Abstract
BackgroundWe have previously demonstrated that acidic preconditioning of human endothelial colony-forming cells (ECFC) increased proliferation, migration, and tubulogenesis in vitro, and increased their regenerative potential in a murine model of hind limb ischemia without baseline disease. We now analyze whether this strategy is also effective under adverse conditions for vasculogenesis, such as the presence of ischemia-related toxic molecules or diabetes, one of the main target diseases for cell therapy due to their well-known healing impairments.MethodsCord blood-derived CD34+ cells were seeded in endothelial growth culture medium (EGM2) and ECFC colonies were obtained after 14–21 days. ECFC were exposed at pH 6.6 (preconditioned) or pH 7.4 (nonpreconditioned) for 6 h, and then pH was restored at 7.4. A model of type 2 diabetes induced by a high-fat and high-sucrose diet was developed in nude mice and hind limb ischemia was induced in these animals by femoral artery ligation. A P value < 0.05 was considered statistically significant (by one-way analysis of variance).ResultsWe found that acidic preconditioning increased ECFC adhesion and the release of pro-angiogenic molecules, and protected ECFC from the cytotoxic effects of monosodium urate crystals, histones, and tumor necrosis factor (TNF)α, which induced necrosis, pyroptosis, and apoptosis, respectively. Noncytotoxic concentrations of high glucose, TNFα, or their combination reduced ECFC proliferation, stromal cell-derived factor (SDF)1-driven migration, and tubule formation on a basement membrane matrix, whereas almost no inhibition was observed in preconditioned ECFC. In type 2 diabetic mice, intravenous administration of preconditioned ECFC significantly induced blood flow recovery at the ischemic limb as measured by Doppler, compared with the phosphate-buffered saline (PBS) and nonpreconditioned ECFC groups. Moreover, the histologic analysis of gastrocnemius muscles showed an increased vascular density and reduced signs of inflammation in the animals receiving preconditioned ECFC.ConclusionsAcidic preconditioning improved ECFC survival and angiogenic activity in the presence of proinflammatory and damage signals present in the ischemic milieu, even under high glucose conditions, and increased their therapeutic potential for postischemia tissue regeneration in a murine model of type 2 diabetes. Collectively, our data suggest that acidic preconditioning of ECFC is a simple and inexpensive strategy to improve the effectiveness of cell transplantation in diabetes, where tissue repair is highly compromised.
Highlights
We have previously demonstrated that acidic preconditioning of human endothelial colony-forming cells (ECFC) increased proliferation, migration, and tubulogenesis in vitro, and increased their regenerative potential in a murine model of hind limb ischemia without baseline disease
While higher levels of TGFβ1, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), IL-8, IL-4, vascular endothelial growth factor (VEGF), and IL-10 were detected in supernatants from preconditioned ECFC, epidermal growth factor (EGF) showed no significant differences between treatments (Fig. 1e)
TNFα-induced apoptosis of ECFC was significantly increased under high glucose conditions and these effects were observed in both untreated and ECFC angiogenic activity is enhanced by acidic preconditioning under proinflammatory and high glucose conditions in vitro We analyzed whether acid preconditioning, in addition to its cytoprotective effect, improved the angiogenic responses of ECFC exposed to inflammatory but nontoxic concentrations of TNFα in the presence or Regarding ECFC proliferation, TNFα alone induced a significant antiproliferative effect in nonpreconditioned cells, which was more pronounced when combined with high glucose, whereas almost no inhibition was observed in preconditioned ECFC (Fig. 4a)
Summary
We have previously demonstrated that acidic preconditioning of human endothelial colony-forming cells (ECFC) increased proliferation, migration, and tubulogenesis in vitro, and increased their regenerative potential in a murine model of hind limb ischemia without baseline disease. Effective endothelial repair and development of new vessels requires the contribution of both angiogenesis, mediated by mature endothelial cells in nearby tissues, and vasculogenesis, supported by bone marrow-derived cells that have the potential to differentiate into mature endothelial cells and are collectively referred to as endothelial progenitor cells [1]. Different cell populations have been reported to play roles in vasculogenesis, but only one population commonly known as endothelial colony-forming cells (ECFC; known as late outgrowth endothelial progenitor cells) represents an endothelial cell type with potent intrinsic angiogenic capacity and the capability of contributing to vascular repair and de novo blood vessel formation [2, 3]. ECFC proliferate and differentiate into mature endothelial cells and are organized into threedimensional structures that, together with support cells, form the new functional vessel
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