Fig. 5.

ADAM12 upregulation in ischemic hindlimb muscles is primarily in CD31-expressing cells, and this upregulation is more robust in CD31⁺ cells from C57Bl/6 mice compared with CD31⁺ cells from Balb/c. A: CD31-enriched fraction of cells from ischemic (Isch) hindlimbs showed upregulation of ADAM12 mRNA expression compared with CD31-enriched fraction of cells from nonischemic (Non-Isch) hindlimbs (n = 6/group, *P = 0.02). EC, endothelial cell. B: CD31-depleted cells from ischemic hindlimbs show no upregulation of ADAM12 mRNA expression compared with CD31-depleted cells from nonischemic hindlimbs (n = 6/group, NS = P = 0.7). C and D: representative flow cytometry histograph showing less upregulation of ADAM12 on CD31⁺ cells isolated from Balb/c mice (C) compared with CD31⁺ cells from C57Bl/6 mice (D; x-axis shows ADAM12 expression as shown in mean fluorescence). Analysis was gated to exclude dead cells and cell debris and to include only single-cell populations with CD31 expression. E: graph showing ADAM12 expression on CD31⁺ cells from ischemic and nonischemic hindlimbs from C57Bl/6 and Balb/c mice (n = 4/group, **P < 0.01). F: analysis of ADAM12 mRNA expression in the collateral vessels dissected from C57Bl/6 mice, 3 days post-HLI, shows no upregulation of ADAM12 in vessels from post-HLI limbs compared with vessels from the control limbs (n = 6/group, NS = P > 0.5). G: human umbilical vein ECs (HUVECs) exposed to simulated ischemia (Sim-Isch; hypoxia and nutrient deprivation) for 48 h show robust upregulation of ADAM12 mRNA expression compared with control cells in normoxia and normal growth media (n = 6/group, **P < 0.01). H: simulated ischemia induced increased hypoxia-inducible factor 1-α (HIF-1α) mRNA expression, and this is knocked down by small interfering (si)RNA targeting HIF-1α (n = 4/group, **P < 0.01). I: the knocking down of HIF-1α impairs upregulation of ADAM12 mRNA (n = 4/group, *P < 0.05, **P < 0.01).