Both authors contributed equally.
Synergistic effects of telmisartan and simvastatin on endothelial progenitor cells
Article first published online: 23 JUN 2009
© 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Journal of Cellular and Molecular Medicine
Volume 14, Issue 6b, pages 1645–1656, June 2010
How to Cite
Steinmetz, M., Brouwers, C., Nickenig, G. and Wassmann, S. (2010), Synergistic effects of telmisartan and simvastatin on endothelial progenitor cells. Journal of Cellular and Molecular Medicine, 14: 1645–1656. doi: 10.1111/j.1582-4934.2009.00829.x
- Issue published online: 23 JUN 2009
- Article first published online: 23 JUN 2009
- Received: November 22, 2008; Accepted: June 16, 2009
- endothelial progenitor cells;
- angiotensin receptor blockers;
Circulating endothelial progenitor cells (EPC) contribute to endothelial replenishment. Telmisartan is an angiotensin-receptor blocker with PPARγ-agonistic properties. PPARγ-agonists and HMG-CoA reductase inhibitors have been shown to enhance EPC number and function. We focused on the effects of telmisartan alone or in combination with simvastatin on EPC. EPC were isolated from healthy human volunteers, cultured and stimulated with telmisartan, simvastatin, or the combination of telmisartan and simvastatin. Telmisartan significantly increased the number of acLDL/lectin double-positive early EPC, the number of colony forming units (EC-CFU) as well as EPC migratory capacity, inhibited TNFα-induced EPC apoptosis and reduced glucose-induced oxidative stress. The telmisartan effect was dose-dependent and could be inhibited by GW9662, indicating a PPARγ-dependent mechanism. The combination of telmisartan and simvastatin led to a significant additive increase in EPC count and function. In wild-type mice, systemic treatment with either telmisartan or simvastatin elevated the number of sca-1/flk-1-positive EPC in bone marrow and peripheral blood, spleen-derived acLDL/lectin double-positive EPC, EPC migration and EC-CFU. Consistent with the in vitro findings, the combination of telmisartan and simvastatin resulted in a further enhancement of EPC counts. Re-endothelialization after carotid injury was significantly enhanced by telmisartan, simvastatin and the combination. Telmisartan increases EPC number and function mediated by a PPARγ-dependent mechanism. This effect is further enhanced by combination with simvastatin, suggesting a synergistic activation of potentially diverse intracellular pathways.
Endothelial progenitor cells (EPC) have recently emerged as a pivotal cell type in endothelial homeostasis and endothelial dysfunction, which is an early and crucial stage of atherosclerosis [1, 2]. Mature endothelial cells have only limited regenerative capacities, therefore EPC have been assumed to essentially determine endothelial replenishment. EPC are defined according to their surface markers and functional properties. One recent classification distinguishes between early and late outgrowth EPC by differentiating between cellular functional capacities and time of appearance. After culture of mononuclear cells for 4 to 7 days, early EPC are detectable, which are closely related to the EPC defined by Asahara and colleagues, and feature paracrine improvement of neovasculogenesis, endothelial repair and endothelial vasomotor function. Early EPC are subdivided into circulating angiogenic cells (CAC) and endothelial cell colony forming units (EC-CFU) or CFU-Hill, by specific cell culture protocols and their functional properties . Late outgrowth EPC are found in cobblestone shape after 14 to 21 days. They have a high proliferative potential and contribute to tubulogenesis and neovasculogenesis [4–9]. The precise role of these EPC subsets in atherosclerosis is not yet entirely understood.
In clinical investigations, number and function of circulating and early EPC correlate negatively with cardiovascular risk factors, and have been found to be decreased and functionally impaired in association with severity of coronary heart disease and myocardial infarction. EPC are positively influenced by various physiological stimuli, for example physical activity and weight reduction [2, 10–14]. Moreover, several drugs have been identified to beneficially affect EPC number and function in vivo and in vitro, for example angiotensin converting enzyme inhibitors, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), and thiazolidinediones (TZD) [15–18].
The multiple extra- and intracellular signalling pathways involved in EPC mobilization, recruitment, activation and function are only partly identified. In addition to the phosphatidylinositol-3-kinase (PI3K) pathway, which mediates the effects of statins on EPC, TZD exert their effects via a peroxisome proliferator-activated receptor γ (PPARγ)-dependent mechanism [18, 19].
