Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia

Abstract

Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.

Figures

Fig. 1.

A: diagram of surgery. Left femoral artery was ligated between the inguinal ligament and the bifurcation, which divides the artery into saphenous and popliteal arteries. Branching vessels between these ligatures were tied off, and the femoral artery was removed. B: diagram of in vivo perfused hindlimb preparation. To create a circuit solely perfusing the left femoral artery, the right iliac, caudal, left internal iliac, and pubic-epigastric arteries were ligated.

Fig. 2.

A: flow recovery in females is less compared with male mice (P = 0.007). L/R ratio, left (ligated) leg perfusion/right (nonligated) leg perfusion. B: while there was no significant difference in average appearance score, females had a higher average use score, indicating impaired hindlimb function (P = 0.02). Foot appearance score (index of ischemia): 0, normal; 1–5, cyanosis or loss of nail(s), where the score is dependent on number of nails affected; 6–10, partial or complete atrophy of digit(s), where the score reflects number of digits affected; and 11, partial atrophy of forefoot. Hindlimb use score (index of muscle function): 0, normal; 1, no toe flexion; 2, no plantar flexion; and 3, dragging foot. A higher score indicates impaired use or appearance.

Fig. 3.

A: representative images of cellular infiltration and CD31 staining in Matrigel plugs collected from male and female mice. B: males had a greater number of red blood cell-containing vessels (P = 0.04), and higher cellular infiltration (P = 0.01) compared with females. C: aortic endothelial cells (AECs) isolated from male mice showed a greater proliferative capacity as measured by MTT assay than female AECs (P = 0.005). D: 7 days postligation, male mice had a greater number of α-actin-positive vessels in the semimembranousus muscle compared with females (P = 0.03).

Fig. 4.

A and B: Western blot of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein (p-VASP) in calf muscle at baseline. Females had higher expression of VEGF and eNOS (P = 0.03 and 0.0,3 respectively). C and D: Western blots on day 7 show that males have higher levels of VEGF, eNOS, and p-VASP postischemia (P = 0.02, 0.035, and 0.02, respectively).

Fig. 5.

Vascular reactivity immediately following femoral artery ligation. A: vasoreactivity to acetylcholine (ACh). There is no difference between genders unless NG-nitro-l-arginine methyl ester (l-NAME) is used to block eNOS activity, and then males exhibit greater vasodilatation (P = 0.006). B: there is no difference in baseline vasoreactivity to nitroglycerin. C: females have a greater %vasoconstriction to phenylephrine (P = 0.03).

Fig. 6.

A: day 10 vasoreactivity to ACh. Males exhibit greater collateral vasodilatation in response to ACh compared with females (P = 0.016), but this difference is obliterated in the presence of l-NAME. B: males have a greater collateral vasodilation response to nitroglycerin following ischemia (P = 0.0003). C: females have greater %vasoconstriction to phenylephrine postischemia (P = 0.01).