Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Am J Cardiol. 2010 Sep 21;106(11):1606–1608. doi: 10.1016/j.amjcard.2010.07.039

Circulating Levels of Endothelial Progenitor Cell Mobilizing Factors in The Metabolic Syndrome

Ishwarlal Jialal a, Gian Paolo Fadini b, Kari Pollock a, Sridevi Devaraj a
PMCID: PMC3052293  NIHMSID: NIHMS246577  PMID: 21040691

Abstract

Endothelial progenitor cells (EPCs) appear to be an emerging biomarker of vascular health. However, there is scanty data on EPC biology and its mobilizing factors in the Metabolic syndrome (MS). In this study, we assayed EPC mobilizing factors including granulocyte colony stimulating factor (G-CSF), stem cell factor/c-kit ligand (SCF), vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) levels in patients with MS (n =36) and age and gender-matched controls (n = 38). There was a significant reduction in G-CSF levels (83% decrease) in the patients with MS. Also, there was a decrease in SCF and SCF soluble receptor levels. However, there was no significant difference in SDF-1 levels and paradoxically, VEGF levels were increased consistent with resistance. In conclusion, in addition to progenitor cell exhaustion as a mechanism for the decrease in EPC in patients with MS, we suggest that they also have a mobilization defect as manifest by decreased levels of G-CSF and SCF resulting in a decrease in EPC.

Keywords: Progenitor cells, endothelium, metabolic syndrome, prediabetes, mobilizing factors

Introduction

Endothelial Progenitor Cells (EPC), defined by dual positivity of CD34 and KDR, have been shown to correlate with endothelial function, risk factors for coronary artery disease (CAD), cardiovascular disease (CVD) severity and incident cardiovascular events (CVE) 1–5. There is scant data on EPC number and functionality in metabolic syndrome (MS) 6. Two studies on patients with MS without manifest CVD or diabetes showed a decrease in EPC number, and the larger study (MS n=46) also documented impaired EPC functionality 7,8. However, they contrasted with respect to quantitative levels of CD34+ progenitor cells (PC), since the smaller study in obese males (n=19) showed no significant decrease whilst the larger study, conducted in both males and females, showed a significant decrease in CD34+ cells in MS patients. This PC decrease accord with that reported by Fadini et al. in MS patients with diabetes or peripheral arterial disease (PAD) 9 and argues for bone marrow exhaustion as one explanation for the decrease in CD34+KDR+ EPCs. In the study of obese males there was a decrease in plasma concentration of the mobilizing factor stem cell factor/c-kit ligand (SCF), but not of vascular endothelial growth factor (VEGF). These preliminary data point to a possible defect in bone marrow mobilization of EPC in MS patients. Due to the paucity of data on EPC mobilizing factors6 and their critical role in determining EPC status, we undertook a more comprehensive study and report on granulocyte colony stimulating factor (G-CSF), SCF, VEGF and stromal cell-derived factor-1 (SDF-1) levels in patients with MS compared to matched controls, as modulators of PC and EPC mobilization.

Method

All subjects were recruited from Sacramento County through fliers and advertisements in the newspaper. Subjects (age 21–70 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-III10. Control subjects needed to have ≤ 2 features of MS and not be on blood pressure (BP) medications. Other selection criteria have been published previously8. None of the subjects had diabetes, cardiovascular disease or were on medications known to affect EPC biology11. This protocol was approved by the Institutional Review Board at University of California Davis.

After history and physical examination, fasting blood was obtained. Enumeration of peripheral blood PC and EPCs were performed by flow cytometry as described previously8. Cells positive for both CD34 and KDR were characterized as EPCs. Also, the number of PC was quantified as CD34 positive cells. We have previously shown decrease in PC and EPC in this cohort8.

Plasma SDF-1 and VEGF levels were measured by sandwich ELISA according to the manufacturer’s protocol (R&D Systems) and expressed in pg/mL. SCF, SCF-sR (SCF-soluble receptor) and G-CSF levels were measured in serum samples by ELISA according to the manufacturer’s protocol (R&D Systems). SCF-sR levels were expressed in pg/mL and SCF and G-CSF levels were expressed in pg/mL. Inter-assay coefficient of variation (CV) for all the ELISAs was < 10% except for G-CSF with a CV of 14%. HsCRP levels were measured in serum using the Beckman DxI8.