The impact of nuclear transcription factor PPARγ on biological processes remains controversial. PPARγ has mainly been studied in adipocytes, but is expressed in all relevant cell types of the cardiovascular system, including endothelial cells. After binding of specific ligands to the ligand-binding domain, the DNA-binding site of PPARγ initiates the expression of its target genes. Physiological ligands include the prostaglandin D2 derivative, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and oxidized linoleic acid (9[S]- and 13[S]-HODE) as components of oxidized LDL .
The previously mentioned TZD are one group of pharmacological PPARγ stimulators and have been shown to have beneficial effects on EPC and to improve endothelial function in type 2 diabetes mellitus [18, 21, 22]. Several angiotensin-receptor blockers (ARB) also stimulate PPARγ activity, namely irbesartan, losartan and telmisartan . Among them, the highly lipophilic telmisartan appears to have the strongest activating effect on the PPARγ pathway .
In this study, we investigated in vitro and in a murine in vivo model whether (i) the ARB telmisartan enhances early EPC number and function, (ii) this effect is mediated by PPARγ, and (iii) the combination with simvastatin partly leads to an additional enhancement of EPC number and function by potentially activating alternative pathways.
Angiotensin II, simvastatin, GW9662, PD123319, TNFα, salts and other chemicals were purchased from Sigma. Simvastatin was chemically activated by alkaline hydrolysis. Telmisartan was kindly provided by Boehringer Ingelheim.
Peripheral blood samples
Human peripheral blood samples were obtained from adult healthy volunteers by venopuncture and collected into sodium citrate containing tubes. All samples were obtained with informed consent and in accordance with the Declaration of Helsinki.
Isolation of EPC
EPC were isolated from peripheral blood or murine spleen and cultured in accordance with previously published methods . Briefly, mononuclear cells (MNC) were isolated using Ficoll density gradient (Biocoll, Biochrom, Berlin, Germany) and seeded into 24-well tissue culture plates pre-coated with human fibronectin (Sigma, Steinheim am Albuch, Germany) at 4 × 106 (acLDL/lectin staining) or 1 × 106 (EC-CFU) cells/ml in endothelial basal medium (EBM) and supplements as recommended by the manufacturer (Promocell, Heidelberg, Germany).
Stimulation of EPC
Cells were incubated for 72 hrs with telmisartan in concentrations of 1, 10 or 100 μmol/l; angiotensin II 1 μmol/l; telmisartan 10 μmol/l and angiotensin II 1 μmol/l; simvastatin 1 μmol/l; telmisartan 10 or 100 μmol/l and simvastatin 1 μmol/l; GW9662 5 μmol/l; PD123319 1 μmol/l; TNFα 25 ng/well.
After 7 days of culture, fibronectin-adherent cells were washed and incubated with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (Dil)-labelled acetylated low-density lipoprotein (acLDL; CellSystems, St. Katharinen, Germany) and stained with FITC-labeled Ulex europaeus agglutinin (lectin; Sigma). Cells double-positive for acLDL and lectin staining were judged to be EPC . At least three high-power field fluoroscopy microscopic scans of each well were analyzed, and mean values were calculated.
Endothelial cell colony forming units (EC-CFU)
For EC-CFU, cells were isolated and cultured as described previously. After 2 days of initial culture, 1 × 106 cells derived from the supernatant were re-seeded per well, cultured for additional 7 days in EBM on human fibronectin pre-coated wells [11, 14] and stimulated as described previously. The number of colonies was manually counted using light microscopy at the end of the incubation period.
EPC migratory function was evaluated using a modified Boyden chamber (BD Biosciences, Heidelberg, Germany). A polycarbonate filter with 8-μm pore size (BD Biosciences) was placed between the upper and lower chambers. After 4 days of initial culture, cell suspensions (1 × 105 cells per well) were placed in the upper chamber, and the lower chamber was filled with EBM containing 50 ng/ml of human recombinant stromal cell-derived factor-1 (SDF-1; R&D Systems, Minneapolis, MN, USA). Cells were then incubated for additional 24 hrs at 37°C and 5% CO2. The migrated cells on the lower side of the filter were fixed and stained for acLDL/lectin. Migration activity was evaluated by the mean number of cells counted in at least three high-power fields per filter.
