012 Accepted 30 June 2012 Available online 16 July 2012 Keywords: Stem cells Angiogenesis Angiotensin II Akt
a b s t r a c t Background: Angiotensin II (AngII) reportedly enhances stem cell proliferation, and type 1 angiotensin II receptor (AT1R) antagonists reduce angiogenesis in a rodent hindlimb ischemic model. Whether AT1R antagonists can alter the angiogenic activity of bone-marrow mesenchymal stem cells (BMSCs) is unknown. The purpose of this study is to investigate whether AT1R antagonists can alter the angiogenic activity of BMSCs and explore the potential mechanism for such an action. Methods: Mouse BMSCs were isolated and treated with AngII, an AT1R antagonist, and a type 2 angiotensin II receptor (AT2R) antagonist. Angiogenic activity of BMSCs was detected by vascular endothelial growth factor (VEGF) secretion and tube formation of human umbilical vein endothelial cells (HUVECs). BMSCs isolated from enhanced green fluorescent protein (eGFP)-transgenic mice were allografted into ischemic hindlimbs in mice. Results: The BMSCs constitutively expressed AT1Rs and AT2Rs. AngII treatment significantly increased VEGF secretion by BMSCs. Valsartan (AT1R antagonist) but not PD123319 (AT2R antagonist) treatment attenuated the AngII-induced promotion of VEGF synthesis by BMSCs. The AngII and AngII receptor antagonist control of angiogenic activity of BMSCs were confirmed by tube formation of HUVECs. AngII treatment promoted phosphorylated Ser473 Akt abundance in BMSCs. RNA interference of an isoform of AT1R, valsartan, and wortmannin treatments attenuated AngII-induced Akt phosphorylation. Allograft of BMSCs significantly increased blood flow and VEGF expression in the gastrocnemius muscles of ischemic hindlimbs, which was attenuated after valsartan treatment. Conclusions: AT1R antagonists, via AT-1R/PI3K/Akt pathways, impair the AngII-induced promotion of angiogenic activity of mouse BMSCs. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cell therapies with bone marrow stem have been employed clinically in recent years as an adjuvant therapy for ischemic cardiomyopathy [1]. Bone marrow mesenchymal stem cells (BMSCs) produce
☆ Sources of funding: This work was supported by the grants from the Taiwan National Science Council (95-2745-B-182A-005) and Kaohsiung Chang Gung Memorial Hospital (CMRPG 860521). ☆☆ Disclosures: No author has any commercial associations or interests, including consultancies, stock ownership, or other competing equity interests or patentlicensing arrangements. ⁎ Corresponding author at: Department of Medical Research, Chang Gung Memorial Hospital, Kaohsiung, 123, Ta-Pei Road, Niao-Song District, Kaohsiung City, 83301, Taiwan. Tel.: +886 7 7317123; fax: +886 7 7322402. E-mail address: [email protected] (F.-S. Wang). 0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2012.06.128
proangiogenic factors, such as vascular endothelial growth factor (VEGF) [2], and chemotaxic factors, such as stromal-derived factor1α (SDF-1α) [3] and stem cell factor (SCF) [4,5] to attract endothelial progenitor cells. Mesenchymal stem cells also differentiate into the endothelial phenotype in vitro [6]. Transplantation of bone marrow stem cells enhances in vivo angiogenesis and results in functional improvement in both the hindlimb ischemic model and myocardial infarction (MI) model in animal studies [7,8]. These findings indicate that BMSCs are potential candidates for the treatment of ischemic disease. Systemic activation of the renin–angiotensin system (RAS) is the hallmark of severe cardiovascular disease, such as heart failure [9] and MI [10]. In patients with hypertension, advanced atherosclerosis, or heart failure, the plasma level of angiotensinogen increases. Angiotensinogen is converted to angiotensin I, which is in turn converted to the effecter peptide of RAS, angiotensin II (AngII), by
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Fig. 2. In vitro angiogenesis assay. (A) Abundance of SCF (left panel), SDF-1α (middle panel), and VEGF (right panel) in culture medium from BMSCs in different treatment groups. Culture media were harvested 7 d after treatment. Data were expressed as mean ± standard error calculated from at least 3 repeated experiments. *: pb 0.05 vs. control. (B) Representative microscopic photographs of capillary tube and vascular ring formation in HUVECs. Cells were cultured in Matrigel (100×) and treated BMSC culture medium for 5 h. Scale bar = 50 μm. (C) Number of capillary tube (left panel) and vascular ring (right panel) in HUVECs. Data were expressed as mean ± standard error calculated from 5 random fields (100×) in each experiment and at least 3 repeated experiments. *: p b 0.05 vs. CM-control.
