CARDIOVASCULAR RISK
Effects of Obesity and Weight Loss on the Functional Properties of Early Outgrowth Endothelial Progenitor Cells
Nana-Maria Heida, MD*,
Jan-Peter Müller, MD*,
I.-Fen Cheng, MSc*,
Maren Leifheit-Nestler, PhD*,
Vivien Faustin, PhD ,
Joachim Riggert, MD ,
Gerd Hasenfuss, MD*,
Stavros Konstantinides, MD*,* and
Katrin Schäfer, MD*
* Department of Cardiology and Pulmonology, University of Göttingen, Göttingen, Germany
Department of Nutritional Psychology, University of Göttingen, Göttingen, Germany
Department of Transfusion Medicine, University of Göttingen, Göttingen, Germany
Manuscript received April 23, 2009;
revised manuscript received August 20, 2009,
accepted September 1, 2009.
* Reprint requests and correspondence: Dr. Stavros Konstantinides, Department of Cardiology, University General Hospital, 68100 Alexandroupolis, Greece (Email: skonst{at}med.duth.gr).
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Abstract
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Objectives: The purpose of this study was to examine the impact of obesity and weight loss on the angiogenic and regenerative capacity of endothelial progenitor cells (EPCs).
Background: EPCs participate in angiogenesis and tissue repair. Several cardiovascular risk factors are associated with EPC dysfunction.
Methods: Early outgrowth EPCs were isolated from 49 obese (age 42 ± 14 years; body mass index 42 ± 7 kg/m2) normoglycemic participants in a professional weight reduction program and compared with those from 49 age-matched lean controls. EPC function was tested both in vitro and in vivo.
Results: EPCs expanded from the obese possessed reduced adhesive, migratory, and angiogenic capacity, and mice treated with obese EPCs exhibited reduced EPC homing in ischemic hind limbs in vivo. EPCs from the obese subjects failed to respond to conditioned medium of lean controls or to potent angiogenic factors such as vascular endothelial growth factor. Although no differences existed between lean and obese EPCs regarding the surface expression of vascular endothelial growth factor or chemokine receptors, basal p38 mitogen-activated protein kinase (MAPK) phosphorylation was elevated in obese EPCs (3.7 ± 2.1-fold increase; p = 0.006). These cells also showed reduced secretion of the angiogenic chemokines interleukin-8 (p = 0.047) and monocyte chemoattractant protein-1 (p = 0.012). By inhibiting p38 MAPK, we could restore chemokine levels to those of lean control EPCs and also improve the angiogenic properties of obese EPCs. Accordingly, 6-month follow-up of 26 obese persons who achieved significant weight reduction revealed normalization of p38 MAPK phosphorylation levels and improved EPC function.
Conclusions: Obesity is associated with a reversible functional impairment of EPCs. This involves reduced secretion of angiogenic chemokines and increased basal phosphorylation of signaling molecules, notably p38 MAPK.
Key Words: endothelial progenitor cells • obesity • signal transduction • weight loss
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Abbreviations and Acronyms
| | BMI = body mass index | | EPC = endothelial progenitor cell | | MAPK = mitogen-activated protein kinase |
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Clinical studies have identified a relationship between increased body weight and cardiovascular disease including coronary atherosclerosis, congestive heart failure, arrhythmias, and stroke (1–3). In fact, obesity is frequently accompanied by a cluster of comorbidities and metabolic disturbances such as elevated blood pressure, dyslipidemia, and insulin resistance (4). However, in addition to these established cardiovascular risk factors, systemic inflammation, increased oxidative stress, and altered hemodynamics associated with excess weight may directly contribute to endothelial injury and dysfunction and thus to the pathogenesis of atherosclerosis in obese individuals (5).