To determine EPC apoptosis, the Cell Death Detection ELISA Kit (Roche, Mannheim, Germany) was used according to the instructions of the manufacturer. Briefly, EPC were cultured and stimulated as mentioned previously. Cells were pelleted, lysed and transferred to a streptavidin-coated microplate well. Anti-histone (biotin-labelled) and anti-DNA (peroxidase-conjugated) antibodies were added leading to the binding of nucleosome complexes to streptavidin. Samples were incubated with peroxidase substrate (ABTS) and the coloured product was measured spectrophotometrically. Apoptosis was calculated in enrichment factor, which equals the millimass unit of sample (dying or dead cells) distributed to millimass unit of corresponding control (viable cells); mU = absorbance (10−3). The results are shown in relative proportion to the control group.
Measurement of reactive oxygen species
Intracellular reactive oxygen species (ROS) production in cultured EPC was measured by 2’,7’-dichlorofluorescein (DCF; 10 μmol/l) fluorescence using fluorescence microscopy techniques. EPC were stimulated with telmisartan 100 μmol/l; angiotensin II 1 μmol/l; glucose 30 mmol/l; telmisartan 100 μmol/l and angiotensin II 1 μmol/l; simvastatin 1 μmol/l; telmisartan 100 μmol/l and simvastatin 1 μmol/l.
Stimulation was followed by incubation with DCF for 30 min. in the dark at 37°C. Images were collected by single rapid scans and identical parameters, such as contrast and brightness, for all samples. Five groups of 40 cells for each sample were randomly selected from the image and fluorescent intensity was taken.
C57BL/6 (wild-type) mice (Charles River, Wilmington, NC, USA) were used for this study. The animals were maintained in a 22°C room with a 12-hr light/dark cycle and received drinking water ad libitum. The animals were divided into four groups. Group 1 served as control group and received standard diet (Ssniff, Soest, Germany) and saline injections (equal volume as simvastatin injections). Group 2 was fed standard diet supplemented with telmisartan (40 mg/kg body weight/d). Group 3 was fed standard diet and received simvastatin injections subcutaneously (20 mg/kg body weight/d). Group 4 was treated with a combination of telmisartan diet and simvastatin injections. Pharmacological treatment was performed for 10 days. The mice were killed after the indicated treatment. Spleen, blood and bone marrow samples (femur and tibia) were collected and processed immediately after sacrifice. All animal experiments were performed in accordance with institutional guidelines and the German animal protection law.
Murine blood samples were analyzed as previously described . Following red cell lysis, the viable lymphocyte population was analyzed for sca-1-FITC (Becton Dickinson, Heidelberg, Germany) and flk-1-PE (Becton Dickinson). Isotype-identical antibodies and unstained samples served as controls in every experiment (Becton Dickinson). Cell fluorescence was measured immediately after staining using a FACSCalibur instrument (Becton Dickinson). Data were analyzed using CellQuest software (Becton Dickinson). Units of all measured components are specific events obtained after measuring 50,000 (bone marrow) or 20,000 (peripheral blood) events in a pre-specified lymphocyte gate during FACS analysis.
Carotid artery denudation
Carotid artery injury was induced as described previously . Briefly, a subset of the mice was anaesthetized with 150 mg/kg body weight ketamine hydrochloride (ketanest®, Pharmacia, Karlsruhe, Germany) and 0.1 mg/kg body weight xylazine hydrochloride (rompun®2%, Bayer, Leverkusen, Germany). The common carotid artery was exposed and submitted to an electric injury starting at the bifurcation and continuing to the proximal part of the artery (in total 4 mm denudation). The denuded area was determined at day 3 after surgery following intravenous injection of 50 μl Evans blue in an enface preparation of the vessel. Evans blue stained denuded areas and the complete vessel area was measured using AxioVision (Zeiss, Munich, Germany) version 4.5.0 software. The percentage of re-endothelialization 3 days after injury is provided.
Data are expressed as mean ± SEM. Groups were compared by a one-way analysis of variance (ANOVA) with post hoc analyses for pair wise comparisons (Newman–Keuls multiple comparison). Statistical significance was assumed when a null hypothesis could be rejected at P < 0.05. Statistical analysis was performed with the use of SPSS software (SPSS Inc., Chicago, USA), version 11.5, for Microsoft Windows.
