Fig. 6.
TUDCA-treated CKD-hMSCs enhanced neovascularization in the mouse model of CKD-associated hindlimb ischemia through upregulation of mitochondrial PrPC. (A) The scheme of the mouse model of CKD-associated hindlimb ischemia. CKD-associated hindlimb ischemia mouse model was fed with 0.25% adenine for 1 week, and then we administered hindlimb ischemia in each mouse group surgically. Each CKD-associated hindlimb ischemia mouse model was injected with PBS or Healthy-hMSCs, CKD-hMSCs, TUDCA-treated CKD-hMSCs (TUDCA+CKD-hMSC), TUDCA-treated CKD-hMSCs pretreated with si-PRNP (si-PRNP+CKD-hMSC), or TUDCA-treated CKD-hMSCs pretreated with si-Scr (si-Scr+CKD-hMSC) at 0, 5, and 10 days via a tail vein (n = 10 mice/group). To measure tissue apoptosis and neovascularization at postoperative day 3, we euthanized some mice in each group. (B) At postoperative day 3, western blot analysis quantified the cell apoptosis associated protein in each their hindlimb ischemia tissue. The expression levels were determined by densitometry relative of α-tubulin. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. PBS, #p < 0.05 and ##p < 0.01 vs. Healthy-hMSC, $p < 0.05, and $$p < 0.01 vs. CKD-hMSC, && p < 0.01 vs. si-PRNP+CKD-hMSC. (One-way ANOVA, using Tukey's post-hoc test). (C) Expression of hVEGF, hHGF, and hFGF in each their hindlimb ischemia tissue lysis group was analyzed by ELISA. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. PBS, #p < 0.05 and ##p < 0.01 vs. Healthy-hMSC, $p < 0.05, and $$p < 0.01 vs. CKD-hMSC, && p < 0.01 vs. si-PRNP+CKD-hMSC. (One-way ANOVA, using Tukey's post-hoc test). (D) The CKD associated hindlimb ischemia mouse model improved in blood perfusion by laser Doppler perfusion imaging analysis of the ischemic-injured tissues of PBS, Healthy-hMSC, CKD-hMSC, TUDCA+CKD-hMSC, si-PRNP+CKD-hMSC, and si-Scr+CKD-hMSC at 0 day, and 25 days postoperation. (E) The ratio of blood perfusion (blood flow in the left ischemic limb/blood flow in the right non-ischemic limb) was measured in each mouse of the five groups. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. PBS, #p < 0.05 and ##p < 0.01 vs. Healthy-hMSC, $p < 0.05, and $$p < 0.01 vs. CKD-hMSC, && p < 0.01 vs. si-PRNP+CKD-hMSC. (One-way ANOVA, using Tukey's post-hoc test). (F) Representative image illustrating the different outcomes (foot necrosis, toe loss, and limb salvage) of CKD-associated ischemic limbs injected with five treatments at postoperative 25 days. (G) Distribution of the outcomes in each group at postoperative day 25. (H, I) At postoperative 25 days, capillary density (αSMA; Red), and arteriole density (CD31; Red) were analyzed by immunofluorescence staining, respectively. Scale bar = 50 μm, 100 μm. Capillary density was quantified as the number of human αSMA or CD31 positive cells. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. PBS, #p < 0.05 and ##p < 0.01 vs. Healthy-hMSC, $p < 0.05, and $$p < 0.01 vs. CKD-hMSC, && p < 0.01 vs. si-PRNP+CKD-hMSC. (One-way ANOVA, using Tukey's post-hoc test).