Over the past decade, a role for endothelial progenitor cells (EPCs) in cardiovascular homeostasis has emerged. EPCs are released from the bone marrow into the circulation in response to cytokines and other stimuli signaling tissue injury (6). They can be isolated from the peripheral blood and are characterized by the ability to differentiate into endothelium-like cells in culture (7). In particular, early outgrowth EPCs may contribute to endothelial repair and the neovascularization of ischemic tissue acting, at least in part, as potent paracrine stimulators of angiogenesis (8–10). These cells are functionally distinct from leukocytes, although they may share some surface markers with the latter cell type (7,11,12). A correlation between circulating EPC numbers and endothelial function has been observed in patients with various degrees of cardiovascular risk (13), and EPC numbers seemed to predict future cardiovascular events (14,15). Importantly, however, cardiovascular risk factors may also alter the functional capacity of EPCs. For example, EPCs cultivated from persons with diabetes exhibited impaired proliferation, adhesion, and integration in vascular structures (16,17), suggesting that EPC dysfunction may participate in the vascular complications associated with this condition. We therefore hypothesized that obesity may also modulate the functional properties of circulating EPCs and in particular their potential to promote tissue repair.
In the present study, we examined the adhesive, migratory, and angiogenic capacity of early outgrowth EPCs isolated from the peripheral blood of 49 consecutive obese (body mass index [BMI]  35 kg/m2), nondiabetic individuals participating in a professional weight reduction program and compared it with that of age-matched lean volunteers. Obese persons were reexamined 6 months later, and the effects of weight loss on functional parameters of their EPCs were determined. Our findings reveal novel potential mechanisms linking obesity to increased cardiovascular risk and also provide evidence supporting the beneficial effects of weight loss.
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Methods
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Study design and population.
Peripheral venous blood samples were collected in the morning from fasting obese individuals attending a professional weight reduction program (OPTIFAST-52, Nestlé Health Care Nutrition) at the University of Göttingen. The program begins with a 12-week period of complete meal replacement by a formula-based, predominantly liquid diet. This is followed by a 6-week transition to solid food and continues with a regular diet, which is maintained thereafter. The program features nutritional and behavioral counseling; mild physical activity (1 h/week) is encouraged. Participants presenting with a BMI of 35 kg/m2 and fasting blood glucose levels <110 mg/dl were included; we excluded smokers (100 cigarettes during lifetime) and persons with drug- or insulin-dependent diabetes or known cardiovascular disease. Age- and sex-matched normal-weight (BMI 20 to 25 kg/m2) healthy volunteers were recruited as controls. Six months later, study participants were again examined for follow-up analysis of EPC function if sufficient weight reduction (defined as current BMI <35 kg/m2 and/or 10% loss of body weight compared with baseline) had been achieved. The study protocol was approved by the institutional ethics committee, and written informed consent was obtained from all study participants.
Plasma parameters.
Plasma lipid profile was determined enzymatically (MODULAR P/D, Roche Diagnostics, Mannheim, Germany). Plasma high-sensitivity C-reactive protein was measured using turbidimetry after agglutination with antibody-coated latex particles (COBAS INTEGRA 800, Roche Diagnostics). Plasma asymmetrical dimethylarginine (DLD Diagnostika, Hamburg, Germany), E-selectin (RayBiotech, Inc., Norcross, Georgia; Hölzel Diagnostika, Köln, Germany), interleukin-6 (RayBiotech), leptin (R&D Systems, Minneapolis, Minnesota), and tumor necrosis factor- (R&D Systems) levels were determined using specific immunoassays.
Isolation, cultivation, and characterization of EPCs.
Mononuclear cells were isolated from venous blood by density-gradient centrifugation over Histopaque-1077 (Sigma-Aldrich, St. Louis, Missouri) as described (8). On day 7, cultivated cells were characterized based on the uptake of acetylated low-density lipoprotein and binding of Ulex europaeus agglutinin-I (lectin) as well as endothelial cell marker expression (8). Overall, 70 ± 17% of the cells were positive for CD31, 68 ± 12% for CD144, and 89 ± 11% for vascular endothelial growth factor receptor 2. Based on the isolation and cultivation protocol, the adherent mononuclear cells were identified as early outgrowth EPCs (7).
