Suppressed hindlimb perfusion in Rac2-/- and Nox2-/- mice does not result from impaired collateral growth

Abstract

While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.

Fig. 1.

Primary collateral pathways in the mouse hindlimb 14 days after femoral artery ligation or excision. A: intravital microscopy and Microfil vascular casting in the experimental limbs of C57BL/6J (BL6) and Rac2-null (Rac2^−/−) mice identified four primary pathways that provided a collateral bypass. Arrows show collateral pathways. B: for all collateral pathways in the experimental limbs, an identical pathway composed of the same preexisting vessels could be identified in the contralateral control limbs of the same animal (identified by arrowheads), as shown in the top left versus right images. The boxes indicate the areas enlarged in the bottom images. FA, femoral artery; PF, profunda FA; P, popliteal artery; S, saphenous artery; II, internal iliac artery branch.

Fig. 2.

Vascular compensation to femoral arterial ligation in BL6 and Rac2^−/− mice. A: experimental-to-control limb perfusion ratios for BL6 and Rac2^−/− mice at days 1, 7, and 14 postligation demonstrated that relative perfusion was increased after day 1 and was similar between strains at all days (BL6 mice, n = 6; and Rac2^−/− mice, n = 7). B: diameters of control and collateral artery 14 days after femoral arterial ligation demonstrated significant increases in collateral vessel diameters in both BL6 and Rac2^−/− mice (BL6 mice, n = 6; and Rac2^−/− mice, n = 7). C: average intimal cell numbers of arterial cross sections showed an increase in collateral arteries of both BL6 and Rac2^−/− mice, as shown in D (BL6 mice, n = 6; and Rac2^−/− mice, n = 7). D: representative vessel cross sections (stained with Lee's methylene blue) demonstrated typical luminal expansion and increased intimal cell numbers in collateral arteries compared with control arteries from BL6 and Rac2^−/− mice at 14 days postligation. Arrows identify some of the intimal cell nuclei. *Microfil in the lumen of the vessels, which was sometimes lost during sectioning (bottom right image).

Fig. 3.

Vascular compensation to femoral arterial excision in BL6 and Rac2^−/− mice. A: limb perfusion ratios demonstrated an increase in perfusion after day 1 that was greater in BL6 than Rac2^−/− mice (BL6 mice, n = 7; and Rac2^−/− mice, n = 14). B: collateral diameters in BL6 and Rac2^−/− mice were similarly increased at 7 and 14 days after arterial excision (BL6 mice, n = 6 after 7 days and n = 8 after 14 days; and Rac2^−/− mice, n = 4 after 7 days and n = 5 after 14 days). C: average intimal cell numbers were increased significantly in collaterals of both BL6 and Rac2^−/− mice at 14 days after femoral excision (BL6 mice, n = 5; and Rac2^−/− mice, n = 5).

Fig. 4.

HEMAVET and FACS analysis of total white blood cells (WBCs; A) and CD11b⁺ cells (B). There were significantly more total WBCs and CD11b⁺ cells in Rac2^−/− than BL6 mice both before surgery and during the 14 days after femoral artery excision. The number of WBCs was increased from baseline at day 3 in BL6 mice and days 3–14 in Rac2^−/− mice. The number of CD11b+ cells was increased from baseline at days 1 and 3 postligation in Rac2^−/− mice but not in BL6 mice (BL6 mice, n = 7; and Rac2^−/− mice, n = 6).

Fig. 5.

Vascular compensation to femoral arterial excision in NADPH oxidase 2 (Nox2)-null (Nox2^−/− mice. A: laser-Dopper perfusion imaging (LDPI) ratios for Nox2^−/− mice (n = 9). B: collateral vessel diameters were significantly increased (n = 5).