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

Abstract

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 (1.0×107) 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 bioluminescence 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 functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.

Adipose stromal cell and sarpogrelate orchestrate the recovery of inflammation‐induced angiogenesis in aged hindlimb ischemic mice

“…1C). mADSCs GFP+ possessed multipotency and other characteristics of mADSCs as previously described (Fan et al, 2012). Laser Doppler perfusion imaging (LDPI) spatiotemporally visualized the change in peripheral blood perfusion (Fig.…”

“…Reporter gene imaging was performed to monitor Vegfr2-luc transcription level and mADSCs survival in vivo (Cao et al, 2006;Fan et al, 2012). For in vitro BLI, mADSCs…”

“…Murine ADSCs isolated from young donors with constitutive expression of enhanced green fluorescent protein (eGFP, mADSCs GFP+ ) or firefly luciferase (Fluc, mADSCs Fluc+ ) reporter were used for hindlimb ischemia therapy. Multimodality imaging strategies were employed to visualize the survival and therapeutic efficacy of engrafted mADSCs as previously described (Cao et al, 2006;Fan et al, 2012). Sarpogrelate (Sarp), a 5-hydroxytryptamine(2A) ] receptor antagonist with proven anti-inflammatory and pro-survival properties (Rajesh et al, 2006), was also used to improve functional survival of mADSCs in vivo.…”

Multipotency and secretome: the mechanisms behind the regenerative potential of adipose-derived stem cells

“…Similarly, these cells show low survival rates when implanted, which once again supports that the regenerative effects could be due to their paracrine abilities. [26] There are also several studies which investigated the effects of ASCs administration on renovascular diseases. Administration of ASCs in pigs with renal artery stenosis restored renal hemodynamics and function.…”

Mechanistic molecular imaging of cardiac cell therapy for ischemic heart disease

“…Under hypoxic stress in ischemic myocardium, the vascular endothelial growth factor (VEGF), basic fibroblast growth factor, hepatocyte growth factor, hypoxia inducible factor-1, and stromal cell-derived factor-1 are significantly increased in injured hearts treated with MSCs or multipotent stromal cells (30). With multimodality BLI/BLT/microCT imaging, we recently demonstrated multipotent adipose stromal cells therapy promoted recovery from hindlimb ischemia through enhancing proangiogenic signal in vivo, though the cells failed to incorporate into the host microvasculature as functional components (24). Using in vivo BLI, a recent study reported that porcine PSC-derived endothelial cells could also improve cardiac function after MI via release of proangiogenic and antiapoptotic factors in the ischemic microenvironment (31) (Fig.…”