Data were expressed as mean±SD or, for skewed variables as median and interquartile range. Log transformations were applied to skewed data prior to parametric analyses. Comparisons between the control and MS groups were made with two-sample t-tests. Spearman’s rank correlation coefficients were computed to assess the association between mobilizing factors and both PC and EPC numbers. Data were analyzed using SAS version 9.1.3 (SAS Institute, Cary, NC, USA).

Results

The 2 groups were matched for age and gender. All metabolic features including homeostasis model assessment (HOMA) and hsCRP were significantly increased in patients with MS and HDL-C levels were significantly decreased (Table 1). Also, both levels of progenitor cells (PC) and endothelial progenitor cells (EPC) were significantly decreased in MS patients (p<0.05).

Table 1.

Baseline Characteristics

Variable Controls (n=38) Metabolic Syndrome(n=36)
Age (year) 49 ± 12 53 ± 11
Waist circumference (inches) 36 ± 6 43 ± 5*
Male: Female 7:31 10:26
Blood Pressure
 Systolic (mmHg) 118 ± 13 132 ± 12*
 Diastolic (mmHg) 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
Triglycerides (mg/dl)1 72 140 *
Insulin (mIU/mL) 3.5 ± 0.7 7.5 ± 1.2*
HOMA-IR 1.8 (0.91,2.9) 4.1 (2.5,6.3)*
hsCRP (mg/L)1 1.4 3.5*
Progenitor Cells (CD34+, mfi) 12.1 (9.9, 21.4) 8.4 (6.5, 11.7)*
EPC (CD34+/KDR+, mfi) 5.1 (3.3,8.9) 3.6 (2.5,6.7)*
Open in a new tab

Unless otherwise noted, data were mean ± SD;

1

Data were medians;

*

p<0.05,

**

p<0.01,

***

p<0.001, compared to Controls

Abbreviations: EPC: endothelial progenitor cells; HDL-High density lipoprotien; HOMA_IR: Homeostasis Model Assessment of Insulin Resistance; hsCRP: High sensitivity C-reactive protein; LDL-Low density lipoprotein

With respect to the mobilizing factors, the most significant difference was seen with G-CSF levels with an 83% decrease in patients with MS (Figure 1). Also levels of both SCF and SCF-sR were significantly decreased. However SDF-1 levels were not different between the 2 groups (Table 2).

Figure 1. EPC Mobilizing Factors in Metabolic Syndrome.

Figure 1

Open in a new tab

SCF and G-CSF levels were measured in serum samples of Controls and MS patients by ELISA as described in Methods. *p<0.001 compared to controls

Table 2.

Endothelial Progenitor Cell Mobilizing Factors

Variable Controls (n=38) Metabolic Syndrome (n=36)
SCF-sR (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*
Open in a new tab

Data were mean ± SD.

*

p<0.05,

**

p<0.01, compared to Controls

Abbreviations: SCF-sR: stem cell factor/c-kit ligand soluble receptor; SDF: stromal cell-derived factor; VEGF-vascular endothelial growth factor

Levels of VEGF were significantly increased. Correlations were undertaken between mobilizing factors and CD34+ PC or CD34+KDR+ EPC numbers, both of which we have previously reported to be decreased in MS patients8. The only significant correlations were between EPC and G-CSF (r=0.26; p=0.041) and between PC and VEGF(r=0.42; p=0.044). There were no significant correlations between mobilizing factors and HOMA and hsCRP levels.

