Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo

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.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0872-7) contains supplementary material, which is available to authorized users.

Anticoagulants Interfere With the Angiogenic and Regenerative Responses Mediated by Platelets

“…However, in combination with the chemoattractant stimulus fMLP, both inflammatory cellular responses were synergistically increased in the presence of glucose (Oldenborg and Sehlin, 1999). In addition, a previous work published by our group has demonstrated that inhibition of in vitro angiogenic responses (including endothelial proliferation, migration, survival and tubule formation) induced by TNF-α is increased in combination with glucose (Mena et al, 2018). These results suggest that glucose synergizes with other stimuli to enhance proinflammatory and anti-angiogenic responses.…”

“…Human microvascular endothelial cells (HMEC-1) were grown in MCDB-131 supplemented with fetal bovine serum (FBS) (10%), L-glutamine (10 mM) and streptomycin (100 µg/mL), penicillin (100 U/mL), EGF (10 ng/mL), and hydrocortisone (1 µg/mL) at 37 • C in a humidified 5% CO 2 incubator (Etulain et al, 2018). Human umbilical vein endothelial cells (HUVEC) were obtained from four different donors as previously described (Mena et al, 2018) and were expanded and maintained in Endothelial Growth Medium 2 (EGM2; Lonza, Walkersville, MD, United States) containing Endothelial Basal Medium 2 (EBM2) supplemented with FBS, hydrocortisone, human fibroblast growth factor (hFGF), VEGF, human long R3, insulin-like growth factor-1 (R3-IGF-1), ascorbic acid, human epidermal growth factor (hEGF), gentamicin, amphotericin-B and heparin (Mena et al, 2014). After washing, growth factor-enriched medium was totally replaced by PRPr.…”

Therapeutic Potential of Endothelial Colony-Forming Cells in Ischemic Disease: Strategies to Improve their Regenerative Efficacy

“…Fourth, ECFCs’ angiogenic activity could be further reduced by the harsh microenvironment of ischemic tissues. For instance, ECFC proliferation and tube formation are affected in the presence of elevated pro-inflammatory signaling [ 129 ], oxidative stress [ 130 ], hypoxia [ 131 , 132 ], and damage-associated molecular patterns (DAMP), such as extracellular histones [ 133 ] and monosodium urate [ 109 ].…”

“…Consistently, a human platelet lysate gel has been recently designed to provide a 2D matrix enriched with growth factors and able to promote ECFC amplification and networking formation [ 141 ]. Likewise, ex vivo proliferation is enhanced by pre-conditioning ECFCs with the pro-angiogenic cues fucoidan [ 142 , 143 , 144 ] and SDF-1α [ 145 , 146 ], with the hexapeptide SFLLRN, which mimics the thrombin effect on PAR-1 [ 53 , 147 , 148 ], with the erythroid growth factor EPO [ 78 , 106 , 149 , 150 , 151 , 152 ], with the isoflavone genistein [ 153 , 154 ], with nicotine [ 155 ], and with an acidic growth medium [ 129 , 156 ]. Hypoxic pre-conditioning has also been proposed as an alternative approach to accelerate the ex vivo expansion of primary ECFCs [ 157 , 158 ], although subsequent studies demonstrated that their angiogenic activity is compromised under hypoxia [ 131 , 132 ].…”

“…This approach has long been used to search for suitable bioactive compounds able to counteract ECFC dysfunction. For instance, ceramide 1-phosphate and acidic pre-conditioning prevented tumour necrosis factor α (TNFα) and monosodium urate crystal-induced ECFC death [ 109 , 129 ]. Of note, acidic pre-conditioning rescued ECFC proliferation, migration, and tube formation also in the presence of high inflammatory (TNFα) and high-glucose conditions [ 129 ].…”