Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, China.
Institute of Cardiovascular Diseases, Fudan University, Shanghai, China.
Stem Cell Res Ther. 2017 Dec 13;8(1):280. doi: 10.1186/s13287-017-0732-x.
The stiffness of the myocardial extracellular matrix (ECM) and the transplanted cell type are vitally important in promoting angiogenesis. However, the combined effect of the two factors remains uncertain. The purpose of this study is to investigate in vitro the combined effect of myocardial ECM stiffness postinfarction with a bone marrow-derived cell subset expressing or not expressing CD34 on endothelial lineage commitment.
Myocardial stiffness of the infarct zone was determined in mice at 1 h, 24 h, 7 days, 14 days, and 28 days after coronary artery ligation. Polyacrylamide (PA) gel substrates of different stiffnesses were prepared to mechanically mimic the myocardial ECM after infarction. Mouse bone marrow-derived CD34 and CD34 cells were seeded on the flexible PA gels. The double-positive expression for DiI-acetylated low-density lipoprotein (acLDL) uptake and fluorescein isothiocyanate-Ulex europaeus agglutinin-1 (FITC-UEA-1) binding, the endothelial lineage antigens CD31, von Willebrand factor (vWF), Flk-1, and VE-cadherin, as well as cytoskeleton were measured by immunofluorescent staining on day 7. Cell apoptosis was evaluated by both immunofluorescent staining and flow cytometry at 24 h after culture.
We found that the numbers of the CD34 cell subset adherent to the flexible substrates (4-72 kPa) was much larger than that of the CD34 subset. More double-positive cells for DiI-acLDL uptake/FITC-UEA-1 binding were seen on the 42-kPa (moderately stiff) substrate, corresponding to the stiffness of myocardial ECM at 7-14 days postinfarction, compared with those on substrates of other stiffnesses. Similarly, the moderately stiff substrate showed benefits in promoting the positive expressions of the endothelial lineage markers CD31, vWF, Flk-1, and VE-cadherin. In addition, the cytoskeleton F-actin network within CD34 cells was organized more significantly at the leading edge of the adherent cells on the moderately stiff (42 kPa) or stiff (72 kPa) substrates as compared with those on the soft (4 kPa and 15 kPa) substrates. Moreover, the moderately stiff or stiff substrates showed a lower percentage of cell apoptosis than the soft substrates.
Infarcted myocardium-like ECM of moderate stiffness (42 kPa) more beneficially regulated the endothelial lineage commitment of a bone marrow-derived CD34 subset. Thus, the combination of a CD34 subset with a "suitable" ECM stiffness might be an optimized strategy for cell-based cardiac repair.
心肌细胞外基质（ECM）的僵硬程度和移植细胞类型对于促进血管生成至关重要。然而，这两个因素的综合影响尚不确定。本研究旨在体外探讨梗死区心肌 ECM 僵硬程度与骨髓来源细胞亚群 CD34 表达与否对内皮谱系定向的联合作用。
在冠状动脉结扎后 1 h、24 h、7 d、14 d 和 28 d，确定小鼠梗死区心肌的僵硬程度。制备不同硬度的聚丙稀酰胺（PA）凝胶底物，以机械模拟梗死心肌 ECM。将鼠骨髓来源的 CD34 和 CD34 细胞接种于柔性 PA 凝胶上。第 7 天，通过免疫荧光染色测量 DiI-乙酰化低密度脂蛋白（acLDL）摄取和荧光素异硫氰酸酯-荆豆凝集素-1（FITC-UEA-1）结合的双阳性表达、内皮谱系抗原 CD31、血管性血友病因子（vWF）、Flk-1 和 VE-钙粘蛋白以及细胞骨架。培养 24 h 后，通过免疫荧光染色和流式细胞术评估细胞凋亡。
我们发现，附着在柔性基底（4-72 kPa）上的 CD34 细胞亚群数量明显多于 CD34 亚群。与其他硬度的基底相比，在 42 kPa（中度硬）基底上观察到更多的 DiI-acLDL 摄取/FITC-UEA-1 双阳性细胞，这与梗死 7-14 天后心肌 ECM 的硬度相对应。同样，中度硬基底在促进内皮谱系标志物 CD31、vWF、Flk-1 和 VE-钙粘蛋白的阳性表达方面具有优势。此外，与软基底（4 kPa 和 15 kPa）相比，在中度硬（42 kPa）或硬（72 kPa）基底上，附着细胞的 CD34 细胞中的细胞骨架 F-肌动蛋白网络在前沿处更为明显。此外，与软基底相比，中度硬或硬基底的细胞凋亡率较低。
中度僵硬（42 kPa）的梗死心肌样 ECM 更有利于调节骨髓来源 CD34 亚群的内皮谱系定向。因此，CD34 亚群与“合适”ECM 硬度的结合可能是细胞心脏修复的优化策略。
