Experimental Thrombosis Laboratory, Institute of Experimental Medicine (IMEX), National Academy of Medicine-CONICET, Pacheco de Melo, 3081, Buenos Aires, Argentina.
Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Stem Cell Res Ther. 2018 May 2;9(1):120. doi: 10.1186/s13287-018-0872-7.
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. 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.
Cord 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).
We 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.
Acidic 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.
我们之前的研究表明，对人内皮祖细胞（ECFC）进行酸性预处理可增加其体外增殖、迁移和管状形成能力，并在没有基线疾病的小鼠后肢缺血模型中增加其再生潜能。我们现在分析这种策略在血管生成的不利条件下是否也有效，例如存在与缺血相关的毒性分子或糖尿病，由于其众所周知的愈合损伤，糖尿病是细胞治疗的主要目标疾病之一。
从脐血中分离 CD34 细胞，接种于内皮生长培养基（EGM2）中，14-21 天后获得 ECFC 集落。将 ECFC 在 pH 6.6（预处理）或 pH 7.4（未预处理）下孵育 6 小时，然后将 pH 恢复至 7.4。通过高脂肪和高蔗糖饮食诱导 2 型糖尿病裸鼠模型，并通过股动脉结扎诱导这些动物后肢缺血。通过单向方差分析，认为 P 值 <0.05 具有统计学意义。
我们发现酸性预处理可增加 ECFC 黏附及促血管生成分子的释放，并保护 ECFC 免受尿酸单钠晶体、组蛋白和肿瘤坏死因子（TNF）α 的细胞毒性作用，尿酸单钠晶体、组蛋白和肿瘤坏死因子（TNF）α 分别诱导细胞坏死、焦亡和细胞凋亡。非细胞毒性浓度的高葡萄糖、TNFα 或其组合可降低 ECFC 增殖、基质细胞衍生因子（SDF）1 驱动的迁移以及基底膜基质上的管状形成，而预处理的 ECFC 几乎没有抑制作用。在 2 型糖尿病小鼠中，与磷酸盐缓冲盐水（PBS）和未预处理的 ECFC 组相比，静脉注射预处理的 ECFC 可显著通过多普勒测量诱导缺血肢体的血流恢复。此外，接受预处理的 ECFC 的动物的比目鱼肌组织学分析显示血管密度增加，炎症迹象减少。
酸性预处理可改善 ECFC 的生存和在缺血环境中存在的促炎和损伤信号下的血管生成活性，即使在高葡萄糖条件下也是如此，并增加了它们在 2 型糖尿病小鼠后肢缺血模型中组织再生的治疗潜力。总的来说，我们的数据表明，ECFC 的酸性预处理是一种简单且经济的策略，可提高糖尿病中细胞移植的效果，因为组织修复受到高度损害。