angiotensin converting enzyme (ACE) [11]. By acting on type 1 AngII receptors (AT1Rs) in different organs, AngII promotes vasoconstriction, salt and water retention, secretion of aldosterone, and activation of the sympathetic nervous system [12,13]. Oxidative stress in endothelial cells is also increased due to the additional superoxide production after AT1R stimulation [14]. Various AT1R antagonists have been developed for the treatment of hypertension and heart failure by blocking the activated downstream signaling of AT1R [15]. Although suppression of the over-activated RAS is clinically beneficial for patients with heart failure and hypertension, the application of AT1R antagonists would increase the plasma AngII level in a negative feedback loop [16], leading to activation of type 2 AngII receptors (AT2Rs). Chronic subcutaneous delivery of AngII enhances
angiogenesis after the femoral artery is ligated in the mice hindlimb ischemic model; this phenomenon is blunted when an AT1R antagonist is orally administrated to mice [17]. Although an AT1R antagonist suppresses the angiogenesis effect, an ACE inhibitor has the opposite effect in the hindlimb ischemic model [18]. Stem cell therapy using bone marrow stem cells in animals with myocardial infarction [19] or hindlimb ischemia [20] has resulted in only modest functional improvement, and the improvements in survival and left ventricular systolic function are insignificant in most clinical trials [21,22]. Unlike patients who receive AT1R antagonists for heart failure or hypertension, experimental animals with ischemic disease are seldom administered with pharmaceutical agents when the effects of stem cell transplantation are evaluated. AT1R
Fig. 1. Characteristics and expression of angiotensin receptors in BMSCs harvested from eGFP mice. (A) BMSCs displayed fibroblast morphology (100×) in association with (B) green fluorescence eGFP reaction. Scale bar = 100 µm. (C) Expression of surface markers in BMSCs. Negative and positive immunostaining are expressed in open and red areas as detected by PE-conjugated isotype IgG and antibodies of interest, respectively. Percentage of positively-stained cells is bar scaled in each graph. (D) BMSCs had myocardiac differentiation capacity as evidenced by positive troponin-I immunoflorescence reactions (400×). Scale bar = 25 µm. (E) BMSCs had osteogenic differentiation potency as demonstrated by alizarin red staining (200x). Scale bar = 50 µm. (F) BMSCs had adipogenic differentiation capacity as evidenced by oil-red O staining (100×). Scale bar = 100 µm. (G) BMSCs expressed AT1R and AT2R in the presence or absence of AngII treatment.
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expression occurs in human bone marrow hematopoietic and stromal cells [23]. Local activation of RAS also occurs in the left ventricle after acute MI [10], which is speculated to be crucial for homing of hematopoietic stem cells into the infarcted area to repair damaged myocardium [24]. Additionally, AngII is now known to promote proliferation of HSCs [25]. Therefore, antagonism of AT1R may potentially cause biologic changes in stem cells, and we hypothesize that AT1R antagonists may alter the AngII-mediated angiogenic activity of BMSCs. In this study, we designed in vivo and in vitro experiments to elucidate the potential effects of AngII and AT1R on angiogenesis promoted by BMSCs. 2. Materials and methods
pluripotency of BMSCs, in vitro differentiation into osteogenic, cardiomyogenic, and adipogenic phenotypes was performed. A total of 5 × 104 mice BMSCs were seeded onto 24 well-plates and cultured in respective condition medium. Alizarin red staining, Oil-Red-O staining, and immunofluorescent (IF) staining of anti-mouse cardiac troponin-I (Tn-I) were performed to confirm osteogenesis, adipogenesis, and cardiomyogenesis, respectively. The details of primary culture, flow cytometry, and in vitro differentiation are provided in the Supplemental data. 2.3. Flow cytometry BMSCs were trypsinized and concentrated to 1 × 106 cells/100 μL PBS. Then they were incubated at 4 °C with 5 μL phycoerythrin-conjugated antibodies against mice for 30 min in the dark, rinsed twice with 2 mL PBS and fixed with freshly prepared, cold 1% paraformaldehyde (Sigma-Aldrich). Detection of phycoerythrin labeling was accomplished on a Cytomics 500 equipped with 570 nm argon laser (Beckman Coulter, Fullerton, CA) using CXP software with a minimum of 10,000 events counted.
