The PPAR-γ agonist pioglitazone increases neoangiogenesis and prevents apoptosis of endothelial progenitor cells

Atherosclerosis 192 (2007) 67–74

The PPAR-␥ agonist pioglitazone increases neoangiogenesis and prevents apoptosis of endothelial progenitor cells Christoph Gensch, Yvonne P. Clever, Christian Werner, Milad Hanhoun, Michael B¨ohm, Ulrich Laufs ∗ Klinik f¨ur Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universit¨atsklinikum des Saarlandes, 66421 Homburg/Saar, Germany Received 19 February 2006; received in revised form 5 June 2006; accepted 14 June 2006 Available online 28 July 2006

Abstract PPAR-␥ agonists (thiazolidinediones, TZDs) may improve endothelial function independently of insulin sensitizing. Bone marrow-derived endothelial progenitor cells (EPC) contribute to neoangiogenesis. Mice were treated with pioglitazone, 20 mg/kg/day for 10 days. Treatment with TZD upregulated circulating Sca-1/VEGFR-2 positive EPC in the blood (235 ± 60%) and the bone marrow (166 ± 30%), cultured spleen-derived DiLDL/lectin positive EPC increased to 231 ± 21% (n = 24 per group). Upregulation of EPC was persistent after 20 days. TZD increased SDF-1-induced migratory capacity per number of EPC by 246 ± 73% and increased expression of telomere repeat-binding factor 2 by 320 ± 50%. In vivo neoangiogenesis was increased two-fold (214 ± 42%, 20 days). The NOS inhibitor l-NAME did not inhibit the TZD-induced upregulation of EPC. EPC from TZD-treated animals showed reduced in vivo apoptosis (65 ± 2.8% of vehicle). In cultured human EPC, pre-treatment with pioglitazone prevented H2 O2 -induced apoptosis. Inhibition of EPC apoptosis by TZD was abolished in the presence of wortmannin but not by LNMA. In summary, TZD upregulates both number and functional capacity of endothelial progenitor cells. Pioglitazone prevents apoptosis of EPC in mice as well as in human EPC in a PI3K-dependent but NO-independent manner. Reduction of EPC apoptosis by TZD may be a potentially beneficial mechanism for patients with vascular diseases. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelial progenitor cells; Apoptosis; Angiogenesis; Thiazolidinediones; PPAR

1. Introduction Peroxisome proliferator-activated receptor-␥ (PPAR-␥) agonists (thiazolidinediones, TZDs) lower serum glucose levels in patients with diabetes mellitus type 2. In addition to their insulin sensitizing effects, increasing evidence suggests that these drugs improve endothelium-dependent vascular function and inflammatory biomarkers of arteriosclerosis [1–3]. Interestingly, these vascular effects seem to occur independently of glucose lowering and have been demonstrated in non-diabetic, healthy individuals [1,3–5]. Furthermore, experiments on cultured vascular cells support a direct and ∗

Corresponding author. Tel.: +49 6841 162 3000; fax: +49 6841 162 1331. E-mail address: [email protected] (U. Laufs).

0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.06.026

beneficial modulation of key regulators of arteriosclerosis such as cellular adhesion molecules, tissue factor, plasminogen activator inhibitor and matrix metalloproteinases (for review see [6–8]). These findings have led to the hypothesis that TZDs may exert vasculoprotective effects independent of their metabolic action [7]. Recent evidence has shown that cardiovascular function and angiogenesis is significantly modulated by circulating premature cells derived from the bone marrow [9]. A subset of these stem cells named endothelial progenitor cells [10], EPC, enhances angiogenesis, promotes vascular repair and improves endothelial function [11–17]. The circulating numbers of EPC are regulated. Vascular risk factors such as diabetes and hypertension have been shown to reduce EPC suggesting that vascular health

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and repair require increased numbers of this beneficial population of cells [12,18–21]. On the other hand, lipid lowering with statin drugs or physical activity can be employed to raise EPC numbers and improve their function [11,13,19,22,23]. Recently it was shown that reduced levels of circulating EPCs represent a cellular marker that independently predicts outcome in patients with vascular disease [20,24]. We hypothesized that the PPAR-␥ agonist pioglitazone may modulate vascular function through regulation of endothelial progenitor cells. The level of CD34 + VEGFR2 + mononuclear cells was determined because this cell population predicts the occurrence of cardiovascular events and death from cardiovascular causes [24]. Mice were treated with pioglitazone alone and in the presence of an inhibitor of nitric oxide synthase, and the number and function of EPC as well as neoangiogenesis were quantitated.

