Endothelial nitric oxide synthase deficiency causes collateral vessel rarefaction and impairs activation of a cell cycle gene network during arteriogenesis

Abstract

Rationale: The collateral circulation is tissue- and life-saving in obstructive arterial disease. Disappointing outcomes in clinical trials aimed at augmenting collateral growth highlight the need for greater understanding of collateral biology.

Objective: The role of endothelial nitric oxide synthase (eNOS) in forming native (preexisting) collaterals and remodeling in obstructive disease are unknown or controversial issues, respectively.

Methods and results: We compared the native collateral circulation in healthy tissue and collateral remodeling after femoral artery ligation (FAL) in wild-type and eNOS-knockout (KO) mice. Perfusion after FAL fell further in adult eNOS-KOs, in association with fewer native collaterals in hindlimb (confirmed in brain). This was not attributable to impaired collateral formation in the embryo-neonate, but rather from collateral loss during growth to adulthood. Compared to wild-type, eNOS-KOs evidenced reduced collateral remodeling, angiogenesis, and flow-mediated dilation of the arterial bed supplying the collaterals, resulting in lower perfusion and greater ischemic injury at all time points over 21 days following FAL. To probe the mechanism for impaired remodeling, we performed genome-wide expression profiling of isolated, remodeling hindlimb collaterals 24 hour after FAL. Upregulation of genes encoding cytokines/chemokines, inflammatory, stress response, and cell cycle proteins was evident in wild-type mice. In contrast, expression was lower in 40 of 44 cell cycle genes in eNOS-KO mice, in association with impaired proliferation of vascular wall cells.

Conclusions: Our findings suggest a novel role for eNOS in maintaining native collateral density during natural growth to adulthood and in collateral remodeling in obstructive disease, the latter through regulation of cell proliferation.

Figure 1. Impaired recovery of perfusion in eNOS-KO mice after FAL

A, MicrofilP-casted mouse hindlimb illustrating vasculature before (left) and 21 days after (right) FAL. 1, Proximal femoral artery. 2, LCFA. 3, Superior epigastric artery. 4 and 5, Gracilis collaterals. 6, Distal femoral artery. Green arrows and bars indicate ligation sites. Red arrow indicates higher ligation site in experiments with the second group of mice. White arrows indicate direction of flow before and after FAL. Black arrows indicate the collateral segments microdissected for RNA extraction in microarray analysis. B and C, Laser Doppler perfusion images of adductor and plantar, with region of interest marked with dotted lines (right), and summary data (left). Data in this and all subsequent figures are means±SEM. Two-way ANOVA followed by Dunn–Bonferroni t test; *P<0.05, **P<0.01, ***P<0.001.

Figure 2. Worse ischemic injury in eNOS-KO mice after FAL

A, Representative images of mouse hindlimb after FAL. B and C, Appearance score (B) and use score (C) numerically reflect hindlimb ischemic injury after FAL. Two-way ANOVA followed by Dunn–Bonferroni t test; *P<0.05, ***P<0.001.

Figure 3. Impaired collateral remodeling and reduced hindlimb collateral density in eNOS-KO mice

A, Representative images of MicrofilP-casted mouse gracilis collaterals 21 days after FAL in wild-type (left) and eNOS-KO (right) mice. Insets, Higher magnification images of boxed regions. B, Summary of lumen diameters measured from images represented in A. C and D, Representative images of x-ray angio-grams and number of hindlimb collaterals in eNOS-KO mice, and summary data. E and F, Representative images of alcohol-methyl salicylate–based tissue clearing of MicrofilP-casted hindlimb showing remodeled (left) and native (right) collaterals in eNOS-KO mice, and summary data. **P<0.01, ***P<0.001: t test compared to wild-type.

Figure 4. Increased Doppler-determined flow velocity and flow-mediated dilation of the LCFA, which supplies the gracilis collaterals and other adductor collaterals, in wild-type and eNOS-KO mice

A, Representative images of LCFA (dotted lines show segment where lumen diameter and velocity were measured). B and C, eNOS-KO mice showed much reduced responses. No significant difference in diameter in eNOS-KO and wild-type mice before ligation (not shown). **P<0.01, ***P<0.001: t test compared to wild-type.

Figure 5. Cerebral cortical pial collateral number and diameter at different developmental stages and different genotypes

A, Representative image of MicrofilP-casted adult eNOS-KO mice showing pial artery network and collaterals. Red arrowheads indicate collaterals between middle cerebral (MCA) and anterior cerebral artery (ACA) branches counted and diameters determined. B, representative image of E18.5 eNOS-KO mouse embryonic brain. C and D, Collateral number and diameter. E, No significant differences in pial collateral number in P28 pups crossed to harboring indicated alleles. *P<0.05, **P<0.01; 2-tailed t test.

Figure 6. Comparable expression of leukocyte recruitment and inflammatory response genes during early collateral remodeling (24 hour after FAL or after acute ligation (control group)) in eNOS-KO and wild-type mice

A and B, Comparison of upregulated chemo-attractant and cytokine receptor genes (A) and inflammatory/stress responsive genes (B). SAM indicates significantly upregulated genes identified by SAM analysis (red squares in WT or KO column); d, delta score obtained by SAM analysis; FC, fold changes of gene expression calculated on log(base 2) data. *P<0.05, ***P<0.001 for 2-tailed t test of log(base 2) expression data between eNOS and wild-type mice. Full-sized images of A and B are available in Online Figures VI and VII. C, Representative images of IHC staining for CD45⁺ leukocytes (brown stain), with light hematoxylin counterstaining.

Figure 7. Impaired activation of cell cycle genes in eNOS-KO mice during early (per Figure 6 legend) collateral remodeling

A, Comparison of upregulated cell cycle regulatory genes between eNOS-KO and wild-type mice. Color-highlighted genes belong to various functional categories described in Results. Full-sized image of A is available in Online Figure VIII. B, Representative images of BrdUrd incorporation by proliferating vascular cells in remodeling collaterals 7 days after ligation. Arrows indicate BrdUrd⁺ cells. C, Summary of proliferating cells/collateral averaged from 10 consecutive sections of each genotype. *P<0.05, **P<0.01, ***P<0.001; 2-tailed t test.

Figure 8. Real-time quantitative RT-PCR confirmation of genes identified in the microarray studies and activation of a cell cycle gene network during early collateral remodeling in wild-type mice and absence of its activation in eNOS-KO mice

A, Quantitative RT-PCR showing expression of 8 representative genes from each functional gene category identified in the microarray studies. Fold increase in remodeling collaterals is compared to nonligated collaterals (n=3; *P<0.05, **P<0.01). B, Network identified by IPA of upregulated genes detected by arrays (Figure 7A). Color-filled symbols reflect genes in functional categories described in the legend for Figure 7 and in Results. Full-sized image of B is available in Online Figure IX.