2.1. Experimental animals 2.4. Reagents and in vitro cell treatment The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. All experimental protocols for animal studies were approved by the Animal Care Committee at our institute. Transgenic enhanced green fluorescent protein (eGFP) mice were kindly provided by Dr. Shinn-Chih Wu (Department of Animal Science and Technology, National Taiwan University). eGFP mice were generated by homologous recombination in embryonic stem cells and backcrossed 10 times into the ICR background, and eGFP is tagged on β-tubulin. Wild type ICR mice were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan), as control. 2.2. Identification of BMSCs Eight-week-old eGFP mice were euthanized by carbon dioxide gas inhalation followed by cervical dislocation, and BMSCs were isolated as described previously [26]. The cell cultures within 7 passages were used for the experiment. Flow cytometry with anti-mouse CD11b (BioLegend, Santa Fe, CA), CD29 (BioLegend), CD31 (eBioscience, San Diego, CA), CD34 (eBioscience), CD44 (BioLegend), CD45 (eBioscience), CD73 (BD Biosciences, San Diego, CA), CD86 (eBioscience), CD105 (eBioscience), CD117 (BD Biosciences), CD133 (BD Biosciences), CD166 (BD Biosciences), MHC-I (BD Biosciences), MHC-II (BD Biosciences), and Sca-1 (eBioscience) antibodies conjugated with phycoerythrin were used to identify BMSCs. To evaluate the
BMSCs were seeded at the density of 1 × 105/cm2 and treated with 10−7 M AngII (Sigma-Aldrich, St. Louis, MO), 10−5 M AT1R antagonist valsartan (Novartis, Basle, Switzerland), 10−5 M AT2R antagonist PD123319 (Sigma-Aldrich), or 10−8 M wortmannin (PI3K inhibitor) (Sigma-Aldrich) as indicated. For the experiments investigating AngII effects on Akt phosphorylation and VEGF transcription, undifferentiated BMSCs were cultured in serum-free for 3 h and subjected to the treatment of AngII and various pharmacological inhibitors as indicated. The expression of AT1aR (an isoform of AT1R) was knocked down by delivering small interfering RNA (siRNA) (Ambion, Austin, TX) into BMSCs using electroporation protocols. Scramble RNA was used as scrambled controls. As previously described [27], BMSCs were harvested at 24, 48, 72, and 96 h after electroporation to examine the expression of AT1aR by real time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The details of electroporation and RNA interfering are described in the Supplementary data. 2.5. Western blot Protein extracts were obtained from cell lysates of BMSCs and homogenized gastrocnemius muscles. The samples were probed with goat anti-AT1R (1:1000 dilution) (Abcam), goat anti-AT2R (1:2000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA),
Fig. 3. Effects of AngII and valsartan on phosphorylated Ser473-Akt and VEGF expression in BMSCs. (A) Representative immunoblotting and density plot of phosphorylated Ser473Akt in BMSCs under AngII with or without valsartan and wortmannin as indicated. *: pb 0.05 vs. baseline. (B) Expression of VEGF after treatment with AngII, valsartan or wortmannin for 6 h as indicated. Transcription of VEGF mRNA was determined by quantitative RT-PCR. *: p b 0.05 vs. control. (C) Representative immunoblotting and density plot of phosphorylated Ser473-Akt in BMSCs under AngII with or without PD123319 and wortmannin as indicated. *: pb 0.05 vs. baseline. . #: p b 0.05 vs. AngII. (D) Expression of VEGF after treatment with AngII, PD123319, or wortmannin for 6 h as indicated. Transcription of VEGF mRNA was determined by quantitative RT-PCR. *: p b 0.05 vs. control. #: p b 0.05 vs. AngII.