Analysis of EPC adhesion, migration, and angiogenesis; flow cytometry; and Western blot and assessment of cytokine secretion into the conditioned medium of EPCs are described in the Online Supplemental Methods.
Statistical analysis.
For quantitative data, results are presented as mean ± SD (except for the figures, where they appear as mean ± SEM), and for qualitative data, they are presented as absolute numbers and relative frequencies. The modified D'Agostino and Pearson test was used to test for normal distribution of continuous variables, and the paired t test was used for comparison of values between obese individuals and matched lean controls (except for the hind limb ischemia experiments, in which the unpaired t test was used). For comparison of obese individuals after weight loss with values at baseline and matched lean controls, repeated-measures analysis of variance was performed followed by the Bonferroni test. For comparison of categorical parameters, the McNemar test was used. A p value <0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism software 4.01 (GraphPad Software, Inc., La Jolla, California).
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Results
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Baseline parameters of obese and lean subjects.
EPCs were cultivated from the mononuclear cell fraction of 49 severely obese individuals (31 women), age 42 ± 14 years at the time of enrollment; the control group consisted of 49 lean healthy volunteers (28 women), age 38 ± 11 years. As shown in Table 1, obese persons exhibited elevated high-sensitivity C-reactive protein (p < 0.001) and leptin (p < 0.001) concentrations compared with their lean counterparts. Conversely, plasma levels of interleukin-6, tumor necrosis factor-, or markers of endothelial dysfunction such as E-selectin, asymmetrical dimethylarginine, and the number of circulating endothelial cells did not differ between the 2 groups.
EPCs from obese persons are functionally impaired.
Fewer acetylated low-density lipoprotein, lectin-double positive EPCs could be cultivated from the obese subjects compared with lean controls (70 ± 22% vs. 100 ± 26%; p = 0.006; 10 subjects per group). Of note, we detected no differences in the degree of EPC proliferation as assessed by immunohistochemical analysis of bromodeoxyuridine incorporation (<1% positive cells in either group) or in apoptosis rate as assessed by flow cytometry for annexin V binding in conjunction with the vital dye propidium iodide (not shown). Conversely, our experiments revealed impaired adhesion of EPCs from obese persons to fibronectin compared with lean controls (p = 0.018; 8 per group). Representative findings are shown in Figure 1A (left vs. middle) (cumulative results shown in Fig. 1D, left). Similar differences were observed regarding the adhesion of EPCs to vitronectin (11 ± 5.8 vs. 36 ± 11; p = 0.001; not shown). Moreover, EPCs from the obese subjects were characterized by reduced migratory capacity (p < 0.001; 8 per group) (Fig. 1B, left vs. middle) (cumulative results shown in Fig. 1D, middle). In further experiments using the Matrigel angiogenesis assay, EPCs from the obese subjects exhibited an impaired ability to incorporate into network-like structures provided by cocultivated human umbilical vein endothelial cells (p < 0.001; 7 per group) (Fig. 1C) (cumulative results shown in Fig. 1D, right).
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Figure 1 Effects of Obesity and Weight Loss on Functional Properties of EPCs In Vitro
Chloromethylbenzamido-DiI–labeled endothelial progenitor cell (EPC) (red; 4,6-diamino-2-phenylindole–positive cell nuclei appear blue in A and B), isolated from the peripheral blood of lean or obese individuals, were analyzed for their capacity to adhere to fibronectin (A), migrate in the modified Boyden chamber assay (B), or incorporate into network-like structures provided by human umbilical vein endothelial cells (translucent cells) in the Matrigel angiogenesis assay (C; arrows point to EPCs). In some obese individuals, the studies were repeated after weight loss. (D) Cumulative results. *p < 0.05 and ***p < 0.001 versus lean; #p < 0.05, ##p < 0.01, and ###p < 0.001 versus obese.
