Adipose Stromal Cells Amplify Angiogenic Signaling via the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A 3D Multimodality Imaging Study

Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.06107) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc+-eGFP+, mADSCsFluc+GFP+), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (,5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and biolumines-cence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells ’ distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/ mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functiona