C.-I. Cheng et al. / International Journal of Cardiology 167 (2013) 2765–2774 rabbit anti-Akt (1:1000 dilution) (Cell Signaling Technology, Danvers, MA), rabbit antiphospho-Akt (Ser473) (Cell Signaling Technology), and mouse anti-VEGF (1:1000 dilution) (Abcam) antibodies, respectively, as described in the Supplementary data. Mouse anti-β-actin antibody (1:2000) (Chemicon International) was used as an internal control.
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dishes overnight in triplicate, and then different treatment conditions (i.e., control, AngII, AngII with valsartan) were applied for 7 days. The supernatant at days 0, 1, 4, and 7 was collected for ELISA using mouse VEGF, SDF-1α, and SCF Immunoassay Kit (R&D System Inc., Minneapolis, MN) according to the manufacturer's instructions. Optical density at 405 nm was determined by a microplate reader, and the concentrations of different samples were determined from the standard curves.
2.6. Quantitative real time RT-PCR Total RNA was extracted from cell lysates of BMSCs using the RNeasy Mini Kit (Qiagen, Qiagen AB, Solona, Sweden) according to the manufacturer's instructions. Reverse transcription was then performed with Roche Transcription First Structure cDNA Synthesis Kit (Roche Diagnostics) by using 1 μg of total RNA according to the manufacturer's instructions. Quantitative real time PCR was performed as described in the Supplemental data. The sequences of primers are listed in the Supplementary table. 2.7. Enzyme-linked immunosorbent assay (ELISA) The concentration of VEGF, SDF-1α, and SCF in the supernatant was measured by ELISA at indicated days. A total of 1 × 105 eGFP mice BMSCs were seeded to 6 well-
2.8. Capillary tube and vascular ring formation assay Matrigel-induced capillary tube and vascular ring formation using human umbilical vein endothelial cells (HUVECs) were performed to assess the angiogenic activity of BMSCs in triplicate. Briefly, Matrigel (BD Biosciences) was diluted to 4 mg/mL with cold PBS and added to 24-well plates in a total volume of 200 μL per well. Plates stood at 37 °C for 30 min to form a gel layer. After gel formation, 2 × 105 cells of human umbilical vein endothelial cell line were applied to each well, along with the addition of 1 mL supernatant from different treatment conditions described above for ELISA (day 7). The plates were then incubated at 37 °C for 24 h with 5% CO2. Following incubation, HUVECs were observed, and the formation of capillary
Fig. 4. Effect of AT1aR RNA interference on Akt phosphorylation and VEGF expression in BMSCs. (A) AT1aR RNA interference decreased AT1aR mRNA expression. mRNA expression in cell cultures was detected by quantitative RT-PCR. *: p b 0.05 vs. scramble control. Representative immunoblotting of (B) AT1R expression and (C) phosphorylated Ser473-Akt expression in AT1aR RNA interference- (upper panel) and scramble control-transfected (lower panel) BMSCs. (D) AT1aR RNA interference decreased VEGF mRNA expression in BMSCs. mRNA expression in cell cultures was detected by quantitative RT-PCR 8 h after treatment. Data was expressed as mean ± standard error calculated from at least 3 repeated experiments. *: p b 0.05 vs. vehicle group.
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tubes and vascular rings was quantified in 3 randomly chosen fields in triplicate at 100× magnification.
2.9. Hindlimb ischemic model and transplantation of eGFP BMSCs Six of eight-week-old male ICR mice in each group were pretreated for 2 weeks with 20 mg/kg/day of valsartan (provided by Novartis) or vehicle dissolved in drinking water. A total of 3 × 105 BMSCs from eGFP mice suspended in 50 μL phosphate-buffered saline (PBS) (separated into 2 injections) (n = 6 with valsartan; n = 6 without valsartan) or PBS alone (n = 6 with valsartan; n = 6 without valsartan) were injected into the left gastrocnemius muscle 24 h after ligation of the femoral artery under the anesthesia with intraperitoneal 200 mg/kg ketamine and 10 mg/kg xylazine. An analgesic of 0.1 mg/kg buprenorphine was administered subcutaneously to relieve pain when observed. Laser Doppler blood flow (LDBF) imaging was then performed as described in the Supplementary data.