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Next, we analyzed the effects of obesity on the angiogenic capacity of EPCs in vivo. Chloromethylbenzamido-DiI (a carbocyanine dye)–labeled EPCs from obese subjects were less frequently detected within the ischemic hind limb musculature 10 days after injection into nude mice (28 ± 25 vs. 88 ± 85 chloromethylbenzamido-DiI–positive cells/mm2; p = 0.017; 11 mice per group) (Fig. 2A). Moreover, mice treated with EPCs from obese subjects revealed a reduced angiogenic response assessed by the number of CD31-positive cells per square millimeter (p = 0.036 vs. lean) (Fig. 2B).
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Figure 2 Effects of Obesity on the Angiogenic Properties of EPCs In Vivo
After induction of hind limb ischemia and injection of chloromethylbenzamido-DiI (CM-DiI)–labeled endothelial progenitor cells (EPCs) from either lean or obese subjects, 5-µm-thick serial cryosections were analyzed for the presence of EPCs (red signal; arrows in A, top); costaining of cell nuclei with 4,6-diamino-2-phenylindole (DAPI) (blue signal) demonstrates that no artificial signals were counted (A, bottom). (B) To assess angiogenesis after ischemia, the density of CD31-positive capillaries per square millimeter (green signal; 4,6-diamino-2-phenylindole–positive cell nuclei appear blue) was determined in the ischemic (right) and the contralateral nonischemic (left) lower hind limb. *p < 0.05 and #p < 0.05 versus lean; **p < 0.01 versus nonischemic leg.
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Altered paracrine potency of EPCs in obesity.
To investigate whether alterations of the secretion profile may contribute to the impaired adhesive, migratory, and angiogenic properties of EPCs in obesity, crossover experiments using conditioned medium from either lean or obese EPCs were performed. The Matrigel angiogenesis assay after a 24 hour-incubation of obese EPCs with homologous (i.e., derived from obese EPCs) conditioned medium confirmed the reduction of their incorporation into endothelial networks compared with lean EPCs incubated with homologous (from lean EPCs) conditioned medium (p = 0.009) (first column vs. third column in Fig. 3A). Interestingly, preincubation with heterologous conditioned medium (from obese EPCs) reduced the angiogenic capacity of lean EPCs (p = 0.003 vs. lean EPCs with homologous conditioned medium; n = 9) (first column vs. second column in Fig. 3A), indicating a disturbed secretion of angiogenic mediators in EPCs from obese individuals. Further analysis of the paracrine potency of EPCs derived from obese as opposed to lean individuals supported these results: these studies revealed a reduced ability of conditioned medium from obese EPCs (compared with that from lean controls) to enhance migration of mature human umbilical vein endothelial cells in the modified Boyden chamber assay (p = 0.026; 4 per group; not shown) or to stimulate sprouting of mature human umbilical vein endothelial cells in the spheroid assay (p = 0.004; 6 per group) (Fig. 3B).
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Figure 3 Paracrine Effects and Secretion Profile of EPCs From Obese Versus Lean Individuals
(A) Conditioned medium was harvested, and its effects on the angiogenic properties of EPCs were tested using the Matrigel assay. (B) The effect of conditioned medium alone on the sprouting of human umbilical vein endothelial cells was analyzed using the spheroid angiogenesis assay. (C) Cytokines in conditioned medium from lean versus obese individuals, before and after weight loss: representative findings of cytokine antibody arrays (3 independent experiments, each using conditioned medium pooled from 4 persons per study group; interleukin (IL)-6 and tumor necrosis factor (TNF)- were not detected by the assay) together with the corresponding quantitative analysis using specific enzyme-linked immunosorbent assays. *p < 0.05 and **p < 0.01 versus lean. CdM = conditioned medium; HUVEC = human umbilical vein endothelial cell; MCP = monocyte chemoattractant protein; WL = weight loss; other abbreviation as in Figure 2.
