Extracorporeal Shock Wave Therapy Salvages Critical Limb Ischemia in B6 Mice through Upregulating Cell Proliferation Signaling and Angiogenesis.

ABSTRACT

(1) This study tests hypothesis whether extracorporeal shock wave (ECSW) therapy effectively salvages mouse critical limb ischemia (CLI). In vitro result demonstrated that the angiogenesis parameters (i.e., tubular length/cluster/network formation) and protein expressions of EGFR/VEGFR2/RAS/c-Raf/MEK/ERK/VEGF/p-PI3K/p-Akt/p-m-TOR were significantly and progressively increased with stepwise augmentation of ECSW energy (0.1/0.14/0.20 mJ/mm2/140 impulses). On the other hand, they were suppressed by administration of Avastin (20 µM). Adult male B6 mice (n = 24) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 [CLI + ECSW (0.12 mJ/mm2/120 impulses/at days 1/3/7 after CLI induction)] and group 4 [CLI + ECSW (0.12 mJ/mm2/120 impulses) + Avastin (1 mg/intramuscular-injection)] at days 1/3/7 after CLI induction] and quadriceps were harvested by day 14. The laser Doppler result showed that the ratio of left (ischemia) to right (normal) limb blood flow was highest in group 1, lowest in group 2, and significantly higher in group 3 than in group 4 by days 7/14 after the CLI procedure (p < 0.0001). The protein expressions of cell proliferation/migration/angiogenesis receptors (EGFR/VEGFR2), angiogenesis biomarkers (VEGF/CXCR4/SDF-1) and cell proliferation/growth/survival (Ras/c-Raf/MEK/ERK)/(PI3K/Akt/m-TOR) and cell motility/proliferation (p-FAK/p-Scr) signaling biomarkers were significantly higher in group 3 than in groups 1/2/4, and significantly lower in group 1 than in groups 2/4, but they did not show a difference between groups 2 and 4 (all p < 0.001). The small vessel density and cellular levels of endothelial cell surface marker (CD31+) exhibited an identical pattern of blood flow, whereas the angiogenesis (CXCR4+/VEGF+) displayed an identical pattern of VEGFR2 among the groups (all p < 0.0001). The in vitro and in vivo studies found ECSW salvaged the CLI mainly through upregulating Ras-Raf-MEK/ERK/cell motility, cell proliferation/growth pathways and angiogenesis.