EPC numbers in vitro
The uptake of acLDL and presence of lectin is a specific characteristic of EPC. AcLDL/lectin double-positive cells were counted to assess the number of EPC in vitro. Compared to the vehicle-treated control group (100%), the number of EPC was decreased after stimulation with angiotensin II (64 ± 14% of control, P < 0.05 versus control). This effect was antagonized by co-incubation with telmisartan (134 ± 9% of control, P < 0.05 versus angiotensin II), and EPC numbers appeared to be higher than in the control group (P < 0.05 versus control). Telmisartan dose-dependently increased EPC numbers in the absence of angiotensin II (1 μmol/l: 121 ± 11% of control; 10 μmol/l: 184 ± 12% of control, P < 0.05 versus control; 100 μmol/l: 275 ± 9% of control, P < 0.05 versus control) (Fig. 1A).
Stimulation with 1 μmol/l simvastatin increased the absolute EPC number to 198 ± 16% of control (P < 0.05 versus control). Combined stimulation with telmisartan and simvastatin further enhanced EPC count, with a dose-dependent effect for telmisartan (simvastatin 1 μmol/l and telmisartan 10 μmol/l: 258 ± 15% of control, P < 0.05 versus control; simvastatin 1 μmol/l and telmisartan 100 μmol/l: 509 ± 11% of control, P < 0.05 versus control, P < 0.05 versus telmisartan 10 and 100 μmol, simvastatin 1 μmol, simvastatin 1 μmol and telmisartan 10 μmol) (Fig. 1A).
Co-treatment of EPC with telmisartan and the specific PPARγ antagonist GW9662 abolished the telmisartan-induced effect on EPC counts (telmisartan 10 μmol/l: 133 ± 7% of control, P < 0.05 versus control; telmisartan 100 μmol/l: 185 ± 8% of control, P < 0.05 versus control; GW: 104 ± 7% of control; telmisartan 10 μmol/l and GW: 81 ± 6% of control, P < 0.05 versus telmisartan 10 μmol/l; telmisartan 100 μmol/l and GW: 104 ± 26% of control), whereas co-stimulation with the specific AT2-receptor antagonist PD123329 did not affect the telmisartan effect on EPC numbers (PD: 103 ± 6% of control; telmisartan 10 μmol/l and PD: 138 ± 11% of control, P < 0.05 versus control; telmisartan 100 μmol/l and PD: 149 ± 10% of control, P < 0.05 versus control) (Fig. 1B).
Endothelial cell colony forming units in vitro
Compared to the vehicle-treated control group (100%), the number of EC–CFU was significantly diminished by angiotensin II (48 ± 23% of control, P < 0.05 versus control). This was neutralized by co-treatment with telmisartan 10 μmol/l (120 ± 30% of control). Stimulation with telmisartan alone dose-dependently enhanced CFU count (1 μmol/l: 122 ± 19% of control; 10 μmol/l: 168 ± 19% of control; 100 μmol/l: 225 ± 14% of control, P < 0.05 versus control). The increase of CFU counts by stimulation with simvastatin (148 ± 20% of control) was significantly enhanced by co-incubation of simvastatin with telmisartan 10 μmol/l (194 ± 16% of control, P < 0.05 versus control) and telmisartan 100 μmol/l (367 ± 10% of control, P < 0.05 versus control) (Fig. 2).
Migration in vitro
The migratory capacity was assessed to determine a functional parameter of EPC. Compared to the vehicle-treated control group (100%), angiotensin II deteriorated migratory capacity (74 ± 8% of control, P < 0.05 versus control). This effect was antagonized by co-stimulation with telmisartan 10 μmol/l (125 ± 9% of control, P < 0.05 versus angiotensin II). Treatment with telmisartan alone dose-dependently improved the migratory capacity of EPC (1 μmol/l: 115 ± 8% of control; 10 μmol/l: 158 ± 13% of control, P < 0.05 versus control; 100 μmol/l: 178 ± 12% of control, P < 0.05 versus control). The significant effect of simvastatin treatment (154 ± 7% of control, P < 0.05 versus control) was further enhanced by the co-application of telmisartan (10 μmol/l: 168 ± 12% of control, P < 0.05 versus control; 100 μmol/l: 191 ± 10% of control, P < 0.05 versus control) (Fig. 3).