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To characterize the secretion profile of EPCs from obese compared with lean individuals, conditioned medium of cells was examined using cytokine antibody arrays. Semiquantitative analysis (3 independent experiments of pooled conditioned medium from 4 subjects per group) revealed differentially expressed levels of interleukin-8 and monocyte chemoattractant protein-1. Subsequent quantitative analysis by enzyme-linked immunosorbent assay confirmed a significant reduction of the angiogenic factors interleukin-8 (p = 0.047; 12 per group) and monocyte chemoattractant protein 1 (p = 0.012; n = 7) concentrations in the conditioned medium from obese compared with that from lean EPCs (Fig. 3C). Conversely, we detected similar levels of interleukin-6 (p = 0.345; 10 per group), stromal cell-derived factor-1 (p = 0.793; n = 16; not shown), and tumor necrosis factor- (p = 0.795; n = 7), whereas vascular endothelial growth factor levels were below the detection limit (<5 pg/ml).
Possible signaling defects of EPCs in obesity.
Apart from alterations of the secretion profile of EPCs in obesity, our crossover experiments also showed that the impaired incorporation of obese EPCs into endothelial structures could not be rescued by the addition of conditioned medium from lean EPCs (p = 0.614 versus the addition of obese conditioned medium to obese EPCs; 9 per group) (third column vs. fourth column in Fig. 3A). This finding indicated that obese EPCs also exhibited an impaired response to factor(s) present in conditioned medium from lean EPCs. To begin to clarify this finding, we compared the responsiveness of lean and obese EPCs to stimulation with a potent angiogenic growth factor such as vascular endothelial growth factor. These experiments revealed a reduced angiogenic response of obese EPCs to vascular endothelial growth factor in the Matrigel assay compared with lean EPCs (p = 0.012 after stimulation with 10 ng/ml; 6 per group [Fig. 4A] and p = 0.017 with 100 ng/ml; not shown). Similar results were obtained using the spheroid angiogenesis assay (p = 0.003 after stimulation with 10 ng/ml vascular endothelial growth factor, 6 per group [Fig. 4B] and p = 0.018 with 100 ng/ml; not shown).
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Figure 4 Response of EPCs From Lean and Obese Persons to Angiogenic Factors
The angiogenic response of EPCs to stimulation with vascular endothelial growth factor (VEGF) (10 ng/ml for 24 h) was analyzed using the Matrigel (A) and the spheroid (B) angiogenesis assays. Results are expressed as percentage of unstimulated cells. (C), Expression of the indicated surface receptors was analyzed using flow cytometry: representative histograms (green line, lean EPCs; red line, obese EPCs; gray line, IgG control). *p < 0.05 and **p < 0.01 versus unstimulated cells; #p < 0.05 and ##p < 0.01 versus stimulated cells from lean individuals;  p < 0.01 versus stimulated cells from obese individuals. CCR2 = chemokine (C-C motif) receptor 2; CXCR2 = chemokine (C-C motif) receptor 2; PE = phycoerythrin; VEGFR = vascular endothelial growth factor receptor; other abbreviations as in Figures 1 and 3.
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In light of these results, we first analyzed the expression of the 2 major vascular endothelial growth factor receptors on EPCs (Fig. 4C). However, flow cytometry revealed no differences between EPCs from obese individuals (n = 12) and lean controls (n = 12) regarding the expression of either vascular endothelial growth factor receptor 1 (receptor-positive EPCs, 62 ± 13% vs. 65 ± 9.2%; p = 0.813) or vascular endothelial growth factor receptor 2 (94 ± 6.5% vs. 89 ± 9.4%; p = 0.053). Moreover, expression of the counterreceptors for monocyte chemoattractant protein-1 (chemokine [C-C motif] receptor 2) and interleukin-8 (chemokine [C-X-C motif] receptor 2) also did not differ significantly between lean and obese EPCs (Fig. 4C).
