Extracorporeal shock wave-assisted adipose-derived fresh stromal vascular fraction restores the blood flow of critical limb ischemia in rat

Abstract

We tested the hypothesis that extracorporeal-shock-wave (ECSW)-assisted adipose-derived stromal vascular fraction (SVF) therapy was better than either one for restoring the blood flow in critical limb ischemia (CLI). Adult male-SD rats were categorized into group 1 (sham-operated-control), group 2 (CLI), group 3 [CLI + ECSW (280 impulses/0.10 mJ/mm2) applied to left inguinal area at 3 h after CLI], group 4 [CLI + SVF (1.2 × 106) implanted into CLI area at 3 h after CLI], group 5 (CLI + ECSW-SVF). In vitro studies showed that ECSW significantly enhanced angiogenesis in human umbilical-vein endothelial cells and carotid-artery ring, and SVF significantly suppressed inflammation (TNF-α/NF-Κb/IL-1ß/MMP-9) in smooth-muscle cells treated by LPS (all p < .001). By day 14 after CLI, the ratio of ischemic/normal blood flow (INBF) was highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but no difference was shown between the latter two groups (all p < .001). The fibrotic area in CLI region exhibited an opposite pattern of INBF ratio (all p < .0001). Protein (CD31/vWF/eNOS) and cellular (CD31/vWF) expressions and number of small vessels in CLI area exhibited an identical pattern, whilst protein expressions of apoptotic (caspase3/PARP/mitochondrial-Bax) fibrotic/DNA-damaged (Samd3/TFG-ß/γ-H2AX) biomarkers exhibited an opposite pattern to INBF among five groups (all p < .0001). The numbers of angiogenetic cells in CLI region (SDF-1α/VEGF/CXCR4) and endothelial-progenitor cells (C-kit/CD31+//Sca-1/CD31+//CD34/KDR+/VE-cadherin/CD34+) in circulation significantly and progressively increased from groups 2 to 5 (all p < .0001). In conclusion, ECSW-SVF therapy effectively enhanced angiogenesis and restoration of blood flow in CLI area.