2.10. Immunohistochemistry (IHC) staining and IF staining IHC staining was performed using gastrocnemius muscles in the legs with hindlimb ischemia, and IHC staining was performed with rabbit anti-von Willebrand factor (vWF) antibody (1:250 dilution) (Chemicon International, Temecula, CA) as previously described [28]. Capillary density was then calculated as capillaries per field and per muscle fiber in 3 randomly chosen fields of each specimen (6 mice in each group) at 200× magnification. The details of immunocytochemistry and IHC staining are described in the Supplemental data. To elucidate the in vivo endothelial differentiation and paracrine function of eGFP mice BMSCs after transplantation, the tissue from gastrocnemius muscles was processed as described in the IHC procedure, and tissue sections were subjected to IF staining with mouse anti-VEGF antibody (1:500 dilution) (Abcam, Cambridge, MA), and rabbit anti-vWF antibody (1:250 dilution). Images were generated by Zeiss LMS5 Live confocal microscope (Zeiss, Oberkochen, Germany). The details of the IF are shown in the Supplementary data.
2.11. In vivo fluorescence tracking In order to confirm whether transplanted eGFP BMSCs were retained in the gastrocnemius muscles, mice receiving eGFP BMSCs transplantation after hindlimb ischemia were examined by Lumazone In Vivo Imaging System (MAG Biosystems, Pleasanton, CA) at day 15 after anesthesia with intramuscular ketamine 50 mg/kg and xylocaine 50 mg/kg. 2.12. Statistical analysis Data were presented as mean ± SEM and analyzed using a two-tailed t test or one-way ANOVA processed by the operating SPSS 11.0 computer program (SPSS Inc., Chicago, IL). A probability value b0.05 is considered statistically significant.
3. Results 3.1. Constitutive expression of AT1R and AT2R in BMSCs We verified whether bone marrow stromal cells (BMSCs) in eGFP mice expressed AT1R or AR2R. Microscopic observation showed that BMSCs displayed spindle-shape, fibroblast-like morphology (Fig. 1A) and green fluorescence (Fig. 1B). Flow cytometric analyses revealed that BMSCs were positive for CD29 (86.2%), CD44 (98.0%), CD73 (75.2%), CD105 (90.7%), Sca-1 (93.8%) and MHC-I (93.3%), but negative for CD11b (0.5%), CD31 (0.3%), CD34 (0.5%), CD45 (0.4%), CD86 (0.3%), CD117 (0.5%), CD133 (0.7%), CD166 (0.9%), and MHC-II (0.5%) (Fig. 1C). Additionally, BMSCs differentiated toward myogenic, osteogenic, and adipogenic lineages, as demonstrated by positive stain
Fig. 5. Angiogenesis in ischemic limbs with or without BMSCs or valsartan treatment. (A) Representative laser Doppler images of ischemic hindlimb at 15 days after BMSCs transplantation into ischemic mice with or without valsartan administration. (B). Treatment with valsartan reduced the promoting effect of BMSCs graft on blood flow in ischemic limbs. *: p b 0.05 vs. PBS group. (C) Representative images of capillary vessels as demonstrated by positive vWF immunostaining in gastricnemius muscle of ischemic limb at 14 days after BMSCs or valsartan treatment (200×). Scale bar = 50 µm. (D) Administration of valsartan attenuated the promotional effects of BMSCs on capillary vessel distribution in ischemic limbs. Data was calculated from 3 randomly-selected fields (left panel) and 30 random muscle fibers (right panel) from 6 animals in each group. *: P b 0.05; **: P b 0.001; ****: P b 0.0001 vs. PBS group.