In contrast to the unaltered expression of specific receptors on EPCs in obesity, when intracellular signaling molecules were investigated by Western blot analysis, significantly higher baseline p38 MAPK phosphorylation levels (3.7 ± 2.1-fold) were found in EPCs from obese compared with lean individuals (p = 0.006) (representative blot in Fig. 5A) (summarized results from 9 persons per group in Fig. 5B). These results suggested that increased phosphorylation of signaling molecules and modulation of post-receptor signal transduction pathways may contribute to the reduced angiogenic capacity of EPCs in obesity. In support of this notion, we could also show that inhibition of p38 MAPK (using 20 µM of SB203580) significantly increased interleukin-8 (p = 0.04) and monocyte chemoattractant protein-1 (p = 0.02) levels in the conditioned medium of obese EPCs, resulting in levels similar to those found in conditioned medium from lean EPCs (Fig. 5C). Moreover, in the Matrigel assay, p38 MAPK inhibition increased the incorporation of obese EPCs into endothelial networks (p = 0.035) (Fig. 5D).
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Figure 5 Effects of Weight Loss and p38 MAPK Inhibition on Basal p38 MAPK Phosphorylation, Cytokine Secretion, and Angiogenesis
Total protein lysates were prepared from unstimulated EPCs, and the basal phosphorylation of p38 MAPK was analyzed by Western blot. (A) Representative findings. (B) Quantitative analysis of 9 individuals per group. Results were normalized to total p38 protein and expressed as times-fold increase compared with levels found in lean subjects. The effect of p38 MAPK inhibition (using 20 µM of SB203580) or dimethylsulfoxide (DMSO) (negative control) on the interleukin-8 and monocyte chemoattractant protein 1 (MCP1) levels in conditioned medium from lean and obese EPC (C) and their angiogenic properties (Matrigel assay; D) was analyzed. *p < 0.05 and **p < 0.01 versus lean; #p < 0.05 versus control-treated obese EPC; ###p < 0.001 versus obese. MAPK = mitogen-activated protein kinase; other abbreviations as in Figures 2 and 3.
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Weight loss restores the functional properties of EPCs.
To determine whether the alterations of the functional properties of EPCs in obesity as described previously can be reversed by weight loss, we reexamined 6 months later 26 obese participants (12 men, 14 women) in the program who successfully reduced their weight (as defined in the Methods section). The clinical and laboratory parameters of this subgroup at baseline and follow-up are shown in Table 2. Of note, the baseline medication of the obese subjects (Table 1) remained unchanged during this period. Weight loss seemed to restore the number of acetylated low-density lipoprotein, lectin double-positive cells (p < 0.05 vs. initially obese and p > 0.05 vs. lean controls; 10 per group; not shown). Furthermore, weight loss improved the adhesive properties of EPCs from obese persons on matrix proteins and particularly on fibronectin (p < 0.05 vs. initially obese and p > 0.05 vs. lean; 5 per group) (Fig. 1A, right) (cumulative results shown in Fig. 1D, left) and vitronectin (p < 0.01 and p > 0.05, respectively; not shown). The intervention also normalized the migratory activity of EPCs in vitro (p < 0.001 vs. initially obese and p > 0.05 vs. lean; 8 per group) (Fig. 1B, right) (cumulative results shown in Fig. 1D, middle), and restored their angiogenic properties in the Matrigel assay (p < 0.01 and p > 0.05, respectively; n = 7) (Fig. 1C, right) (cumulative results shown in Fig. 1D, right).
In further experiments, weight loss normalized the responsiveness of EPCs to vascular endothelial growth factor, both in the Matrigel (p = 0.008 vs. unstimulated cells, p < 0.01 vs. stimulated obese EPCs, and p > 0.05 vs. lean EPCs) (Fig. 4A) and in the spheroid angiogenesis assay (p = 0.076 vs. unstimulated cells and p > 0.05 vs. lean EPCs) (Fig. 4B). Importantly, the restored responsiveness of EPCs after weight loss was associated with a reduction of basal tyrosine phosphorylation of p38 MAPK-activated protein kinase (from 3.7 ± 2.1-fold to 1.1 ± 1.1-fold compared with baseline; p < 0.001 and p > 0.05 vs. lean controls) (Fig. 5A) (summarized results in 9 persons shown in Fig. 5B). Conversely, analysis of conditioned medium revealed that weight loss did not alter the secretion of interleukin-8 or monocyte chemoattractant protein 1 of EPCs from obese individuals at 6-month follow-up (Fig. 3C).