2. Methods 2.1. Animals Animal experiments were conducted in accordance to institutional guidelines (Tierversuchsgenehmigung K110/180–07). Male C57/Bl6 mice (Charles River) were treated with saline (vehicle) or pioglitazone 20 mg/kg/day s.c. (gift of Takeda Pharma GmbH, Aachen, Germany). For indicated mice, NG-nitro-l-arginine methyl ester (l-NAME) (Sigma) was added to the drinking water (daily dose, ∼50 mg/kg; concentration in drinking water, 1.5 mg /ml) [25]. Serum concentrations of glucose, total cholesterol, HDL-cholesterol or LDL-cholesterol were determined by the Department of Clinical Chemistry, Universit¨atsklinikum des Saarlandes, Homburg, Germany.

pliciforia lectin I (lectin, 10 ␮g/ml) (Vector Laboratories). Apoptotic cell death was quantitated by ELISA of cytoplasmic histone-associated DNA fragments (Cell Death Detection ELISA-plus, Roche). 2.4. Migration assay Migratory capacity of EPCs was determined in a modified boyden chamber [26]. EPC were harvested by centrifugation, resuspended in 500 ␮l EBM with supplements, counted and placed in the upper chamber of a modified boyden chamber (1 × 105 cells/per chamber; BD Biosciences). The chamber was placed in a 24-well culture dish containing EBM with supplements and VEGF (50 ng/ml). After incubation at 37 ◦ C for 24 h, the lower side of the filter was washed, fixed and incubated with DiLDL. Migrated cells at the lower part of the filter were counted manually in four random microscopic fields. 2.5. Real-time RT-PCR Real-time reverse transcription-polymerase chain reaction was performed with the Prism 7700 Sequence Detection System, PE Biosystems. Primers for eNOS were 5 -TTCCGGCTGCCACCTGATCCTAA-3 and 5 -AACATATGTC-

2.2. FACS-analysis Blood and bone marrow were analysed as described [15,16,19,23]. The viable lymphocyte population was analyzed for Sca-1-FITC (E13–161.7, Pharmingen) and VEGFR-2 (Flk-1) (Avas12␣1, Pharmingen) conjugated with the corresponding PE labeled secondary antibody (Sigma). Isotype-identical antibodies served as controls in every experiment (Becton Dickinson). 2.3. Culture of spleen-derived EPC In mice, the spleen functions as hematopoietic organ. Spleen mononuclear cells were isolated and cultured on fibronectin (Sigma) as described [15,16,19,23]. After 7 days in culture, EPC were identified by uptake of 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyaninelabeled acetylated LDL (DiLDL, 2.4 ␮g/ml, CellSystems) and staining with FITC labeled Giffonia (bandeiraea) sim-

Fig. 1. Sca-1/VEGFR2+ endothelial progenitor cells were quantified by FACS analyses in samples of peripheral blood (A) and bone marrow (B) in mice treated with vehicle-(white bars), pioglitazone (Pio, black bars), 20 mg/kg/day and Pio + l-NAME (50 mg/kg/day, grey bars) for 10 days.

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CTTGCTCAAGGCA-3 . For 18S the primers were 5 -TTGATTAAGTCCCTGCCCTTTGT and 5 -CGATCCGAGGGCCTA ACTA. 2.6. Disc angiogenesis model A disc of polyvinyl alcohol sponge (Rippey), covered with nitrocellulose cell-impermeable filters (Millipore), allows capillaries to grow only through the rim of the disc [23,27]. The discs were subcutaneously implanted. After 20 days, space-filling fluorescent microspheres (0.2 ␮m; Molecular Probes) were injected into the left ventricle to deliver them to the systemic microvasculature. The area of the disc invested by fibrovascular growth was assessed using Lucia Measurement Version 4.6 software.

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2.7. Culture of human EPC and quantification of apoptosis Mononuclear cells were isolated by density gradient centrifugation with Biocoll from peripheral blood of healthy human volunteers (n = 6) as described [24]. Following isolation, 106 MNCs/ml medium were plated on culture dishes coated with human fibronectin (Sigma) and maintained in endothelial basal medium (CellSystems) supplemented with EGM SingleQuots and 20% fetal calf serum. After 3 days in culture, nonadherent cells were removed by thorough washing with phosphatebuffered saline and adherent cells were incubated in fresh medium with or without pioglitazone, H2 O2 (Sigma), Ly294002 (Biomol) and LNMA (Alexis). EPCs were char-

Fig. 2. (A) Representative fluorescence microscopy of lectin and DiLDL-positive endothelial progenitor cells and (B) quantification after treatment with vehicle (white bars, 10 days), pioglitazone 20 mg/kg/day (Pio, black bars, 10 and 20 days), and Pio + l-NAME (50 mg/kg/day, grey bars, 10 days).

