Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells.
Adult BMCs injected into ischemic limbs without immunosuppressant therapy differentiated into blood vessels and skeletal myofibers, and this was associated with accelerated blood flow restoration and increased serum levels of VEGF, FGF-2, TGF-beta, IL-4, and TNF-alpha. Skeletal muscle formation may provide benefits beyond angiogenesis to patients with chronic peripheral arterial disease or to patients with low cardiac output states who also suffer from skeletal muscle atrophy.
BackgroundCell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34+/M-cad+ BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34+/M-cad+ BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD.Methods and FindingsColony-forming cell assays and flow cytometry analysis showed that CD34+/M-cad+ BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE−/− mice, CD34+/M-cad+ BMCs alleviated ischemia and significantly improved blood flow compared with CD34+/M-cad− BMCs, CD34−/M-cad+ BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34+/M-cad+ cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP+ CD34+/M-cad+ cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP+ CD34+/M-cad+ cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34+/M-cad+ progenitor cells. A cytokine antibody array revealed that CD34+/M-cad+ cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34+/M-cad− cell-conditioned medium. The proangiogenic cytokines secreted by CD34+/M-cad+ cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34+/M-cad− cells during hypoxia.ConclusionCD34+/M-cad+ BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE−/− mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34+/M-cad+ BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors.
Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2 minute) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1(COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth endothelial progenitor cells (EPCs) derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P<0.05) and showed higher intrinsic angiogenic capability; conditioned medium from PGI2-EPCs promoted better tube formation than conditioned medium from native EPCs (P<0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine–sensitive K+ current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50±1.59pA/pF, P<0.05.) compared with rSMCs cocultured with native EPCs (3.99±1.26 pA/pF.). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in-vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.
SummaryProstacyclin and stable analogs have shown beneficial effects in treating patients with critical limb ischemia. By using multiple imaging techniques, we have shown here that a stable prostacyclin analog, carbaprostacyclin (cPGI2), improves local perfusion by enhancing the growth of arteriolar networks. Our results provide the first evidence that the cPGI2-peroxisome proliferator-activated receptor β/δ axis positively affects acidic fibroblast growth factor protein expression in skeletal myotubes and smooth muscle cells. Together, the data suggest local delivery of cPGI2 exerts its therapeutic effects at the level of the arteriolar circulation.
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