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for troponin-I expression (Fig. 1D), Alizarin red staining (Fig. 1E), and Oil-Red-O (Fig. 1F). Experiments were conducted to verify whether BMSCs expressed AT1R or AT2R. Immunoblotting showed that BMSCs constitutively expressed AT1R and AT2R (Fig. 1G). 3.2. AT1R antagonists attenuate the AngII-mediated angiogenic activity of BMSCs We tested whether AT1R or AT2R signaling affected angiogenic activities of BMSCs. ELISA results showed that AngII treatment significantly increased VEGF levels in culture medium harvested from BMSCs. The AT1R antagonist valsartan significantly attenuated the AngII-induced promotion of VEGF secretion by BMSCs (Fig. 2A). The AT2R antagonist PD123319 did not change the AngII-induced increase in VEGF. These findings indicated that activation of AT1R enhances the secretion of VEGF. Exposure to AngII, valsartan, or PD123319 did not significantly affect SCF or SDF-1α secretion by BMSCs. Experiments were also conducted to determine whether culture medium harvested from AngII or valsartan-treated BMSCs could affect the angiogenesis of HUVECs. Microscopic observation showed that cells in the Ang II group had increased tube and vessel ringlike morphology. These effects were reduced after valsartan treatment (Fig. 2B). Valsartan treatment significantly attenuated the promotional effects of AngII on the number of tubes and vascular rings in HUVEC. PD123319 did not markedly affect the angiogenic activity of BMSCs (Fig. 2C). 3.3. AT1R antagonists attenuate the AngII-mediated Akt signaling and VEGF expression in BMSCs We investigated whether AngII or an AT1R antagonist could affect Akt phosphorylation or VEGF expression. BMSCs were incubated in the serum-free conditions. Immunoblotting showed that AngII treatment promoted phosphorylated Ser473-Akt expression in 45 min and 60 min, respectively. Valsartan or PI3K inhibitor wortmannin treatment attenuated the AngII-induced enhancement of phosphorylated Ser473-Akt levels (Fig. 3A). Treatment with valsartan or wortmannin alone or combined treatment with valsartan and wortmannin did not significantly affect the baseline levels of phosphorylated Akt (Fig. 3A). Quantitative RT-PCR analyses revealed that treatment with valsartan or wortmannin alone or combined treatment with valsartan and wortmannin significantly reduced the promotional effects of AngII on the VEGF expression in cell cultures (Fig. 3B). Notably, treatment with valsartan alone did not significantly reduce the baseline VEGF expression whereas valsartan and wortmannin synergistically block the AngII-induced VEGF transcription. Treatment with PD123319 (an AT2R antagonist) did not significantly change the promotional effects of AngII on the concentrations of phosphorylated Akt (Fig. 3C) or the expression of VEGF in BMSCs (Fig. 3D). Analytic results indicate that AngII increased VEGF expression through AT1R-dependent Akt signaling in BMSCs. Moreover, compared with the scrambled control, AT1aR RNA interference significantly decreased the expression of AT1R mRNA (Fig. 4A), AT1R1 protein (Fig. 4B), phosphorylated Ser473-Akt (Fig. 4C), and VEGF (Fig. 4D) in the AngII-treated BMSCs.
Fig. 6. In vivo VEGF abundance in the ischemic hindlimb tissues. (A) Representative immunoblot images of VEGF gastrocnemius muscle in ischemic limb at 15 days after BMSCs or valsartan treatment. (B) Intensity of VEGF immunoblots. Treatment with valsartan reduced the BMSC-induced enhancement of VEGF concentrations in ischemic limbs. Data was calculated from 6 animals in each group. *: Pb 0.05 vs. PBS treatment alone group.