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acterized by dual staining for 1,1 -dioctadecyl-3,3,3 3 tetramethylindocarbocyaninelabeled acetylated low-density lipoprotein (DiLDL) and lectin. Detection of apoptosis by FACS analysis: adherent cells were detached with trypsin, washed in PBS, and incubated with fluorescein isothiocyanateconjugated annexin V [28]. IgG 2a-fluorescein isothiocyanate (Pharmingen) served as negative control. FACS analysis was performed immediately after staining using a FACS Calibur instrument (Becton Dickinson) and Cell Quest software (BD Biosciences). Detection of apoptosis by DAPI staining: Cells were cultured on glass slides coated with human fibronectin, fixed in 4% formaldehyde, and stained with 4 ,6-diamidinophenylindole (DAPI; 0.2 ␮g/ml in 10 mmol/l Tris–HCl, pH 7.0, 10 mmol/l EDTA, 100 mmol/l NaCl) for 20 min. Five hundred cells were counted by two independent blinded investigators, and the percentage of apoptotic cells per total number of cells was determined. 2.8. Western blotting Proteins were isolated and prepared as described [23,29]. Immunoblotting was performed using TRF2 (H-300 rabbit polyclonal Ab; Santa Cruz sc-9143; 1:250), p-Akt (Ser 473 rabbit polyclonal Ab; Santa Cruz sc-7985-R; 1:250) and ␤actin (Sigma A5441; 1:500). Immunodetection was accomplished using goat anti-rabbit or goat anti-mouse secondary antibody (1:4000, Sigma) and an enhanced chemiluminescence kit (Amersham).

(20 mg/kg/day, 10 days) did not alter eNOS mRNA expression in the aorta (104 ± 2.4% of vehicle treated mice, n = 4). In order to further test if the upregulation of EPC was mediated by nitric oxide synthase, mice (n = 14) were treated with TZD in the presence of NG-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg [25]). This concentration of l-NAME inhibited exercise-induced upregulation of EPC [23]. However, l-NAME did not inhibit the TZD-induced upregulation of Sca-1/VEGFR-2 positive EPC in the blood nor the bone marrow (Fig. 1). As a second method of EPC quantification, DiLDL/lectin positive EPC were expanded from spleen-derived mononuclear cells. In animals treated with TZD for 10 days, cultured EPC increased to 231 ± 21% of vehicle (n = 24, p < 0.01) (Fig. 2). Upregulation of EPC was persistent after 20 days of treatment (234 ± 19% of vehicle, n = 14, p < 0.01). Again, inhibition of NOS by l-NAME did not block the TZDinduced increase of EPC. Migration is an important characteristic of EPC function. SDF-1-induced migratory capacity was tested in a boyden chamber assay. Treatment with TZD increased migration per number of EPC to 246 ± 73% of control after 10 days (n = 14, p < 0.05) and to 235 ± 19% after 20 days (n = 14, p < 0.01) (Fig. 3). Mechanistically, the capacity of ex vivo expansion and migration of EPC has been shown to depend on the protein expression of telomere repeat-binding factor 2 [29]. We therefore quantitated TRF2 expression by western analysis.

2.9. Statistical analysis Results are presented as mean ± S.E.M. Paired and unpaired Student’s t-tests and ANOVA for multiple comparisons were employed where applicable. Post-hoc comparisons were performed with the Neuman–Keuls test. Values of p < 0.05 were considered significant.

3. Results Treatment with pioglitazone 20 mg/kg/day for 10 days did not change the serum concentrations of glucose, total cholesterol, HDL-cholesterol or LDL-cholesterol. To test the effects of PPAR-␥ agonism on EPC, C57/Bl6 mice were treated with pioglitazone, 20 mg/kg/day or saline for 10 days. Sca-1/VEGFR-2 positive EPC were quantified by FACS analysis. Treatment with TZD increased circulating blood EPC to 235 ± 60% of vehicle-treated mice (n = 24 per group, p < 0.02) (Fig. 1A). TZD not only increased EPC circulating in the peripheral blood but also in the bone marrow (upregulation to 166 ± 30%, p < 0.05) (Fig. 1B). Endothelial nitric oxide synthesis has been shown to be an important regulator of EPC release [14]. Therefore, the effect of Pioglitazone on eNOS expression was studied using real-time RT-PCR. Treatment with pioglitazone

Fig. 3. (A) Effect of treatment with pioglitazone 20 mg/kg/day (Pio, black bars, 10 and 20 days) on EPC migration in a modified boyden chamber using VEGF 50 ng/ml as chemo-attractant and (B) quantification and representative western blot of telomere repeat-binding factor (TRF2) protein expression of murine EPC treated with pioglitazone 10 ␮M for 24 h.

