Loss of Interleukin-21 Receptor Activation in Hypoxic Endothelial Cells Impairs Perfusion Recovery After Hindlimb Ischemia

Abstract

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.

Endothelial interleukin-21 receptor up-regulation in peripheral artery disease

“…9 These data suggest that IL-21R up-regulation and activation contributes to perfusion recovery after HLI. 9,10 …”

“…These data are consistent with our results from the distal muscle in mice with HLI and endothelial cells cultured under hypoxic conditions. 9 …”

“…14 Skeletal muscle biopsy procedures were performed as described previously. 9,15 Briefly, biopsy samples were obtained from the medial side of the gastrocnemius muscle. The biopsy site was anesthetized with a 2% lidocaine solution, and a 1.0-cm incision was made through the skin and gastrocnemius fascia.…”

“…The immunofluorescence procedures used were performed as previously described. 9 Briefly, muscle specimen cryosections were fixed with acetone and then blocked with 5% donkey serum (cat. no.…”

Deficient CDKN2B Expression

“…This adds to other data suggesting that hypoxic endothelium in hind limb muscle may well respond very differently than ECs that are not under hypoxic stress. 12 In vascular SMCs, CDKN2B knockdown has minimal effects on migration and proliferation when grown under hypoxia. When they co-inject CDKN2B deficient ECs and SMCs using a matrigel plug assay, they are able to recapitulate the phenotype observed in vivo , i.e.…”