showed that gastrocnemius muscles in the ischemic legs after BMSC transplantation displayed increased capillary density, as evidenced by vWF expression (Fig. 5C). Histomorphometric analyses showed that treatment with valsartan significantly attenuated the BMSCinduced increase in capillary vessel number (Fig. 5D). Gastrocnemius muscle strongly displayed VEGF expression following BMSC transplantation. The injured tissue weakly expressed VEGF immunoreactivity after valsartan treatment (Fig. 6A and B). 3.5. eGFP BMSCs did not differentiate into endothelial phenotype after transplantation We determine whether BMSCs could differentiate early in the hindlimb ischemic model. In vivo fluorescence observation showed that injured sites strongly displayed green fluorescence at 2 weeks after transplantation of eGFP BMSCs, which suggested the allografted eGFP BMSCs were still viable in injured sites (Fig. 7A). Confocal microscopic images showed that green fluorescent eGFP BMSCs codisplayed red fluorescence VEGF immunoreactivity (Fig. 7B, left panel) but not vWF immunostaining (Fig. 7B, right panel). These effects were attenuated after valsartan treatment. Analytic results suggest that graft with BMSCs enhanced angiogenesis in the ischemic hindlimbs was through induction of VEGF but not endothelial differentiation of BMSCs. 4. Discussion
3.4. AT1R antagonists attenuate the in vivo angiogenesis in the hindlimb ischemic model with intramuscular transplantation of BMSCs
4.1. Mechanism of angiogenesis
We tested whether BMSCs or valsartan treatment changed angiogenesis in mice with ischemic hindlimbs. BMSCs from eGFPtransgenic mice were allografted into the injured site. BMSCs transplantation significantly increased blood flow in injured tissues compared to control group. Administration of valsartan significantly attenuated the BMSC graft-induced enhancement of blood flow in ischemic hindlimbs (Fig. 5A and B). Immunohistochemical observation
The present study demonstrates that activation of AT1R/PI3K/Akt is crucial for the angiogenic effect of BMSCs, which is attenuated by pharmacological inhibition or AT1aR RNA interference. Unlike a previous study in a mice MI model [29] which showed eGFP BMSCs incorporated into vessel after bone marrow transplantation, our study hardly recognized the co-localization of endothelial markers with eGFP expressed in BMSCs in the early phase of in vivo angiogenesis
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Fig. 7. Representative fluorescence photographs of grafted BMSCs in ischemic hindlimb. (A) Localization of exogenous eGFP-BMSC (green fluorescence) in ischemic hindlimb by using an in vivo fluorescence tracing system 15 d after various treatments as indicated. Left panel: white light. Middle panel: green fluorescence. Right panel: merged image. (B) Representative confocal images of VEGF expression (red fluorescence; let panel) and vWF (red fluorescence; right panel) in eGFP-BMSC (green fluorescence) in gastrocnemius muscle. Specimens were probed by using antibodies against VEGF and vWF conjugated with Alexa Fluor 568, respectively. Nuclei in cells were recognized by DAPI (blue fluorescence). Scale bar = 100 μm.
with confocal imaging. Even though BMSCs may potentially express endothelial markers in vivo, formation of functional capillary networks is not feasible during the early stage of angiogenesis, suggesting that trans-differentiation is less likely to be the mechanism of angiogenesis. Increased production of VEGF in muscle proportional to capillary density and increased in vitro secretion of VEGF detected by ELISA in our study suggested that enhanced angiogenesis by BMSCs was mainly due to the paracrine effect and not the direct formation of capillaries by BMSCs. Several pro-angiogenic factors, including VEGF and FGF [30], and chemo-attractants, such as SDF-1α and SCF, are involved in bone marrow stem cell-related angiogenesis [31]. In this study, we demonstrated that the AT1R antagonist attenuates the AngII-mediated VEGF production; the effect of AngII on the secretion of SDF-1a and SCF is neutral. Furthermore, a previous study [25]
suggested that the anti-proliferative effect of the AT1R antagonist could be another mechanism attenuating the angiogenesis of BMSCs. Further investigation is warranted to study the effect of AT1R antagonism on the in vivo survival of BMSCs. 4.2. Angiogenesis and renin–angiotensin system (RAS) In the rodent hindlimb ischemic model, ACEI and the AT1R antagonist demonstrate opposite effects on angiogenesis, although both exert similar effects in the treatment of hypertension and heart failure. The pro-angiogenic effect of ACEI is mediated by the bradykinin B2 receptor-dependent up-regulation of endothelial nitric oxide synthetase (eNOS) and is independent of VEGF expression [32]. On the contrary, the action of AngII on angiogenesis is mediated by the AT1R-related expression of VEGF [33] and does not exist in eNOS-