Discussion

We and others have previously shown that PCs and EPCs are decreased in subjects with MS6–9. This finding was further confirmed in this study with a smaller sample size. This observation might have profound implications in terms of cardiovascular risk, as both CD34+12 and CD34+KDR+ cells4,5 predict incident CVE and regulate cardiovascular homeostasis. However, the causes of (E) PC pauperization in MS remain unclear. Herein, we expand our previous observations on decreased (E)PC level and impaired functionality8 and suggest that an imbalance in the levels of mobilizing factors may be responsible in part for reduced (E)PC. Striking was the reduction of G-CSF associated with MS, which was accompanied by low levels of SCF. The reduced concentration of SCF-sR argues against a decoy effect by soluble c-kit generated by cleavage of the membrane isoform13 and suggests a reduced expression of SCF. Interestingly, G-CSF and SCF are known to synergize in inducing bone marrow PC mobilization14 and plasma levels of SCF have been also taken to represent a measure of PC mobilization15. Given that they are both produced by, among other cells, the endothelium, it is possible that this represents a novel feature of endothelial dysfunction associated with MS. The paradox of increased VEGF levels may reflect VEGF post-receptor resistance, as seen in diabetes and argues for VEGF functional impairment since EPC numbers were significantly decreased 16–18. In this study, the direct PC/VEGF and the inverse EPC/G-CSF correlations suggest that an imbalance in VEGF and G-CSF in MS patients may be responsible for defective EPC generation and mobilization from bone marrow PC. This hypothesis is supported by observation that VEGF does not stimulate bone marrow PC in the absence of G-CSF19. Thus, we hypothesize that since G-CSF, SCF and SCF soluble receptor are significantly decreased coupled with a functional deficiency of VEGF (resistance) that inspite of normal levels of SDF, patients with MS have an imbalance of mobilizing factors supporting a mobilizing defect. In future studies, we will examine VEGF resistance in monocytes by studying VEGF receptors (VEGF R1 and VEGF R2)20, post-receptor signaling and also the status of the SDF receptor CXCR421 on EPCs in MS. Noteworthy, in the present report, the imbalance of mobilizing factors was found in MS without diabetes and CVD, indicating that it may have a role early in the natural history of the disease.

Acknowledgments

We also like to thank Manpreet Kaur, UC Davis Medical Center for manuscript preparation.

