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Expression Profiles of Angiogenesis-Related Genes in Skeletal Muscle from Young and Old Mice Following Acute Hindlimb Ischemia

Jinsong Wang, Xia Liu, Lee Alderman, Justin Tilan, Remi Adenika, James Andrews, Epstein E Stephen, Mary Susan Burnett.
MedStar Research Institute, Washington D.C., D.C.

OBJECTIVES: Decreased levels of vascular endothelial growth factor (VEGF) have been reported to contribute to impaired angiogenesis in aged mice. It is unclear how aging affects other angiogenesis-related genes. Here we investigate the expression profiles of angiogenesis-related genes in young and old C57Bl mice at different time points after acute hindlimb ischemia.

METHODS: Young (4-month, n=22) and old (18-month, n=22) female C57Bl mice were subjected to left hindlimb ischemia by removal of the femoral artery. Peripheral blood, bone marrow, adductor and calf muscles were harvested at baseline, 2 hours, 16 hours, 3 days, and 7 days after hindlimb ischemia. Total RNA was isolated and analyzed by Real-Time PCR for the expression of: matrix metalloproteiniase-9 (MMP9), endothelial nitric oxide synthase (eNOS), stromal derived growth factor-1 (SDF1), hypoxia inducible factor-1α (HIF1α), VEGF receptor-1 (FLT1), VEGF receptor-2 (FLK1), Angiopoietin-1 (ANG1), CD133, CD26 and VEGF. Adductor muscle was stained for CD31 in baseline samples.

RESULTS: In peripheral blood, MMP9 expression peaks earlier in young mice (2 hours) than old (16 hours) (p<0.05). In calf muscle, MMP9 and eNOS were upregulated only in the old mice after 16 hours post acute ischemia (p<0.05). SDF1 and HIF1&alpha; were upregulated in both young and old mice after 16 hours, but levels returned to baseline in the young animals on day 7 and remained elevated in the old mice (p<0.05). While VEGF and FLT1 were down-regulated in old mice (p<0.05), there was no difference in expression of FLK1 and ANG1 between young and old (p>0.05). In the bone marrow, although expression of CD133 was higher in the old mice (p<0.05), the level of CD26, which induces stem cell release, was higher in the young group (p<0.05). Baseline staining for CD31 was less intense in the adductor muscle of old compared to young mice (p<0.05).

CONCLUSIONS: Decreased pre-existing capillary density, delayed systemic responses, and impaired expression of VEGF and FLT1 contribute to greater ischemia in old mice, which leads to increased expression of MMP9, eNOS, SDF1 and HIF1&alpha;. ANG1 and FLK1are not key factors in the impaired blood flow recovery in aged mice. Concurrent elevated CD133 and decreased CD26 in the bone marrow of old animals suggests that stem cell mobilization may be impaired with aging.

AUTHOR DISCLOSURES: J. Wang, NIHRO1; X. Liu, None; L. Alderman, None; J. Tilan, None; R. Adenika, None; J. Andrews, None; E. Stephen, NIHRO1; M. Burnett, NIHRO1.