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deficient mice [34]. This finding suggests that AngII enhanced ischemia-induced angiogenesis through AT1R activation involves the VEGF/eNOS-dependent pathway. Furthermore, impaired angiogenesis in hindlimb ischemia in AT1aR-deficient mice was rescued successfully by peripheral blood mononuclear cells derived from wild type mice rather than those derived from AT1aR-deficient mice [35]. These findings also indicate that the AngII/AT1R/VEGF pathway positively modulates ischemia-induced angiogenesis, and our study confirmed that RAS axis is important for the pro-angiogenic effects of BMSCs. 4.3. AT2R vs. AT1R in angiogenesis of BMSCs Conflicting results have been observed regarding the roles of AT1R and AT2R in AngII-related angiogenesis. AT2R activation by AngII may increase hypoxia-induced endothelial sprouting in mice hearts through the bradykinin B2 receptor/nitric oxide/cGMP pathway [36]. Blockage of AT1R during AngII stimulation may counterregulate the activation of AT2R, subsequently leading to the apoptosis of smooth muscle cells and the pro-angiogenic effect of endothelial cells. On the other hand, AngII was found to increase eNOS expression and cGMP release in bovine aortic endothelial cells overexpressing AT1R, and this effect was further accentuated by the AT1R antagonist [37]. Another study using the hindlimb ischemic model in Agtr2 −/Y mice showed that AT2R may negatively modulate ischemia-induced angiogenesis through an activation process, and the up-regulation of Bcl-2 and eNOS was detected independent of VEGF expression in the ischemic legs of Agtr2 −/Y mice [17]. In our present study, the secretion of VEGF caused by AngII was not affected by PD123319, suggesting that AT2R-mediated anti-angiogenesis was not observed in our model using BMSCs. Similarly, angiogenesis was not altered in AT1R-deficient mice when mice were treated simultaneously with 30 mg/kg/day of PD123319 and implanted with peripheral blood mononuclear cells [35]. Although AT1R and AT2R are both expressed on BMSCs, the effects of AngII on angiogenesis are mainly mediated through AT1R. The self-renewal ability dominantly drives BMSCs away from the AT2R-related anti-proliferative and antiangiogenic effect. 4.4. Akt signaling in BMSCs Signaling of AngII-mediated angiogenesis and proliferation in stem cells occurs via several different mechanisms. PI3K/Akt signaling is important for the maintenance of pluripotency and viability in human embryonic stem cells [38]. One study has shown that AngII stimulates DNA synthesis by increasing cyclin and cyclin-dependent kinase, which is mediated through the AT1R-dependent Ca ++/phosphokinase C pathway and the epidermal growth factor receptor/ PI3K/Akt/mTOR-dependent pathway [39]. Another recent publication also indicates AngII stimulated the synthesis of VEGF in MSCs through the activation of ERK1/2 and the Akt pathway via AT1R activation [40]. Furthermore, Akt overexpression in MSCs promoted cardiac repair more significantly by enhancing paracrine function, including the secretion of VEGF [41]. These findings, along with the results of our present study, indicate that AT1R/PI3K/Akt signaling is crucial for the angiogenesis of BMSCs. Nevertheless, how AngII activates PI3K through AT1R and how Akt induces the transcription of VEGF in BMSCs remain unclear. Future study should focus on exploring these issues. 4.5. Clinical implication AT1R antagonists are widely used to treat a variety of cardiovascular diseases. Additional benefits include halting the progression of atherosclerosis [42], reducing restenosis of intracoronary stents [43], and preventing post-MI remodeling of the left ventricle [44].
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Although direct antagonism of AT1R reduces the bradykinin-related side effects induced by ACEI, these agents do not result in better clinical outcomes (i.e., prevention of MI and cardiovascular death) [45]. As previously mentioned, AT1R antagonists slow ischemiainduced angiogenesis in the hindlimb ischemic model and in in vitro cell survival. Our study also confirms that this agent may impair ischemia-induced angiogenesis of mice BMSCs. Despite the over-activation of RAS in cardiovascular disease, further evaluation is warranted, especially for the patients receiving stem cell therapy for severe ischemic conditions.
5. Conclusion To our knowledge, this is the first time that an AT1R antagonist is demonstrated to attenuate the in vivo angiogenesis of BMSCs. Based on the results of this study, we conclude that AT1R antagonists impair the AngII-related angiogenesis of mice BMSCs by inactivation of the AT-1aR/PI3K/Akt-related pathway. Application of AT1R antagonists in patients receiving stem cell therapy for ischemic conditions needs further evaluation. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijcard.2012.06.128.
Acknowledgments This work was supported in part by the grants from the Taiwan National Science Council (95-2745-B-182A-005) and Kaohsiung Chang Gung Memorial Hospital (CMRPG 860521). The authors also thank the Center for Laboratory Animals for the use of the facilities.
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