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Discussion
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The importance of obesity as a contributor to cardiovascular morbidity and mortality is rapidly increasing worldwide. In the present study, we examined whether the adverse cardiovascular effects of excess body weight may be mediated, at least in part, by a decreased capacity of EPCs to promote endothelial repair and neovascularization. The main findings of our study can be summarized as follows. 1) EPCs from obese subjects were characterized by reduced adhesive, migratory, and angiogenic capacities and were less capable of promoting angiogenesis after induction of hind limb ischemia in vivo in a mouse model. 2) These findings were associated with alterations in the secretion profile of EPCs and with intracellular signaling defects and reduced responsiveness to angiogenic stimuli. 3) The functional deficiency of cultivatable EPCs could be reversed after significant weight reduction.
Reduced numbers of circulating EPC have been reported in persons with cardiovascular risk factors, and EPC levels were found to inversely correlate with waist circumference and BMI (18). Experimental evidence suggests beneficial effects of lifestyle modification including exercise (19) and smoking cessation (20) on EPC numbers, and recently a significant increase in the number of circulating CD34, CD117 double-positive cells was reported after weight loss in obese subjects (18). However, the ability of EPCs to confer vascular protection depends not only on their absolute numbers or concentrations, but also on their functional properties. Using 2 different in vitro assays, we could show that EPCs cultivated from obese subjects were characterized by reduced adhesive and migratory capacity compared with EPCs from lean controls and exhibited an impaired ability to incorporate into network-like structures provided by cocultivated mature endothelial cells. Importantly, these defects of EPCs were reversible after significant weight loss. Of note, early outgrowth EPCs are characterized by a low proliferative potential, which is in contrast to endothelial colony-forming cells (7,21), and we detected no differences in the degree of EPC proliferation or apoptosis between lean and obese individuals.
The ability to home, adhere, and incorporate into vascular structures is a prerequisite for functional competence of EPC and represents an important feature underlying their vasoregenerative potential. In this regard, animal models of diabetes have pointed to an impaired potency of progenitor cells for therapeutic angiogenesis (22–24). The findings of the present study, as obtained in the in vivo mouse model of hind limb ischemia, now suggest that obesity itself (i.e., beyond the contribution of diabetes or insulin resistance) may impair the potential of early outgrowth human EPCs to incorporate into vascular structures and promote angiogenesis. In this regard, we excluded from the present study obese individuals who had a fasting plasma glucose level >110 mg/dl (in accordance with existing criteria for the definition of impaired fasting glucose [4]), as well as those with known diabetes mellitus. Thus, although we cannot completely exclude the possibility that some of the obese EPC donors may have had impaired glucose tolerance, it is unlikely that insulin resistance significantly contributed to our findings. Moreover, we detected no differences between obese individuals and lean controls with regard to plasma E-selectin or asymmetrical dimethylarginine levels or the number of circulating endothelial (CD146, CD31 double-positive) cells. These latter findings support the notion that the functional impairment of the EPCs isolated from obese subjects was independent of the presence of generalized endothelial dysfunction.
After the homing process, EPCs become capable of secreting angiogenic growth factors, which may stimulate vasculogenesis in a paracrine fashion (9). In our experiments, conditioned medium of early outgrowth EPCs stimulated the migration and sprouting of mature endothelial cells. Notably, however, the angiogenic potential of conditioned medium of EPCs cultivated from obese subjects was markedly reduced, and analysis of the supernatant revealed significantly reduced levels of the pro-angiogenic cytokines interleukin-8 and monocyte chemoattractant protein-1.
