Background
Critical limb ischemia (CLI) is characterized by obstruction of lower extremity arteries and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and a dysfunctional BM environment contribute to impaired neovascularization in CLI.
Methods
Levels of primitive (CD34+ and CD133+) progenitors and CD34+KDR+ haemangioblastic EPC were analyzed using flow cytometry in peripheral blood (PB) and BM from 101 CLI patients in the JUVENTAS trial (
NCT00371371
) and healthy controls (n=37 and n=12 for PB and BM, respectively). Endothelial damage markers (sE-selectin, sICAM-1, sVCAM-1, thrombomodulin) and PB levels of progenitor cell mobilizing (VEGF, SDF-1α, SCF, G-CSF) and inflammatory (IL-6, IL-8, IP-10) factors were assessed by ELISA and multiplex. Levels and activity of the EPC mobilizing protease MMP-9 were assessed in BM plasma by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured from PB, and CAC paracrine function was assessed.
Results
Endothelial damage markers were higher in CLI (
p<
0.01). PB levels of VEGF, SDF-1α, SCF, G-CSF (
p<
0.05) and of IL-6, IL-8 and IP-10 were higher in CLI (
p<
0.05). Circulating EPC and CD133+ cells and BM CD34+ cells were significantly lower in CLI (all
p
<0.05), BM levels and activity of MMP-9 were lower in CLI (both
p<
0.01). Multivariate regression analysis showed an inverse association between IL-6 levels and BM CD34+ cell levels (
p=
0.007). CAC outgrowth did not differ significantly between CLI patients and healthy controls (
p=
0.137), however CAC from CLI patients had profoundly reduced migration stimulating potential (
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. Our data suggest that reduced levels and function of circulating progenitor cells and BM dysfunction contribute to the defective neovascularization response in CLI.
Bone Marrow Alterations and Lower Endothelial Progenitor Cell Numbers in Critical Limb Ischemia Patients