Apoptosis in vitro
TNFα-induced apoptosis resulted in the highest rate of cell death and was used as reference (100%). Vehicle-treated cells served as negative control (19 ± 14% of TNFα, P < 0.05 versus TNFα). Angiotensin II significantly enhanced EPC apoptosis, but to a lesser degree than TNFα (69 ± 6% of TNFα, P < 0.05 versus TNFα, P < 0.05 versus negative control). The angiotensin II effect was abolished by co-stimulation with telmisartan 10 μmol/l (24 ± 13% of TNFα, P < 0.05 versus angiotensin II, P < 0.05 versus TNFα). Telmisartan alone dose-dependently inhibited TNFα-induced apoptosis (1 μmol/l: 75 ± 7% of TNFα; 10 μmol/l: 58 ± 5% of TNFα, P < 0.05 versus TNFα; 100 μmol/l: 45 ± 9% of TNFα, P < 0.05 versus TNFα). Expectedly, simvastatin inhibited TNFα-induced apoptosis of EPC (50 ± 19% of TNFα, P < 0.05 versus TNFα). The combined stimulation of EPC with simvastatin and telmisartan led to an additive anti-apoptotic effect (simvastatin and telmisartan 10 μmol/l: 43 ± 16% of TNFα, P < 0.05 versus TNFα; simvastatin and telmisartan 100 μmol/l: 35 ± 14% of TNFα, P < 0.05 versus TNFα) (Fig. 4).
ROS production in vitro
DCF fluorescence was used to measure intracellular ROS production. Compared to vehicle-treated control (100%), ROS formation was significantly enhanced by angiotensin II (145 ± 14% of control, P < 0.05 versus control), which was diminished by the addition of telmisartan (86 ± 4% of control). Because we sought to evaluate the PPARγ-associated telmisartan effect on oxidative stress beyond angiotensin-receptor blocking capacity, we additionally used glucose to generate oxidative stress. Glucose-mediated ROS production (170 ± 14% of control, P < 0.05 versus control) was comparable to that of angiotensin II. Addition of telmisartan normalised oxidative stress to control levels (98 ± 6% of control, P < 0.05 versus glucose), as did simvastatin (114 ± 4% of control, P < 0.05 versus glucose). Co-incubation of telmisartan and simvastatin decreased oxidative stress even further below control levels (79 ± 5% of control, P < 0.05 versus control, P < 0.05 versus glucose, P < 0.05 versus glucose + telmisartan) (Fig. 5).
EPC counts in vivo
Wild-type mice were treated with the study drugs for 10 days, and EPC counts in bone marrow and peripheral blood were analyzed by flow cytometry. Compared to the vehicle-treated control group (100%), both telmisartan and simvastatin treatment significantly increased the number of EPC in bone marrow and peripheral blood (telmisartan: 185 ± 13% of control in bone marrow and 132 ± 8% of control in peripheral blood; simvastatin: 210 ± 9% of control in bone marrow and 159 ± 12% of control in peripheral blood; all P < 0.05 versus control). Consistent with the in vitro findings, the combination of telmisartan and simvastatin led to a synergistic enhancement of EPC counts in bone marrow (439 ± 28% of control, P < 0.05 versus control, P < 0.05 versus telmisartan, P < 0.05 versus simvastatin) and peripheral blood (195 ± 16% of control, P < 0.05 versus control) (Fig. 6).
EPC numbers ex vivo
After cultivation of spleen-derived mononuclear cells for 7 days, the number of acLDL/lectin double-positive EPC was evaluated. Compared to the vehicle-treated control group (100%), telmisartan and simvastatin led to a significant increase of double-positive cells (138 ± 6% and 163 ± 27% of control, P < 0.05 versus control). The combination of telmisartan and simvastatin further enhanced EPC numbers (235 ± 18% of control, P < 0.05 versus control, P < 0.05 versus telmisartan) (Fig. 7).
Endothelial cell colony forming units ex vivo
Compared to control group (100%), the number of murine EC–CFU was significantly increased in telmisartan (162 ± 9% of control, P < 0.05 versus control) or simvastatin (219 ± 15% of control, P < 0.05 versus control) treated mice. Combined administration of telmisartan and simvastatin increased EC–CFU not only compared to control but also beyond single treatment with telmisartan or simvastatin (291 ± 24% of control, P < 0.05 versus control, P < 0.05 versus telmisartan, P < 0.05 versus simvastatin) (Fig. 8).
