Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis

Abstract

Objective: Older patients are thought to tolerate acute ischemia more poorly than younger patients. Since aging may depress both angiogenesis and arteriogenesis, we determined the effects of age on both angiogenesis and arteriogenesis in a model of severe acute limb ischemia.

Methods: Young adult (3-months-old) and aged (18-months-old) C57BL/6 mice underwent right common iliac artery and vein ligation and transection. Data were collected on days 0, 7, and 14. Perfusion was measured with a laser Doppler scan and compared to the contralateral limb. Functional deficits were evaluated with the Tarlov scale. Capillary density and endothelial progenitor cell (EPC) number were determined by direct counting lectin-positive/alpha-actin-negative cells and VEGFR2/CXCR4 dually-positive cells, respectively; angiography was performed to directly assess arteriogenesis.

Results: Young adult and aged mice had a similar degree of decreased perfusion after iliac ligation (young, n = 15: 20.4 +/- 1.9%, vs aged, n = 20: 19.6 +/- 1.3%; P = .72, analysis of variance [ANOVA]); however, young 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). Aged mice had worse functional recovery by day 14 compared to young mice (2.3 +/- 0.3 vs 4.3 +/- 0.4; P = .0021). Aged mice had increased capillary density (day 7, 12.9 +/- 4.4 vs 2.8 +/- 0.3 capillaries/hpf; P = .02) and increased number of EPC incorporated into the ischemic muscle (day 7, 8.1 +/- 0.9 vs 2.5 +/- 1.9 cells; P = .007) compared to young mice, but diminished numbers of collateral vessels to the ischemic limb (1 vs 9; P = .01), as seen on angiography.

Conclusion: After severe hind limb ischemia, aged animals become ischemic to a similar degree as young animals, but aged animals have significantly impaired arteriogenesis and functional recovery compared to younger animals. These results suggest that strategies to stimulate arteriogenesis may complement those that increase angiogenesis, and may result in improved relief of ischemia.

Figure 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±.3 vs. 4.3±.4; p=.0021). E) Similar ischemia score in young adult and aged mice. F) Similar modified ischemia score in young adult and aged mice.

Figure 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, 20X. 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.

Figure 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 (ANOVA, p=0.01); the differences at day 7 and 14 are significant (p=0.007 and 0.02, respectively; post-hoc testing).

Figure 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 and after 1 or 2 weeks of hind limb ischemia. Magnification, 60x. 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<0.0001; ischemic leg, p=0.008; ANOVA); the differences at day 7 and 14 are significant (control leg, p=0.0002 and 0.04, respectively; ischemic leg, p=0.007 and 0.01, respectively; post-hoc testing).

Figure 5

Decreased arteriogenesis in aged mice after hind limb ischemia. A) Representative 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=0.0129). 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 (*).