Grants: NIH K-24 AT00596 (IJ) and ADA grant (IJ).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348 :593–600. doi: 10.1056/NEJMoa022287. [DOI] [PubMed] [Google Scholar]
  • 2.Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001;89:E1–7. doi: 10.1161/hh1301.093953. [DOI] [PubMed] [Google Scholar]
  • 3.Kunz GA, Liang G, Cuculi F, Gregg D, Vata KC, Shaw LK, Goldschmidt-Clermont PJ, Dong C, Taylor DA, Peterson ED. Circulating endothelial progenitor cells predict coronary artery disease severity. Am Heart J. 2006;152(1):190–195. doi: 10.1016/j.ahj.2006.02.001. [DOI] [PubMed] [Google Scholar]
  • 4.Schmidt-Lucke C, Rössig L, Fichtlscherer S, Vasa M, Britten M, Kämper U, Dimmeler S, Zeiher AM. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair. Circulation. 2005;111(22):2981–2987. doi: 10.1161/CIRCULATIONAHA.104.504340. [DOI] [PubMed] [Google Scholar]
  • 5.Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Bohm M, Nickenig G. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005;353(10):999–1007. doi: 10.1056/NEJMoa043814. [DOI] [PubMed] [Google Scholar]
  • 6.Jialal I. Endothelial progenitor cell status in metabolic syndrome. Met Syn and Relat Disord. 2010;8:193–195. doi: 10.1089/met.2010.0803.edi. [DOI] [PubMed] [Google Scholar]
  • 7.Westerweel PE, Visseren FL, Hajer GR, Olijhoek JK, Hoefer IE, de Bree P, Rafii S, Doevendans PA, Verhaar MC. Endothelial progenitor cell levels in obese men with the metabolic syndrome and the effect of simvastatin monotherapy vs. simvastatin/ezetimibe combination therapy. Eur Heart J. 2008;29(22):2808–2817. doi: 10.1093/eurheartj/ehn431. [DOI] [PubMed] [Google Scholar]
  • 8.Jialal I, Devaraj S, Singh U, Huet BA. Decreased number and impaired functionality of endothelial progenitor cells in subjects with metabolic syndrome: Implications for increased cardiovascular risk. Atherosclerosis. 2010 doi: 10.1016/j.atherosclerosis.2010.01.036. [Epub ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, Avogaro A. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol. 2005;45(9):1449–1457. doi: 10.1016/j.jacc.2004.11.067. [DOI] [PubMed] [Google Scholar]
  • 10.Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin BA, Gordon DJ, Krauss RM, Savage PJ, Smith SC, Jr, Spertus JA, Costa F American Heart Association; National Heart, Lung, and Blood Institute. Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation. 2005;112(17):2735–2752. doi: 10.1161/CIRCULATIONAHA.105.169404. [DOI] [PubMed] [Google Scholar]
  • 11.Werner N, Nickenig G. Clinical and therapeutical implications of EPC biology in atherosclerosis. J Cell Mol Med. 2006;10:318–332. doi: 10.1111/j.1582-4934.2006.tb00402.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Fadini GP, de Kreutzenberg S, Agostini C, Boscaro E, Tiengo A, Dimmeler S, Avogaro A. Low CD34+ cell count and metabolic syndrome synergistically increase the risk of adverse outcomes. Atherosclerosis. 2009;207(1):213–219. doi: 10.1016/j.atherosclerosis.2009.03.040. [DOI] [PubMed] [Google Scholar]
  • 13.Blechman JM, Lev S, Brizzi MF, Leitner O, Pegoraro L, Givol D, Yarden Y. Soluble c-kit proteins and antireceptor monoclonal antibodies confine the binding site of the stem cell factor. J Biol Chem. 1993;268(6):4399–4406. [PubMed] [Google Scholar]
  • 14.Duarte RF, Franf DA. The synergy between stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF): molecular basis and clinical relevance. Leuk Lymphoma. 2002;43(6):1179–1187. doi: 10.1080/10428190290026231. [DOI] [PubMed] [Google Scholar]
  • 15.Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002;109(5):625–637. doi: 10.1016/s0092-8674(02)00754-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Waltenberger J. VEGF resistance as a molecular basis to explain the angiogenesis paradox in diabetes mellitus. Biochem Soc Trans. 2009;37(Pt 6):1167–1170. doi: 10.1042/BST0371167. [DOI] [PubMed] [Google Scholar]
  • 17.Sasso FC, Torella D, Carbonara O, Ellison GM, Torella M, Scardone M, Marra C, Nasti R, Marfella R, Cozzolino D, Indolfi C, Cotrufo M, Torella R, Salvatore T. Increased vascular endothelial growth factor expression but impaired vascular endothelial growth factor receptor signaling in the myocardium of type 2 diabetic patients with chronic coronary heart disease. J Am Coll Cardiol. 2005;46(5):827–834. doi: 10.1016/j.jacc.2005.06.007. [DOI] [PubMed] [Google Scholar]
  • 18.Tchaikovski V, Olieslagers S, Böhmer FD, Waltenberger J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes. Circulation. 2009;120(2):150–159. doi: 10.1161/CIRCULATIONAHA.108.817528. [DOI] [PubMed] [Google Scholar]
  • 19.Pitchford SC, Furze RC, Jones CP, Wengner AM, Rankin SM. Differential mobilization of subsets of progenitor cells from the bone marrow. Cell Stem Cell. 2009;4(1):62–72. doi: 10.1016/j.stem.2008.10.017. [DOI] [PubMed] [Google Scholar]
  • 20.Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9(6):669–676. doi: 10.1038/nm0603-669. [DOI] [PubMed] [Google Scholar]
  • 21.Egan CG, Lavery R, Caporali F, Fondelli C, Laghi-Pasini F, Dotta F, Sorrentino V. Generalised reduction of putative endothelial progenitors and CXCR4-positive peripheral blood cells in type 2 diabetes. Diabetologia. 2008;51(7):1296–305. doi: 10.1007/s00125-008-0939-6. [DOI] [PubMed] [Google Scholar]

RESOURCES