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Fig. 4. Effect of treatment with pioglitazone 20 mg/kg/day (Pio, black bars, 20 days) on neoangiogenesis: (A) representative example and (B) quantification of ingrowth of new vessels around the border of the polyvinyl sponge.

Treatment with TZD upregulated TRF2 expression in mouse EPC to 320 ± 50% (n = 5, p < 0.05). Improvement of neoangiogenesis is one of the best characterized functions of EPC [9]. To assess the effects of TZD on neoangiogenesis in vivo, the in-growth of new vessels was quantitated in a subcutaneously implantated polyvinyl sponge (20 days) by perfusion with fluorescent microspheres (0.2 ␮m) via the left ventricle. Treatment with pioglitazone increased the area of neoangiogenesis by two-fold (214 ± 42%, n = 6, 20 days, p < 0.05) (Fig. 4). EPC apoptosis might be a potential mechanism to regulate the number of circulating EPC [11,23,30]. Therefore, the rate of apoptosis was quantitated in EPC isolated from TZD and vehicle treated animals. Fig. 5 demonstrates that TZD decreased EPC apoptosis to 65 ± 2.8% of vehicle treated mice (n = 6, p < 0.05). To extent these finding to human cells, cultured EPC from six healthy volunteers were treated ex vivo with pioglitazone in the presence and absence of H2 O2 . Analysis of apop-

tosis by two independent methods, annexin V FACS and morphological analysis [30], showed significantly decreased apoptosis in the presence of pioglitazone (n = 6, p < 0.05) (Fig. 6). The inhibition of EPC apoptosis by TZD was com-

Fig. 5. Rate of apoptosis in EPC isolated from mice treated with pioglitazone 20 mg/kg/day (Pio, black bars, 20 days) compared to vehicle treated animals.

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Fig. 6. Regulation of apoptosis in human endothelial progenitor cells: (A) detection of apoptosis by annexin V staining. Effect of pioglitazone 10 ␮M alone and in the presence of H2 O2 (500 ␮M, 24 h) on apoptosis in cultured human EPC, (B) representative slides and (C) quantification of apoptotic cell death by morphological analysis. Five hundred cells were counted by two independent blinded investigators, and the percentage of apoptotic cells per total number of human EPC was determined after treatment with pioglitazone 10 ␮M alone and in the presence of H2 O2 (500 ␮M, 24 h) as well as after 30 min pretreatment with LY294002 (Ly, 10 ␮M), wortmannin (Wort, 100 nM) or LNMA (1 mM).

pletely abolished in the presence of the PI3K inhibitors wortmannin (100 nM) or LY294002 (10 ␮M) but not by the NOS-inhibitor LNMA (1 mM) (Fig. 6C). Treatment with pioglitazone (10 ␮M, 24 h, n = 5) did not change the protein expression of phosphorylated Akt, a downstream target of PI3K. Similar to the murine EPC, protein expression of TRF2 was increased in human EPC treated with pioglitazone, 1 ␮M, 72 h, to 344 ± 36%.

4. Discussion Treatment with the PPAR-␥ agonist pioglitazone increased the number of endothelial progenitor cells circulating in the blood as well as in the bone marrow. The upregulation of EPC was observed in healthy wild-type mice, TZD-treatment did not alter glucose or lipid serum levels. Upregulation of EPC persisted at least for 20 days. In addition

