Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

Abstract

BackgroundMicroparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPsShh+) that are able to regulate in vitro angiogenesis.Methodology/Principal FindingsHere, we investigated the ability of MPsShh+ to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPsShh+, MPsShh+ plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPsShh+-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPsShh+-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPsShh+ increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPsShh+ enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPsShh+ on eNOS pathway. Quantitative RT-PCR revealed that MPsShh+ treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles.Conclusions/SignificanceThese findings suggest that MPsShh+ may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis.

Microparticles in Angiogenesis

“…Thus, we have engineered MPs from activated/apoptotic T-cell lines bearing Sonic hedgehog, 54 which evoke nitric oxide production on endothelial cells, restore endothelium-dependent relaxation after ischemiareperfusion injury, and favor in vitro angiogenesis and new vessel formation in an ischemic hind limb model. 48,55,75 These findings suggest that MPs Shhϩ may represent a potent tool in stimulating neovascularization in disease states associated with impaired angiogenesis. In contrast, MPs from apoptotic T-cell line that do not express Sonic hedgehog 48 inhibit angiogenesis, suggesting a potential therapy to reduce tumorigenesis via inhibition of tumor vascularization and possibly tumor development.…”

“…More interestingly, in vivo treatment of hind limb ischemic mice with MPs Shhϩ enhanced the neovascularization, as reflected by the improvement of both blood flow and number of capillaries in the ischemic leg when compared with the nonischemic leg. 55 The effects evoked by MPs Shhϩ were attributable to an interaction ligand (Sonic hedgehog)/receptor (patched/smoothened receptors) and were associated with an increase in nitric oxide production as a consequence of the activation of endothelial nitric oxide synthase and endogenous Sonic hedgehog pathway in ischemic muscles. Moreover, the expression of a several proangiogenic factors was increased by MPs Shhϩ treatment, including fibroblast growth factor-5, fibroblast growth factor-2, and VEGF.…”

Human Umbilical Cord Mesenchymal Stem Cell Exosomes Enhance Angiogenesis Through the Wnt4/β-Catenin Pathway

“…To determine the effects of hucMSC-Ex on endothelial cell growth, we evaluated the viability of EA.hy926 cells treated with various concentrations of exosomes (80 and 160 mg/ml) for different times (24,48,72, and 96 hours). The results of the MTT assay showed that hucMSC-Ex promoted EA.hy926 cell growth in a dose-dependent manner ( Fig.…”

Microparticles, Vascular Function, and Atherothrombosis

“…152 These observations prompted the investigation of the role of MPs in postnatal neovascularization. Such neovascularization is augmented by MPs originating from platelets [153][154][155] or endothelial progenitor cells, 156 as well as by MPs isolated from mouse plasma 157 or ischemic hind-limb muscle. 151 However, conflicting results were obtained using MPs from lymphocytes.…”

“…152 The mechanism implicated depends on the origin of MPs: a binding of MP to progenitor cells that changes their phenotype for platelet MPs; 155 reactive oxygen species for MPs isolated from ischemic muscles; 152 or peroxisome proliferator-activated receptor-␣ for plasma MPs. 157 It should be highlighted that these positive effects of MPs on postischemic neovascularization have been documented not only in vitro but also in vivo in unilateral femoral artery ligation 152,158 and arterial wire-induced injury murine models. 155 …”