Migration ex vivo
EPC migration was significantly improved in mice treated with telmisartan (130 ± 9% of control, P < 0.05 versus control) or simvastatin (135 ± 9% of control, P < 0.05 versus control), as compared to the control group (100%). The combination of telmisartan and simvastatin also significantly enhanced migratory capacity (151 ± 8% of control, P < 0.05 versus control), which was, however, not significantly increased compared to monotherapy with either telmisartan or simvastatin (Fig. 9).
Re-endothelialization in vivo
Carotid arteries were electrically denuded, and the remaining lesion and re-endothelialization were assessed 3 days later. In the control group, 23 ± 3% of the total lesion area were re-endothelialized. In mice treated with telmisartan or simvastatin, this area was increased to 38 ± 3% for telmisartan (P < 0.05 versus control) and 40 ± 4% for simvastatin (P < 0.05 versus control). The combination of telmisartan and simvastatin resulted in a further but not significant improvement of re-endothelialization compared to monotherapy (44 ± 3%, P < 0.05 versus control) (Fig. 10).
Focus of the present study was the cellular effect of the ARB telmisartan and combination of telmisartan with the HMG-CoA reductase inhibitor simvastatin on early EPC number and function in vitro and in vivo. Telmisartan blocked the deleterious effects of angiotensin II on early EPC and dose-dependently enhanced EPC number and function in the absence of angiotensin II. Furthermore, combined treatment of telmisartan with simvastatin led to, or at least suggested a trend towards, an additive, synergistic effect on EPC number and function.
It has been shown, that angiotensin II accelerates EPC senescence via AT1-receptor activation through induction of oxidative stress [27, 28]. In turn, angiotensin II blockade has been associated with improvement of EPC number and function in hypertension via antioxidant mechanisms [29–31]. As expected, the AT1-receptor antagonist telmisartan blocked angiotensin II effects on early EPC number and function, and reduced angiotensin II-induced oxidative stress in our study. Our in vitro data indicate that telmisartan exerts a direct, dose-dependent effect on EPC independent of angiotensin II blockade, namely enhancement of EPC number, colony formation and migratory capacity, and inhibition of TNFα-induced apoptosis. The reduction of glucose-induced oxidative stress likewise points to a mechanism independent of AT1-receptor blockade. Importantly, telmisartan increased EPC numbers in bone marrow, peripheral blood and spleen of wild-type mice, confirming our in vitro data in an in vivo setting.
The direct effect of telmisartan is mediated by a PPARγ-dependent mechanism, because co-incubation of telmisartan with the PPARγ antagonist GW9662 inhibited the effect of telmisartan on EPC numbers in our cell culture experiments. Our finding is in line with studies that demonstrated PPARγ-activating properties of telmisartan and showed an increase of EPC numbers and functional capacities after treatment with PPARγ-activating TZD in vitro and in vivo[18, 21, 23, 24].
Zhao and colleagues reported that angiotensin II induces PPARγ expression and ligand-mediated PPARγ activity via AT2-receptor activation, which is involved in neuronal cell differentiation . Another study demonstrated a PPARγ-dependent down-regulation of the AT1 receptor by telmisartan independent of angiotensin II . Telmisartan is a highly selective AT1-receptor blocker with strikingly less affinity to the AT2 receptor. However, we sought to exclude an involvement of AT2-receptor stimulation in the observed direct effects of telmisartan on EPC. In our analysis, telmisartan increased the number of EPC in the absence of angiotensin II, which could not be blocked by the specific AT2-receptor antagonist PD123319. In contrast to a possible angiotensin II effect on AT2 receptors in the presence of an AT1-receptor blocker, an involvement of AT2-receptor activation in the telmisartan-induced effect on EPC numbers can be excluded in this experimental setting.
