Endothelial progenitor cells (EPCs) are an emerging biomarker of vascular health. However, there are few data on the biology and mobilizing factors of EPCs in metabolic syndrome (MS). The aim of this study was to assay EPC mobilizing factors, including granulocyte colony-stimulating factor, stem cell factor/c-kit ligand (SCF), vascular endothelial growth factor, and stromal cell–derived factor–1 levels, in patients with MS (n = 36) and age- and gender-matched controls (n = 38). There was a significant reduction of 83% in granulocyte colony-stimulating factor levels in patients with MS. Also, there were decreases in SCF and SCF soluble receptor levels. However, there was no significant difference in stromal cell–derived factor–1 levels, and paradoxically, vascular endothelial growth factor levels were increased, consistent with resistance. In conclusion, in addition to progenitor cell exhaustion as a mechanism for the decrease in EPCs in patients with MS, they also have a mobilization defect, as manifested by decreased levels of granulocyte colony-stimulating factor and SCF, resulting in a decrease in EPCs.
Endothelial progenitor cells (EPC), defined by dual positivity of cluster of differentiation 34 (CD34) and kinase insert domain receptor, have been shown to correlate with endothelial function, risk factors for coronary artery disease, cardiovascular disease severity, and incident cardiovascular events. There are few data on EPC number and functionality in metabolic syndrome (MS). Two studies of patients with MS without manifest cardiovascular disease or diabetes showed decreases in EPC number, and the larger study (46 patients with MS) also documented impaired EPC functionality. However, those studies differed with respect to quantitative levels of CD34+ progenitor cells (PCs): the smaller study, in obese men (n = 19), showed no significant decrease, whereas the larger study, conducted in men and women, showed a significant decrease in CD34+ cells in patients with MS. This PC decrease accords with that reported by Fadini et al in patients with MS with diabetes or peripheral arterial disease and argues for bone marrow exhaustion as 1 explanation for the decrease in CD34+ and kinase insert domain receptor–positive EPCs. In the study of obese men, there was a decrease in plasma concentration of the mobilizing factor stem cell factor/c-kit ligand (SCF) but not in vascular endothelial growth factor (VEGF). These preliminary data point to a possible defect in bone marrow mobilization of EPCs in patients with MS. Because of the paucity of data on EPC mobilizing factors and their critical role in determining EPC status, we undertook a more comprehensive study and report on granulocyte colony-stimulating factor, SCF, VEGF, and stromal cell–derived factor–1 levels in patients with MS compared to matched controls, as modulators of PC and EPC mobilization.
Methods
All subjects were recruited from Sacramento County through fliers and advertisements in the newspaper. Subjects (aged 21 to 70 years) with MS (n = 36) and healthy controls (n = 38) were studied. MS was defined using the criteria of the National Cholesterol Education Program Adult Treatment Panel III. Control subjects needed to have ≤2 features of MS and not be taking blood pressure medications. Other selection criteria have been published previously. None of the subjects had diabetes or cardiovascular disease or were taking medications known to affect EPC biology. This protocol was approved by the institutional review board at the University of California, Davis.
After history and physical examination, fasting blood was obtained. Enumeration of peripheral blood PCs and EPCs was performed by flow cytometry, as described previously. Cells positive for CD34 and kinase insert domain receptor were characterized as EPCs. Also, the number of PCs was quantified as CD34+ cells. We have previously shown decreases in PCs and EPCs in this cohort.
Plasma stromal cell–derived factor–1 and VEGF levels were measured by sandwich enzyme-linked immunosorbent assay according to the manufacturer’s protocol (R&D Systems Inc., Minneapolis, Minnesota) and are expressed in picograms per milliliter. SCF, SCF soluble receptor, and granulocyte colony-stimulating factor levels were measured in serum samples by enzyme-linked immunosorbent assay according to the manufacturer’s protocol (R&D Systems Inc.) and are expressed in picograms per milliliter. Interassay coefficients of variation for all enzyme-linked immunosorbent assays were <10%, except for granulocyte colony-stimulating factor, with a coefficient of variation of 14%. High-sensitivity C-reactive protein levels were measured in serum using the Beckman DxI (Beckman Coulter, Inc., Brea, California).
Data are expressed as mean ± SD or, for skewed variables, as medians and interquartile ranges. Log transformations were applied to skewed data before parametric analyses. Comparisons between the control and MS groups were made using 2-sample Student’s t tests. Spearman’s rank correlation coefficients were computed to assess the associations between mobilizing factors and numbers of PCs and EPCs. Data were analyzed using SAS version 9.1.3 (SAS Institute Inc., Cary, North Carolina).
Results
The 2 groups were matched for age and gender. All metabolic features, including homeostasis model assessment and high-sensitivity C-reactive protein, were significantly increased in patients with MS, and high-density lipoprotein cholesterol levels were significantly decreased ( Table 1 ). Also, levels of PCs and EPCs were significantly decreased in patients with MS (p <0.05).
| Variable | Controls (n = 38) | Patients With MS (n = 36) |
|---|---|---|
| Age (years) | 49 ± 12 | 53 ± 11 |
| Waist circumference (inches) | 36 ± 6 | 43 ± 5 ⁎ |
| Men/women | 7/31 | 10/26 |
| Blood pressure (mm Hg) | ||
| Systolic | 118 ± 13 | 132 ± 12 ⁎ |
| Diastolic | 73 ± 8 | 82 ± 10 ⁎ |
| Fasting glucose (mg/dl) | 89 ± 7 | 101 ± 11 ⁎ |
| Total cholesterol (mg/dl) | 188 ± 32 | 200 ± 27 |
| HDL cholesterol (mg/dl) | 54 ± 14 | 40 ± 11 ⁎ |
| LDL cholesterol (mg/dl) | 119 ± 26 | 130 ± 20 |
| Median triglycerides (mg/dl) | 72 | 140 ⁎ |
| Insulin (mIU/mL) | 3.5 ± 0.7 | 7.5 ± 1.2 ⁎ |
| Homeostasis model assessment of insulin resistance | 1.8 (0.91–2.9) | 4.1 (2.5–6.3) ⁎ |
| Median hs-CRP (mg/L) | 1.4 | 3.5 ⁎ |
| PCs (CD34+, mfi) | 12.1 (9.9–21.4) | 8.4 (6.5–11.7) ⁎ |
| EPCs (CD34+ and KDR+, mfi) | 5.1 (3.3–8.9) | 3.6 (2.5–6.7) ⁎ |
With respect to the mobilizing factors, the most significant difference was seen with granulocyte colony-stimulating factor levels, with an 83% decrease in patients with MS ( Figure 1 ). Also, levels of SCF and SCF soluble receptor were significantly decreased. However, stromal cell–derived factor–1 levels were not different between the 2 groups ( Table 2 ).
| Variable | Controls | Patients With MS |
|---|---|---|
| (n = 38) | (n = 36) | |
| SCF soluble receptor (pg/ml) | 20 ± 6 | 15 ± 4 † |
| SDF-1α (pg/ml) | 2.2 ± 0.4 | 2.2 ± 0.4 |
| VEGF (pg/ml) | 41 ± 29 | 85 ± 44 ⁎ |
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