Vascular endothelial growth factor-A specifies formation of native collaterals and regulates collateral growth in ischemia

Abstract

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial growth factor (VEGF), which is important in embryonic vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-1 (VEGFR-1(+/-)), VEGF receptor-2 (VEGFR-2(+/-)), and overexpressing (VEGF(hi/+)) and underexpressing VEGF-A (VEGF(lo/+)). Recovery from hindlimb ischemia was followed for 21 days after femoral artery ligation. All statements below are P<0.05. Compared to wild-type mice, VEGFR-2(+/-) showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-1(+/-) showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGF(lo/+) had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGF(hi/+) mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These differences were confirmed in the cerebral pial cortical circulation where, compared to VEGF(hi/+) mice, VEGF(lo/+) formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-1 are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.

Figure 1

Impaired recovery of perfusion in VEGFR-1^+/- mice after femoral ligation. (a,b) VEGFR-1^+/- mice have greater hindlimb ischemic appearance score. Scale: 0=normal, 1=cyanosis or loss of nail(s), 2=partial or complete atrophy of digit(s), 3=partial atrophy of fore-foot; n=6-20 per data-point. (c) Laser Doppler perfusion images of plantar foot with region of interest (ROI). (d,e) Quantification of plantar perfusion measured over ROI. Data are mean±SEM for this and other figures. Two-way ANOVA followed by Dunn-Bonferonni t-Test; *p<0.05 vs. wild-type CD-1; n=6-16 mice per data-point.

Figure 2

Reduced collateral remodeling (lumen expansion) and leukocyte recruitment in VEGFR-1^+/- mice. (a) Lectin-stained capillaries in gastrocnemius. (d,g) Capillary number-to-muscle fiber number ratios at baseline (before) and 21 days after femoral ligation. (b) Cyano-Massons-elastin-staining of collateral in gracilis. (e,h) Collateral lumen diameter at baseline and 21 days after ligation. Numbers inside bars here and elsewhere are percent increase from baseline. (c,f,i) CD45⁺ leukocytes in a 1-diameter area around anterior gracilis collaterals. Paired t-Test vs. baseline; Un-paired t-Test vs. wild-type; #p<0.05 vs. percentage change from wild-type; n=6-11 mice per data-point.

Figure 3

Quantitative RT-PCR of VEGF isoforms (a-f) and HIF subunits (g-i) in calf (a-c) and adductor (d-i) of wild-type (a,d,g), VEGF^hi/+ (b,e,h), and VEGF^lo/+ (c,f,i) mice. Data for muscle taken from ligated leg are relative to muscle from non-ligated leg and normalized to 18S rRNA. Non-parametric t-Test vs. non-ligated; n=5-6 mice per data-point.

Figure 4

VEGF^hi/+ mice have better and VEGF^lo/+ worse recovery after femoral ligation. (a,b) Lower ischemic appearance score in VEGF^hi/+ and more ischemia in VEGF^lo/+ (see Fig. 1 for scale); n=6-20 per data-point. (c,d,e) Quantification of perfusion measured over plantar ROI shows less reduction immediately after ligation (“Post-Op”) and better recovery in VEGF^hi/+, and opposite effects in VEGF^lo/+. (f) Representative Doppler images of Pre- and Post-Op time points. ANOVA followed by Dunn-Bonferonni t-Test; *p<0.05, **p<0.01, ***p<0.001 vs. wild-type; n=6-16 per data-point. (g) Mean arterial pressure (MAP) was measured under anesthetic conditions used for Doppler measurements.

Figure 5

Collateral remodeling and angiogenesis are attenuated in VEGF^lo/+ mice. Capillary number-to-muscle fiber number ratios (a,b) and collateral lumen diameters (c,d) at baseline and day-21 post-ligation. Paired t-Test vs. Baseline; Unpaired t-test vs. wild-type; #p<0.05 vs. percentage change from wild-type; n=7-11 per data-point. (e) Immunofluorescent localization of VEGF in VEGF^hi/+ mice. Non-collateral arteriole (left panel) and gracilis collateral at day-0 (middle panel) and day-7 post-ligation (right panel).

Figure 6

Collateral density in skeletal muscle and pial circulations correlates with VEGF genotype. (a) Post-mortem X-ray arteriogram of thigh. Vessels were counted (data given in (b)) that crossed a line drawn from the mid-point between femoral ligations through the thigh collateral zone at baseline and day-7 post-ligation. (c) Post-mortem arteriogram of pial circulation. Collaterals were counted (data given in (d)) that interconnected middle and anterior cerebral artery trees at post-natal day-1, day-21 and 12 weeks. Brackets, 1-way ANOVA followed by Dunn-Bonferonni t-Test; *p<0.05 vs. P1; #p<0.05, ###p<0.001 vs. corresponding wild-type time-point; n=8-11 per data-point. (e) TTC staining 24 hours after MCAO. (f) Infarction volumes reported as % cortex volume. (g) Correlation of infarction volume and collateral number.