Figure 7.

TEVs Are Effective in Treating Hind-Limb Ischemia

Aged male Balb/c mice, 26 to 27 months old, were anesthetized, and the external iliac artery was ligated. The mice were divided into 4 groups, and each group had 4 mice. Each group was injected with saline (PBS), EVs derived from young MSCs, aged MSCs, and aged MSCs transduced with miR-126 overexpressing vectors. Intramuscular injection at 3 different sites (gastrocnemius, gracilis, and quadriceps muscles, respectively, 200 μg/200 μl per injection) of the ischemic leg was done daily after ischemia overnight. At post-operative days 0 and 7, mouse limbs were scanned with a laser Doppler. The median perfusion of each limb was determined, and the ratio between the ischemia and the non-operated limb (relative perfusion) was calculated. EVs from young MSCs and TEVs from miR-26–transduced aged MSCs improved blood supply in the ischemic limb (A and B). Ischemic limb tissues were collected, fixed, and stained with DAPI (blue) for nuclei and CD31 antibody (orange) for blood vessel observation. Red arrows point to the examples of CD31 staining for blood vessel. EVs from young MSCs and TEVs from miR-126–transduced aged MSCs significantly increased angiogenesis in the ischemic limb (C and D). (∗p < 0.05 compared with the PBS group). Scale bar: 100 μm. PBS = phosphate-buffered saline; other abbreviations as in Figures 1, 2, and 5.