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to EPC numbers, the migratory capacity per number of EPC, an established parameter of EPC function, was increased after TZD treatment. TZD prevented EPC apoptosis in mice as well as in cultured human EPC. Angiogenesis, defined as sprouting of blood vessels from pre-existing vascular structures, is a central mechanism of tissue protection in all vascular diseases. EPC were originally identified and characterized for their ability to enhance neoangiogenesis and to augment collateral vessel growth [9,31]. Angiogenesis is characterized by the recruitment of EPC to sites of new vessel formation with subsequent differentiation of EPC into mature endothelial cells. The effect on neoangiogenesis in vivo therefore represents a very important parameter to determine EPC function [31]. Here we show that systemic treatment with pioglitazone leads to a robust increase of neoangiogenesis. However, this central finding is in contrast to reports that have shown that application of PPAR-␥ ligands inhibits choroidal and corneal neovascularization as well as vascularization of several tumor models [32–34]. On the other hand, the data are in agreement with recent findings of Wang et al. showing that the PPAR-␥ agonist rosiglitazone promotes the differentiation of bone marrow derived progenitor cells to endothelial cell lineage [35] and the recent report by Pistrosch et al. demonstrating upregulation of EPC in type 2 diabetic patients by rosiglitazone [36]. In addition, Verma et al. showed that rosiglitazone prevents impairment of EPC function induced by treatment with C-reactive protein [37]. In this study, the pro-angiogenic effect of pioglitazone was observed after systemic treatment. In addition, the disk model of neoangiogenesis applied measures the formation and spreading of newly formed microvessels with full functional capacity [38]. The discrepancy of our findings in vivo to the published reports of the antiangiogenic effects of TZD in cell cultures and animal models such as the chick chorioallantoic membrane or the avascular cornea may have parallels to the effects of statins on angiogenesis. Statins potently inhibit angiogenesis in high concentrations and cell culture models, but promote angiogenesis (and EPC) in lower concentrations and in vivo [39]. We propose that TZDs may play a double-edged role in angiogenesis signalling by promoting the number and migration of endothelial progenitor cells at lower tissue concentrations achieved by systemic treatment, whereas the antiangiogenic effects are observed at higher local concentrations. Therefore, our observation of enhanced TZD-mediated angiogenesis is likely to contribute to the improved outcome in mice with post-MI heart failure [40] and after ischemic stroke [41] seen after TZD treatment. In addition to upregulation of EPC numbers, treatment with TZD improved the migratory capacity of EPC, which represents an important marker of EPC function. Dimmeler et al. have recently shown that the impairment of replication and functional capacity in EPCs during propagation in culture can be prevented by treatment with statins via upregulation of the telomere-capping protein TRF2. Overexpression of a dominant negative mutant of the TRF2 protein abro-

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gated statin-induced enhancement of migratory activity down to baseline values. Here we show that pioglitazone potently upregulates TRF2 expression in mice as well as in human EPC. Importantly, these data show a beneficial effect of TZD on telomere biology in EPC. Endothelial nitric oxide synthesis has been shown to be an important regulator of EPC release [14,23,31]. Accordingly, the increase of EPC numbers e.g. induced by physical training in mice is inhibited in eNOS−/− mice or in the presence of the NOS-inhibitor l-NAME [23]. Several effects of TZD such as the improvement of endothelial function suggested that pioglitazone may potentially mediate EPC release via eNOS [1,3]. However, in contrast to our expectations, we did not observe upregulation of vascular eNOS mRNA expression nor reversal of the TZD-induced increase of EPC in the presence of the NOS inhibitor l-NAME. Therefore, TZD regulate EPC by a mechanism independent of eNOS. Importantly, we show that EPC isolated from TZD-treated mice are characterized by a reduced rate of apoptotic cell death. This observation was confirmed in cultured human EPC treated with TZD. Inhibition of apoptosis by TZD in human EPC was not prevented in the presence of a NOSinhibitor. In contrast, the effect of TZD on EPC apoptosis was abolished in the presence of the PI3K inhibitors wortmannin or LY294002 but not by LNMA. These data show that PI3 kinase is a necessary mediator of the effects of pioglitazone on H2 O2 -induced apoptosis, however, the experiments do not support a direct activation of PI3 kinase by pioglitazone since no effect of pioglitazone was observed on Akt phoyphorylation in cultured human EPC. Further studies are needed to characterize the underlying signal transduction in detail. Endothelial progenitor cells have emerged as a new dimension of vascular biology. Increasing evidence suggests that bone marrow derived adult stem cells significantly contribute to vascular and cardiac function. In patients with metabolic syndrome the function of EPC is defective. On the basis of the experiments presented here, further clinical studies are justified to examine the effects of PPAR-␥ agonists on EPC. Improvement of EPC function may represent a relevant effect of thiazolidinediones that could potentially benefit patients with vascular diseases independent of diabetes mellitus. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (U.L.) and the Universit¨at des Saarlandes. The Dept. of Medicine III of the Universit¨atsklinikum des Saarlandes has received an unrestricted grant from Takeda, Aachen, Germany. We thank Simone J¨ager and Ellen Becker for their excellent technical assistance. References [1] Hetzel J, Balletshofer B, Rittig K, et al. Rapid effects of rosiglitazone treatment on endothelial function and inflammatory biomarkers. Arterioscler Thromb Vasc Biol 2005;25:1804–9.

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