In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 (ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2.
Objective
Surgical hindlimb ischemia (HLI) in mice has become a valuable preclinical model to study peripheral arterial disease (PAD). We previously identified that the different phenotypical outcomes following HLI across inbred mouse strains is related a region on the short arm of mouse chromosome 7. The gene coding the interleukin-21 receptor (IL-21R) lies at the peak of association in this region.
Approach and Results
With quantitative RT-PCR, we found that a mouse strain with a greater ability to up-regulate IL-21R following HLI had better perfusion recovery than a strain with no up-regulation after HLI. Immunofluorescent staining of ischemic hind-limb tissue showed IL-21R expression on endothelial cells (EC) from these C57BL/6 mice. An EC-enriched fraction isolated from ischemic hind-limb muscle showed higher Il-21R levels than an EC-enriched fraction from non-ischemic limbs. In-vitro, human umbilical vein EC (HUVEC) showed elevated IL-21R expression after hypoxia and serum starvation. Under these conditions, IL-21 treatment increased cell viability, decreased cell apoptosis, and augmented tube formation. In-vivo, either knockout Il21r or blocking IL-21 signaling by treating with IL-21R-Fc (fusion protein that blocks IL-21 binding to its receptor) in C57BL/6 mice resulted in less perfusion recovery after HLI. Both in-vitro and in-vivo modulation of the IL-21/IL-21R axis under hypoxic conditions resulted in increasedSTAT3 phosphorylation and a subsequent increase in the BCL-2/BAX ratio.
Conclusion
Our data indicate that IL-21R up-regulation and ligand activation in hypoxic endothelial cells may help perfusion recovery by limiting/preventing apoptosis and/or favoring cell survival and angiogenesis through the STAT3 pathway.
Hyperglycaemia in Type 1 DM impairs VEGFR2 protein expression in ischaemic hind limbs, likely due to increased ubiquitination and degradation by the proteasome complex. Glycaemic control allows normal levels of VEGFR2 in ischaemia and improved perfusion recovery.
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