Center for Cardiovascular Research and James A. Haley VA Medical Center, Tampa, FL, USA.
Cell Transplant. 2013;22(9):1637-50. doi: 10.3727/096368912X661427. Epub 2013 Jan 16.
We have previously demonstrated in acute myocardial infarctions that human umbilical cord blood mononuclear cells (HUCBCs), which contain hematopoietic, endothelial, and mesenchymal stem cells, reduce acute myocardial infarction size by ≥50% and preserve LV contractility. We hypothesize that the beneficial effects of HUCBCs are due to secretion of biologically active factors that activate in cardiac endothelial cells and myocytes the cell survival protein Akt. We determined by protein microarrays the growth factors and anti-inflammatory cytokines secreted by HUCBCs into culture media during 12 h of hypoxia (1% O2). We then determined by Western blots the effects of cell-free media from hypoxic-conditioned HUCBCs (HUCM) on activation of the cell survival protein Akt in human coronary artery endothelial cells and cardiac myocytes in culture during 24 h of 1% O2. We also determined in separate experiments endothelial cell and myocyte apoptosis by caspase-3 and Annexin V. In the present experiments, HUCBCs secreted multiple growth factors, anti-inflammatory cytokines, and inhibitors of metalloproteinase during normoxia and hypoxia. Human cord blood cells increased the concentration in culture media of angiopoietin, hepatocyte growth factor, interleukin-4, insulin-like growth factor, placental growth factor, vascular endothelial cell growth factor, angiogenin, and stem cell factor by 100 to >10,000% during 12 h of 1% O2 (p<0.001). HUCM, which contained these biological factors, significantly increased Akt phosphorylation/activation in coronary artery endothelial cells and cardiac myocytes subjected to 24 h of 1% O2 by more than 60% (p<0.05) and increased the antiapoptotic protein Bcl-2 expression by 34-50% in comparison with endothelial cells and myocytes treated without HUCM in 1% O2(p<0.05). HUCM also significantly decreased caspase-3 activity and decreased hypoxic endothelial cell and cardiac myocyte apoptosis by more than 40% in comparison with cells cultured without HUCM (p<0.05). Inhibition of Akt activation in endothelial cells and myocytes by the sensitive and specific antagonist API-1 during 24 h of hypoxia nearly completely prevented the beneficial effects of HUCM on inhibiting caspase-3 activity and apoptosis. We conclude that HUCBCs secrete biologically active factors during hypoxia that activate survival proteins in endothelial cells and myocytes that significantly limit apoptosis.
我们之前已经在急性心肌梗死中证明，人脐血单个核细胞（HUCBC）含有造血细胞、内皮细胞和间充质干细胞，可使急性心肌梗死面积减少≥50%，并保持左心室收缩功能。我们假设 HUCBC 的有益作用是由于分泌生物活性因子，这些因子激活心脏内皮细胞和心肌细胞中的细胞存活蛋白 Akt。我们通过蛋白质微阵列确定了 HUCBC 在 1%O2 缺氧条件下 12 小时分泌到培养基中的生长因子和抗炎细胞因子。然后，我们通过 Western blot 确定了缺氧条件下 HUCBC 产生的无细胞培养基（HUCM）对培养的人冠状动脉内皮细胞和心肌细胞中细胞存活蛋白 Akt 的激活作用，在 1%O2 条件下培养 24 小时。在单独的实验中，我们通过半胱氨酸天冬氨酸蛋白酶-3（caspase-3）和膜联蛋白 V 检测内皮细胞和心肌细胞的凋亡。在本实验中，HUCBC 在常氧和缺氧条件下分泌多种生长因子、抗炎细胞因子和金属蛋白酶抑制剂。人脐带血细胞在 1%O2 条件下培养 12 小时，使血管生成素、肝细胞生长因子、白细胞介素-4、胰岛素样生长因子、胎盘生长因子、血管内皮细胞生长因子、血管生成素和干细胞因子的培养基浓度增加 100 至>10000%（p<0.001）。HUCM 含有这些生物因子，可使在 1%O2 条件下培养 24 小时的冠状动脉内皮细胞和心肌细胞中 Akt 的磷酸化/激活增加超过 60%（p<0.05），并使抗凋亡蛋白 Bcl-2 的表达增加 34-50%，与未经 HUCM 处理的内皮细胞和心肌细胞相比（p<0.05）。HUCM 还可使 caspase-3 活性降低 40%以上，并使缺氧诱导的内皮细胞和心肌细胞凋亡减少 40%以上，与未经 HUCM 培养的细胞相比（p<0.05）。在缺氧 24 小时期间，用敏感和特异的 Akt 拮抗剂 API-1 抑制内皮细胞和心肌细胞中的 Akt 激活，几乎完全阻止了 HUCM 对抑制 caspase-3 活性和凋亡的有益作用。我们的结论是，HUCBC 在缺氧条件下分泌生物活性因子，激活内皮细胞和心肌细胞中的存活蛋白，显著抑制细胞凋亡。
