Diminished Vasculogenic Function of Bone Marrow-Derived CD133+ Endothelial Precursor Cells (EPC) Obtained from Advanced Aged Patients with Coronary Artery Disease

Abstract

Recent studies identify potential diminished number and function of CD133+ EPC with advancing age.. We performed in vitro and in vivo studies directly comparing bone marrow (BM)-derived CD133+ cells from patients with chronic coronary artery disease to those derived from both young adult, volunteer healthy controls and umbilical cord blood (UCB). Selected CD133+ cells from patient- and control-derived blood cells (mononuclear cells (MNC)) from peripheral blood (PB) and BM and from UCB cells were phenotyped and tested for their endothelial progenitor lineage potential by assessing early outgrowth endothelial cell colony formation (CFU-EC), tested functionally by an in vitro transmigration assay, and for their ability to effect neovascularization in a NOD. SCID hindlimb ischemia model. BM-derived EPCs obtained from advanced age cardiovascular patients produced significantly less colonies compared to those obtained from young adult healthy controls and CB: CB, 36/105 MNC plated, young adult PB, 80; young adult BM, 25; vs. patient PB, 6.9, and BM, 0.86/105 MNC. Surface expression of CXCR4 and VEGF-R2 was measured by FACS on normal young adult BM CD133+ cells (23.9 ± 6% and 49.8 ± 11.7%, respectively) and that expressed by patient-derived BM CD133+ cells was noted to be significantly lower (7.9 ± 1.6% and 3.6 ± 1.8%, respectively). UCB was 24.7 ± 3.1% and 11.3 ± 2.6%, respectively, for CXCR4 and VEGF-R2 expression. Transmigration of normal young adult versus advanced age patient-derived BM CD133+ cells was assessed and noted to be higher in response to 200 ng/ml SDF-1 (65% vs. 9.3%, respectively; p<0.05) or 50 ng/ml VEGF (2.4% vs. 0.6%, respectively; p<0.05). Production of both inflammatory cytokines IL8 and RANTES from young adult donor-derived samples were significantly higher when compared to advanced-aged cardiovascular patients (IL-8; 1,886 ± 740 pg/ml vs. 522 ± 209 pg/ml, respectively, p<0.05: RANTES; 405 ± 217 pg/ml vs. 17 ± 16 pg/ml, respectively, p<0.05). Cord blood-derived CD133+ cells produced significantly higher IL-8 and RANTES compared with advanced age cardiovascular patients 2,638 ± 235 pg/ml IL8 and 640 ± 192 pg/ml RANTES (p<0.05), but did not differ compared with that expressed by young adult BM CD133+ EPC. IL8, but not RANTES, showed enhancement under hypoxic conditions by young adult donor-derived CD133+ cells; no enhanced production was observed by advanced aged patient-derived BM CD133+ cells. Basic FGF, HGF, MIP1a, MIP1b, TNFa, and VEGF were measured by Luminex and were noted either to not differ between subject groups or synthesis was not observed above the minimal detection of the assay. To evaluate the ability of cells to secrete factors to modulate inflammatory mechanisms and affect neovascularization, control- or, patient-derived BM- or PB-CD133+ cells, PB mononuclear cells (MNC), or media were injected by direct intra-cardiac route into NOD/SCID mice immediately after femoral artery ligation. Doppler perfusion ratios comparing ischemic/healthy limb perfusion at 28 days were statistically higher in study animals receiving control cord blood CD133+ cells (0.51) compared to those receiving media control (0.37, p<0.05). Mice receiving patient-derived BM CD133+ cells (0.46) or PB MNC (0.37) did not show statistically significant improvement over media control treated animals (p= 0.07, p= 0.94, respectively). In summary, these analyses support the hypothesis that CD133+ EPC in advanced aged patients with cardiovascular disease have reduced vasculogenic biologic functionality demonstrating reduced expression of CXCR4 and VEGF-R2 resulting in reduced ability of these cells to effectively migrate from the marrow to sites of vascular ischemia by their reduced chemoattraction to SDF-1 and VEGF released from ischemic tissues. In addition, reduced local synthesis of inflammatory cytokines and pro-angiogenic cytokines (e.g., IL8 and RANTES) by the limited number of migrating CD133+ EPC may further limit their ability to augment microvascular vasculogenesis within ischemia tissues. Studies to enhance CD133 EPC function in advanced age cardiovascular patients are warranted.