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Tormod S. Westvik, MD, Tamara N

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1 Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis 
Tormod S. Westvik, MD, Tamara N. Fitzgerald, MD, PhD, Akihito Muto, MD, PhD, Stephen P. Maloney, MD, Jose M. Pimiento, MD, Tiffany T. Fancher, MD, Dania Magri, BS, Hilde H. Westvik, MD, Toshiya Nishibe, MD, PhD, Omaida C. Velazquez, MD, Alan Dardik, MD, PhD  Journal of Vascular Surgery  Volume 49, Issue 2, Pages (February 2009) DOI: /j.jvs Copyright © Terms and Conditions

2 Fig 1 Decreased function in aged mice after hind limb ischemia. A, Sketch of the model showing location of iliac artery and vein ligation. B, Comparison of skin incisions needed for the femoral (left panel) and iliac (right panel) ligation models. Note that the iliac model does not require an incision in the ischemic limb. C, Decreased flow in aged mice compared to recovery in young adult mice. Young adult and aged mice had a similar degree of decreased perfusion after iliac ligation (young adult, n = 15: 20.4 ± 1.9%, vs aged, n = 20: 19.6 ± 1.3%; P = .72; however, young adult mice recovered faster and to a greater degree than aged mice (day 7, 35 ± 6% vs 17 ± 4%, P = .046; day 14, 60 ± 5% vs 27 ± 7%, P = .0014). D, Decreased functional status in aged mice compared to recovery in young adult mice. Aged mice had worse functional recovery by day 14 compared to young adult mice (2.3 ± 0.3 vs 4.3 ± 0.4; P = .0021). E, Similar ischemia score in young adult and aged mice. F, Similar modified ischemia score in young adult and aged mice. POD, Postoperative day. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

3 Fig 2 Altered muscle fiber morphology in young adult and aged mice after hind limb ischemia. A, Representative photomicrographs of control and ischemic muscle in young adult and aged mice (n = 12). Magnification, 20×. B, Diminished mean fiber number after ischemia in aged mice (n = 3 mice/group). C, Diminished baseline mean fiber area in aged mice (n = 3 mice/group). D, Similar limb viability in young adult and aged mice. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium; POD, Postoperative day. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

4 Fig 3 Increased capillary density in aged mice after hind limb ischemia. A, Representative photomicrographs of capillary staining of ischemic limbs, day 7, in young adult and aged mice (n = 18). Limb muscles were fixed in 10% formalin and embedded in paraffin. Sections were incubated with FITC-conjugated alpha-actin and TRITC-conjugated lectin antibody. Capillaries were identified by lectin staining (red) in absence of alpha-actin staining (green), and arterioles were detected by alpha-actin staining (green). B, Quantitative analysis of capillary density. Values are expressed as mean ± SEM (n = 2-4). The difference between capillary density in young adult and aged mice is significant (analysis of variance [ANOVA], P = .01); the differences at day 7 and 14 are significant (P = and 0.02, respectively; post-hoc testing). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

5 Fig 4 Increased endothelial progenitor cells in aged mice after hind limb ischemia. A, Representative photomicrographs of immunofluorescence in ischemic tissue of young adult (n = 10; left column) and aged (n = 8; right column) mice at baseline or after 1 or 2 weeks of hind limb ischemia. Magnification, 60×. Red, VEGFR2; green, CXCR4; blue, DAPI; yellow, colocalization of VEGFR2 and CXCR4. Arrows demonstrate representative colocalizing cells; all these cells were noted to be adjacent to the muscle fibers, not within the fibers themselves. B, Bar graph depicts mean number of cells colocalizing for VEGFR2 and CXCR4. The differences between mean number of cells in young adult and aged mice is significant (control leg, P < .0001; ischemic leg, P = .008; analysis of variance [ANOVA]); the differences at day 7 and 14 are significant (control leg, P = and .04, respectively; ischemic leg, P = .007 and .01, respectively; post-hoc testing). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

6 Fig 5 Decreased arteriogenesis in aged mice after hind limb ischemia. A, Representative computed tomography (CT) angiographic images of young adult (left panel) and aged (right panel) mice, 1 week after hind limb ischemia (n = 4). Yellow arrows show collaterals; *, point of ligation; B, bladder. B, Bar graph showing number of collaterals in CT angiograms as determined by pixel densitometry (*, P < .0001). C, Bar graph showing number of collaterals directly counted in CT angiograms (*, P = .0129). D, Representative direct angiographic images of young adult (left panel) and aged (right panel) mice, 1 week after hind limb ischemia (n = 3). Arrows point to lumbar collaterals present (yellow arrows) or absent (red arrows) near the area of iliac ligation (*). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © Terms and Conditions

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