Beyond alterations of the secretion profile we found that, using both the Matrigel and the spheroid angiogenesis assay, EPCs from obese individuals were themselves less capable of responding to conditioned medium from healthy, lean EPCs or to potent angiogenic growth factors such as vascular endothelial growth factor. Flow cytometry showed no abnormalities in vascular endothelial growth factor receptor surface expression and also no general defect in the expression of important chemokine receptors, including those for interleukin-8 and monocyte chemoattractant protein-1, on obese EPCs. However, we did observe altered intracellular signal transduction and particularly increased basal phosphorylation of p38 MAPK. Vascular endothelial growth factor-induced endothelial cell migration seems to require p38 activation (25), and it remains to be clarified how increased basal phosphorylation of the kinase in obesity may impair growth factor signaling. In any case, the pathophysiological relevance of the p38 activation status is supported by our further experiments, which showed that: 1) the p38 increased basal phosphorylation detected in obese individuals was reversible on weight loss; and 2) p38 MAPK inhibition restored both the levels of the angiogenic cytokines interleukin-8 and monocyte chemoattractant protein-1 in the conditioned medium, and the angiogenic properties of obese EPCs to those of lean controls.
Because p38 MAPK-dependent pathways operate downstream of several cytokine and growth factor receptors, the enhanced p38 MAPK phosphorylation levels in obesity may be the result of long-term exposure to elevated levels of the numerous pro-inflammatory mediators in this condition (5). Searching for possible candidates, we found elevated circulating levels of leptin and C-reactive protein in the obese. Indeed, exposure of endothelial (and other) cells to C-reactive protein has been shown to lead to rapid p38 MAPK, and inhibition of p38 MAPK may prevent a C-reactive protein-induced pro-inflammatory phenotype and endothelial dysfunction (26–28). Tumor necrosis factor- and high glucose levels also have been shown to induce p38 MAPK phosphorylation in EPCs (29); in the present study, however, plasma glucose levels of obese individuals were within the normal range, and we found no differences in tumor necrosis factor- concentrations in either the plasma or the EPC conditioned medium of obese versus lean study participants.
Although the obese individuals included in our study had lower high-density lipoprotein cholesterol levels and a higher incidence of arterial hypertension than lean controls, these risk factors were not significantly affected by weight reduction. Moreover, it is unlikely (even though it cannot be completely excluded) that the beneficial effects of weight reduction were partly attributable to increased physical exercise because this particular program includes only mild physical activity (1 h/week), which is far below current recommendations (30).
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Conclusions
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Our findings demonstrate impaired adhesive, migratory, and angiogenic properties of EPCs isolated from the mononuclear cell fraction of obese individuals. These in vitro and in vivo results suggest that defects in EPC-mediated endothelial repair may be involved in the pathogenesis of obesity-associated cardiovascular disease. Although the short duration of follow-up did not allow us to directly assess the impact of EPC (dys)function on the cardiovascular morbidity and mortality of the subjects studied, our findings strongly support the importance of lifestyle modification and particularly weight reduction in the primary prevention of cardiovascular disease.
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Appendix
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For a supplemental Material and Methods section, please see the online version of this article.
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Acknowledgments
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The authors acknowledge the expert technical assistance of Sarah Henkel and Stephanie Minne. They also thank Philipp Stalling, Anja Werner, and Inga Kudlek for their support in the recruitment of obese individuals and Dr. Thomas Korff (University of Heidelberg, Heidelberg, Germany) for his help in setting up the spheroid angiogenesis assay.
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Footnotes
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This study was awarded the 2008 Hans Blömer Young Investigator Award for Clinical Cardiology by the German Cardiac Society to Dr. Heida. The study was supported by a grant from the German Foundation for Heart Research (Deutsche Stiftung für Herzforschung) to Dr. Schäfer.
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References
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J. Am. Coll. Cardiol. 2010 55: A35-A33.
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