Bone marrow alterations and lower endothelial progenitor cell numbers in critical limb ischemia patients

Martin Teraa, Ralf W Sprengers, Peter E Westerweel, Hendrik Gremmels, Marie-José T H Goumans, Tom Teerlink, Frans L Moll, Marianne C Verhaar, JUVENTAS study group, M Teraa, R W Sprengers, M C Verhaar, F L Moll, R E G Schutgens, I C M Slaper-Cortenbach, Y van der Graaf, P A Doevendans, W P Th M Mali, Martin Teraa, Ralf W Sprengers, Peter E Westerweel, Hendrik Gremmels, Marie-José T H Goumans, Tom Teerlink, Frans L Moll, Marianne C Verhaar, JUVENTAS study group, M Teraa, R W Sprengers, M C Verhaar, F L Moll, R E G Schutgens, I C M Slaper-Cortenbach, Y van der Graaf, P A Doevendans, W P Th M Mali

Abstract

Background: Critical limb ischemia (CLI) is characterized by lower extremity artery obstruction and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and alterations in the BM contribute to impaired neovascularization in CLI.

Methods: Levels of primitive (CD34(+) and CD133(+)) progenitors and CD34(+)KDR(+) EPC were analyzed using flow cytometry in blood and BM from 101 CLI patients in the JUVENTAS-trial (NCT00371371) and healthy controls. Blood levels of markers for endothelial injury (sE-selectin, sICAM-1, sVCAM-1, and thrombomodulin), and progenitor cell mobilizing and inflammatory factors were assessed by conventional and multiplex ELISA. BM levels and activity of the EPC mobilizing protease MMP-9 were assessed by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured and their paracrine function was assessed.

Results: Endothelial injury markers were higher in CLI (P<0.01). CLI patients had higher levels of VEGF, SDF-1α, SCF, G-CSF (P<0.05) and of IL-6, IL-8 and IP-10 (P<0.05). Circulating EPC and BM CD34(+) cells (P<0.05), lymphocytic expression of CXCR4 and CD26 in BM (P<0.05), and BM levels and activity of MMP-9 (P<0.01) were lower in CLI. Multivariate regression analysis showed an inverse association between IL-6 and BM CD34(+) cell levels (P = 0.007). CAC from CLI patients had reduced paracrine function (P<0.0001).

Conclusion: CLI patients have reduced levels of circulating EPC, despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34(+)-cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. The results of this study suggest that inflammation-induced BM exhaustion and a disturbed progenitor cell mobilization response due to reduced levels and activity of MMP-9 in the BM and alterations in the SDF-1α/CXCR4 interaction contribute to the attenuated neovascularization in CLI patients.

Conflict of interest statement

Competing Interests: Author Marie-José T.H. Goumans is a PLOS ONE Editorial Board Member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Lower circulating progenitor cell levels…
Figure 1. Lower circulating progenitor cell levels in CLI patients.
Data represent median and P75. Circulating progenitor cell numbers in CLI patients (n = 101) and healthy controls (HC; n = 37). The number of circulating CD133+ and CD34+KDR+ EPC was significantly reduced in CLI patients (* P<0.05). No significant (ns) differences in circulating numbers were observed for CD34+ cells.
Figure 2. BM CD34 + progenitor cell…
Figure 2. BM CD34+ progenitor cell are reduced in CLI patients.
Data represent median and P75. BM progenitor cell numbers in CLI patients (n = 101) and healthy controls (HC; n = 12). CD34+ cells were significantly reduced (* P<0.05) in CLI patients compared to health controls. No significant (ns) differences in CD133+-cells and CD34+KDR+ EPC in the BM.
Figure 3. Altered expression of CD26 and…
Figure 3. Altered expression of CD26 and CXCR4 in blood and BM of CLI patients.
Data represent median and P75. The percentage of CXCR4 expressing lymphocytes was higher in the blood of CLI patients (n = 101) compared to healthy controls (HC; n = 37), while a significant reduction was observed in the BM (n = 101 and n = 12 for CLI patients and healthy controls, respectively). The percentage of CD26 expressing lymphocytes was not different in the PB, while a lower percentage of lymphocytes in the BM expressed CD26 in CLI patients. * P

Figure 4. Conditioned medium obtained from CAC…

Figure 4. Conditioned medium obtained from CAC cultured from CLI patients has impaired paracrine effects.

Figure 4. Conditioned medium obtained from CAC cultured from CLI patients has impaired paracrine effects.
Data represent median and P75. Percentage of scratch wound closure stimulated with conditioned medium (CM) obtained from CLI derived CAC (n = 33) was significantly reduced (P
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The reported work was supported by the “Stichting Vrienden UMC Utrecht” on behalf of the Dirkzwager-Assink foundation (The Netherlands, grant CS 06.007, www.vriendenumcutrecht.nl), The Dutch Heart Foundation (The Netherlands, grant 2008B094, www.hartstichting.nl), the Netherlands Organisation for Scientific Research (The Netherlands, ZonMw-TAS grant 116001026, www.zonmw.nl) and foundation “De Drie Lichten” (The Netherlands, grant 10/06). M.C. Verhaar is supported by the Netherlands Organisation for Scientific Research (NWO) (The Netherlands, Vidi grant 016.096.359, www.nwo.nl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Figure 4. Conditioned medium obtained from CAC…
Figure 4. Conditioned medium obtained from CAC cultured from CLI patients has impaired paracrine effects.
Data represent median and P75. Percentage of scratch wound closure stimulated with conditioned medium (CM) obtained from CLI derived CAC (n = 33) was significantly reduced (P

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