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

Abstract

arteriogenesis; hindlimb ischemia; necrosis; regeneration; NADPH oxidase 2 IN THE PERIPHERAL CIRCULATION, the dilation and enlargement of preexisting vessels that form collateral pathways subsequent to arterial occlusion are the primary vascular compensations that preserve tissue viability and maintain function. These vessels dilate within seconds (25,39,45,70) and undergo expansion for weeks (20,25,45,69). In various species, the preexisting vessels are the size of the smallest arteries, and they enlarge ϳ100% (14,20,35,45,56). Available clinical studies have indicated that subsequent to arterial occlusion in the peripheral circulation, the primary vessels that enlarge as collaterals are preexisting arteries (4,29,57). The largest of the preexisting vessels are typically those that become the dominant collaterals (35,45,51). Combined anatomic and modeling studies have predicted that hindlimb flow subsequent to femoral artery occlusion is primarily determined by these collaterals (22,56). This is consistent with studies of segmental resistances that demonstrated that compensation in the collateral vessels is of significantly greater hemodynamic importance than adaptations in the distal microvasculature (43,71,75). Nevertheless, few studies investigating the mechanisms of vascular compensation subsequent to arterial occlusion in mice have specifically identified and studied these preexisting vessels that form the primary collateral pathways. Such investigations are needed because angiogenesis and collateral growth are initiated by different stimuli, and differences exist in the molecules and mechanisms that mediate these important processes (7, 11).Leukocytes, especially lymphocytes (63, 74) and macrophages (2,33,37), have been shown to have an important role in vascular compensation to hindlimb ischemia. Recent studies by Tojo et al. (66) and Urao et al. (72) have established that NADPH oxidase 2 (Nox2)-derived ROS from bone marrowderived cells (BMDCs) have an important role in neovascularization in the ischemic mouse hindlimb. The initial report (66) concluded from microsphere data that collateral growth was impaired, but did not specifically identify collateral bypass vessels or measure their diameters.To investigate the hypothesis that Nox2-derived NAD(P)H oxidase mediates primary collateral growth subsequent to arterial occlusion, the present study used Rac2-null (Rac2 Ϫ/Ϫ ) and Nox2-null (Nox2 Ϫ/Ϫ ) mice and a novel method of identifying primary hindlimb collaterals. Rac2 is expressed primarily, if not exclusively, in hematopoietic cells (38, 52) and binds to and activates Nox2-containing NAD(P)H oxidase (24,40). In addition, leukocytes from Rac2 Ϫ/Ϫ and Nox2 Ϫ/Ϫ mice have impaired function related to reduced ROS production (47,66,72). We present a method to identify the dominant or primary collateral that should be the major collateral supplying flow to

Trans-illuminated laser speckle imaging of collateral artery blood flow in ischemic mouse hindlimb

“…2 was chosen. This particular ligation pattern has been previously shown to produce consistent arteriogenesis in the collateral arteries of the gracilis adductor muscles [26][27][28][29] with minimal heterogeneity in the baseline collateral structure and with the predicted changes in flow direction from baseline. Mice, 9 to 13 weeks of age, were anesthetized (i.p.…”

DNA Methyltransferase 1–Dependent DNA Hypermethylation Constrains Arteriogenesis by Augmenting Shear Stress Set Point

“…We used a previously detailed FAL scheme6, 20, 21 that produces consistent arteriogenesis in the collateral arteries of the gracilis adductor muscles,6, 22, 23, 24, 25, 26, 27 along with minimal heterogeneity in the baseline collateral structure and known changes in flow direction from baseline. Male mice (10 to 12, 20 to 21, or 70 to 80 weeks of age for C57BL/6, Balb/c, and aged Balb/c studies, respectively) were anesthetized (intraperitoneal [IP] 120 mg/kg ketamine, 12 mg/kg xylazine, and 0.08 mg/kg atropine), depilated, and prepped for aseptic surgery.…”

“…For analysis of luminal diameters in the gracilis collateral arteries and to enable sectioning at specific regions, vascular casting was performed using an opaque polymer that allows for accurate luminal diameter measurements 24. At the determined point of harvest after FAL, mice were anesthetized (IP 120 mg/kg ketamine, 12 mg/kg xylazine, and 0.08 mg/kg atropine) and euthanized via an overdose of pentobarbital sodium and phenytoin sodium (Euthansol, Virbac), and then the abdominal aorta was cannulated.…”

Toward Functional Revascularization: Regulation of Vascular Network Remodeling Through Altered Hemodynamics

“…Femoral arterial ligation (FAL) was performed to induce collateral remodeling in the gracilis adductor muscle and produce a moderate level of ischemia in the downstream tissue in a method similar to that previously described (99,112,154,155). Briefly, age-matched mice (5-7 months of age, >8 weeks post BMT) were anesthetized (i.p 120 mg/kg ketamine, 12 mg/kg xylazine, and 0.08 mg/kg atropine) and prepped for aseptic surgery.…”

“…Limbs were then perfused with 4% paraformaldehyde in PBS and allowed to fix for 30 minutes, followed by perfusion with a radio-opaque vascular casting agent (Microfil®, FlowTech Inc, 2:1:0.15 ratio of color:dilutent:curing agent). Once casting agent was visible in the saphenous artery distal to the ligation site, perfusion was discontinued to limit filling only to the primary collaterals, as previously described (155). The casting agent was allowed to cure overnight at 4 o C followed by decalcification for 5 days.…”