Enhanced notch signaling modulates unproductive revascularization in response to nitric oxide‐angiopoietin signaling in a mouse model of peripheral ischemia

Figures

• 

  Figure 1. VEGF inhibits NO‐Tie‐mediated revascularization in C57Bl6 mice. (A) Laser speckle images from representative animals over the course of recovery. (B) Two‐way repeated measures ANOVA of the ischemic/contralateral ratio reveals that virus had a significant effect in the variation of recovery profiles (5.89% of total variance). This was particularly important at 3 and 7 days after surgery, when NO‐Tie mice (injected with Ad.eNOS+Ad.Ang1) had faster blood flow recovery compared with control mice (injected with Ad.eGFP), while there was no difference between control and NO‐Tie‐VEGF mice (injected with Ad.eNOS+Ad.Ang1 + Ad.VEGF). n = 12 mice/group. Two‐way repeated measures analysis of variance followed by Tukey's multiple comparison test: *P < 0.05, **P < 0.01. (C) Confocal images from representative muscle sections of ischemic adductors stained with isolectin B4 for endothelial cells (green) and α‐smooth muscle actin for vascular smooth muscle cells (red). Scale bar = 25 μm. (D) Capillary density is reduced in NO‐Tie‐VEGF mice when compared to both control and NO‐Tie mice. (E) Arteriole density is increased in NO‐Tie mice relative to control mice. n = 5‐6 mice/group. Kruskal‐Wallis…

• 

  Figure 2. Increases in VEGF‐A/KDR and Notch1 signaling are concomitant with poor blood flux recovery. (A) VEGF‐A and KDR transcripts were significantly upregulated in the ischemic leg compared to the contralateral leg of control animals (*P < 0.05, two‐tailed paired t‐test) but this did not happen in the other animal groups. VEGF‐A and KDR transcripts were significantly lower in the ipsilateral adductors of NO‐Tie and NO‐Tie‐VEGF animals than on the ischemic adductors of control animals, although no differences were observed between them (***P < 0.001, ANOVA with Holm‐Sidak post‐test). (B) Although no differences were observed in Dll4 expression, expression of its receptor Notch1 was significantly upregulated in ischemic adductors of NO‐Tie‐VEGF mice when compared to NO‐Tie mice (**P < 0.005, ANOVA with Holm‐Sidak post‐test). (n = 4 animals/group)

• 

  Figure 3. Ad.sDll4 increased sprouting but reduced branching. (A) HDBECs were co‐cultured on top of fibroblasts in an in vitro angiogenesis assay. They were treated with conditioned media for either Ad.eGFP, Ad.VEGF, Ad.sDll4, or Ad.VEGF + Ad.sDll4 every other day for 2 weeks (n = 3 experimental replicates, experiment was repeated three times). Cells were then fixed and stained for the endothelial junctional marker, VE‐Cadherin (green) and the nuclear marker, DAPI (blue). (B) Both sDll4 and VEGF + sDll4 resulted in a significantly higher tube density (sDll4: P = 0.0004, sDll4 + VEGF: P = 0.0005), than the control eGFP, yet shorter mean tube lengths (C). Treatment with sDll4 resulted in tubes with the most sprouts (16.33 ± 0.677 P = 0.0091); however, VEGF alone and in combination with sDll4 also increased this (D). Additionally, all three treatments increased the density of branches, with VEGF inducing the most and sDll4 alone resulting in slightly less than the combination treatment. One‐way ANOVA with a Bonferroni post‐test, with a 95% confidence interval. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

• 

  Figure 4. Ad.sDll4 impairs recovery of blood flux in NO‐Tie mice, even when paralleled by increased angiogenesis and arteriolargenesis. (A) Ad.sDll4 had no effect on recovery of blood flux after ischemia when compared to control mice but (B) a significant effect in the variation of recovery profiles was observed when this was added to NO‐Tie mice (3.22% of total variance, ***P = 0.0004). (C) Addition of Ad.sDll4 further worsened the outcome of NO‐Tie‐VEGF mice (1.80% of total variance, **P = 0.0070). Two‐way ANOVA with Sidak's post‐test: *P < 0.05, n = 12 mice/group. In all animal groups, addition of Ad.sDll4 led to an increase in both capillary (D‐F) and arteriole densities (G‐I). n = 5‐6 mice/group. Two‐tailed Mann‐Whitney test with 95% confidence interval: ****P < 0.0001