Introduction: It has been established that neovascularization is impaired in the aged population, resulting in reduced tissue survival following an ischemic insult. We hypothesize this may be due to an age associated defect in the trafficking of vascular progenitor/stem cells to sites of injury. We have previously demonstrated that stromal cell-derived factor-1 (SDF-1) is a critical determinant of stem cell recruitment and neovascularization at sites of ischemic insult, and is regulated in vivo and in vitro by the transcription factor hypoxia-inducible factor-1 (HIF-1). Here we examine whether defects in SDF-1 and attenuated stem cell recruitment may be responsible for the vascular complications of aging.
Methods: A 2.5cm x 1.25 cm dorsal peninsular flap was raised on young (2-4 months old) and aged (20-24 months old) C57 mice (n=6 per group) to create an in vivo gradient of soft tissue ischemia as previously described by our laboratory. Ischemia was confirmed by laser Doppler flow analysis and direct oxygen measurements. Flaps were harvested 12 hours later, and days 7 and 14, and sectioned into three progressively ischemic zones. RNA and protein were then isolated from all three sections as well as from normal mouse skin. Comparative levels of HIF-1 were obtained by western blot and levels of SDF mRNA expression were quantitated using real-time PCR. Vascular density was determined by sectioning of flap tissue and enumeration of cells staining positive for the CD31 antigen. Mobilization of endothelial progenitor cells was determined by FACS analysis of peripheral blood flk-1+/CD11b- EPCs.
Results: 12 hours post-operatively, SDF-1 mRNA was induced in the distal (most ischemic) area of the flap over 2 fold less (1.9 vs 4.3) in the aged group compared to the young group (p<.001). HIF-1 alpha protein expression was also induced almost two-fold less in the aged group (1.39 vs 2.55) in the distal ischemic flap region. At day 7, the aged group failed to restore normal oxygen tensions and blood flow to ischemic tissue following surgery whereas the young group had near complete restoration. This was accompanied by a corresponding impairment in mobilization of flk-1+/CD11b- EPCs (6 fold reduction, n=3). A failure of neovascularization was appreciated in the aged group (18 CD31+/HPF vs 75 CD31+/HPF) as evidenced by CD31+ vessel counts in ischemic tissue when compared to controls. At day 14, aged group flaps revealed a marked reduction in tissue survival (15-30% vs 100%) compared to the young group.
Conclusion: Age-associated impairment of HIF-1 alpha attenuates the induction of SDF-1 in reponse to ischemia, resulting in impaired stem cell recruitment and vasculogenesis. This may contribute to the increased incidence of vascular disease in the aged population. Restoration of age related deficits in stem cell trafficking or function may then allow for normal neovascularization minimizing the complications associated with vascular disease in the aged population.