Expectedly, simvastatin treatment improved EPC numbers and function in vitro and in vivo in our study. This finding is consistent with previous reports that demonstrated increased EPC mobilization, proliferation and migration as well as decreased EPC apoptosis and senescence after statin treatment [16, 17, 34–38]. The most important intracellular mechanism for these effects seems to be the PI3K/Akt axis. Simvastatin-induced phosphorylation and activation of Akt lead to enhanced EPC numbers, differentiation and inhibition of apoptosis, which can be effectively inhibited by PI3K blockers or over-expression of dominant-negative Akt [16, 19, 36, 38].
Interestingly, in the present study, the combined application of telmisartan and simvastatin led to an additive enhancement of EPC numbers and partly function. As described previously, telmisartan activates PPARγ, whereas simvastatin leads to activation of the PI3K/Akt pathway, indicating stimulation of diverse signalling pathways. In addition, experimental studies have shown that some TZD effects on EPC can be blocked by inhibitors of PI3K, demonstrating that PPARγ activation may lead to additional activation of the PI3K pathway . Moreover, it has been demonstrated that statins inhibit premature EPC senescence by restoration of telomere repeat-binding factor-2 (TRF-2) and preventing telomere uncapping, a process not related to PI3K activation . Both simvastatin and telmisartan attenuate TNFα-mediated apoptosis of EPC, which is in line with recent studies investigating the effect of statins and PPARγ-activating TZD on EPC apoptosis [21, 35, 38]. Again, the involvement of PI3K has been demonstrated for statins. TNFα-mediated apoptosis involves activation of NF-kB, and it has been shown that PPARγ-activating TZD may inhibit the phosphorylation of the p65 subunit of this signalling protein . The anti-apoptotic effect of telmisartan may involve this PPARγ-mediated signalling cascade, too. Finally, it is possible that telmisartan activates additional signalling pathways that have not yet been investigated. Taken together, a synergistic stimulation of multiple intracellular pathways by telmisartan and simvastatin is most likely responsible for the additive effect on EPC.
As anticipated, monotherapy with telmisartan or simvastatin significantly enhanced re-endothelialization after a defined carotid injury in mice. The combination of both substances improved re-endothelialization with a modest trend towards further enhancement. However, this latter effect did not reach statistical significance compared to the single drugs. One may speculate that the regenerative capacity for endothelial damage was already saturated by the sole application of telmisartan or simvastatin in otherwise healthy wild-type mice, and significant differences may be obtained in other models of vascular disease. On the other hand, the combination of telmisartan and simvastatin might primarily affect numbers of early EPC but not further augment functionality such as migratory capacity.
Although telmisartan plasma concentrations of 10 μmol/l, as used in our in vitro experiments, may be achieved with therapeutic doses of this ARB in clinical settings in humans , higher doses may not reflect therapeutic in vivo levels in humans. However, the observed effect of telmisartan was dose-dependent and, importantly, the effect of telmisartan and the synergistic effect of combined treatment with simvastatin was confirmed in vivo in mice. Future clinical trials will have to clarify the relevance of our findings in humans.
EPC are important for endothelial replenishment, endothelial repair after vascular damage, neovasculogenesis, and endothelial function [2, 4–9]. Given the fact that endothelial dysfunction is a marker of early atherosclerosis and predicts future cardiovascular events, and EPC counts correlate not only with endothelial vasomotor function but also inversely with cardiovascular morbidity and mortality, these cells may not only reflect a cellular marker of the severity of vascular disease but may play an important role in the prevention of atherosclerosis [1, 2, 9, 11, 13, 14]. It may be speculated that EPC-enhancing treatment strategies resemble an interesting therapeutic option for the prevention and treatment of vascular disease. Statins, PPARγ agonists, and ARB have been shown to improve endothelial function, inhibit development and progression of atherosclerosis and reduce cardiovascular events, as demonstrated in numerous animal studies and human clinical trials [22, 41–50]. The mobilization, increase of cell numbers and functional improvement of EPC mediated by the aforementioned agents, as shown in our study, may possibly contribute to their therapeutic benefits. Our study demonstrates that the combination of telmisartan and simvastatin exerts synergistic effects on mobilization and specific functional characteristics beyond the respective monotherapies. These results encourage further studies to characterize the pharmacological modulation of EPC and their effect on endothelial health and regeneration.
The excellent technical assistance of Isabel Paez-Maletz, Annika Bohner and Kathrin Paul is greatly appreciated. This study was supported by the Deutsche Forschungsgemeinschaft (DFG).