Center of Aging Sciences and Translational Medicine - CESI-Met and Institute of Cardiology, "G. D'Annunzio" University, Chieti-Pescara, Chieti, Italy; Department of Internal Medicine, Cardiology, The University of Texas Health Science Center at Houston, Houston, Texas, United States; Department of Neurosciences, Imaging and Clinical Sciences, "G. d'Annunzio" University, Chieti-Pescara, Chieti, Italy.
Department of Medical, Oral & Biotechnological Sciences, Dentistry and Biotechnology, and Aging Research Center and Translational Medicine, "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy; Stem TeCh Group, Via L Polacchi 13, Chieti, Italy.
J Mol Cell Cardiol. 2019 Jun;131:171-186. doi: 10.1016/j.yjmcc.2019.04.019. Epub 2019 May 2.
Understanding mechanisms of the therapeutic effects of stem/progenitor cells, among which adipose tissue-derived mesenchymal stromal cells (AT-MSCs), has important implications for clinical use. Since the majority of such cells die within days or weeks after transplantation and do not persist in the transplanted organ or tissue, their effects appear to be largely mediated by paracrine signaling pathways, and are enhanced by overexpression of the antisenescent protein telomerase reverse transcriptase (TERT), and the anti-apoptotic transcription factor myocardin (MYOCD).
By a proteomic approach combining two-dimensional gel electrophoresis (2DE) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry, we aimed at analyzing how soluble and vesicular secretomes of aged murine AT-MSCs and their angiogenic function are modulated by the overexpression of TERT and MYOCD.
We cultured murine mock-transduced AT-MSCs and "rejuvenated" AT-MSCs overexpressing TERT and MYOCD (rTMAT-MSCs) harvested from 1-year-old male C57BL/6 mice. We established proteomes from 3 mock-transduced AT-MSCs and rTMAT-MSCs cultures in serum-free conditions, as well as their corresponding conditioned medium (CM) and exosome-enriched fractions (Exo+).
Proteomic analysis revealed a 2-fold increase of matrix metalloproteinase-2 (MMP-2) and its inhibitor metalloproteinase inhibitor 2 (TIMP2) in the CM - but not in the Exo + - of rTMAT-MSCs as compared to mock-transduced AT-MSCs. At the functional level, rTMAT-MSCs-CM, and - to a lesser extent - its Exo + fraction, increased tube formation of human vein endothelial cells (HUVECs), which could be blocked by anti-MMP2 and enhanced by anti-TIMP2 antibodies, respectively. Altogether, our results identify MMP2 and its inhibitor TIMP2 as novel candidates by which rTMAT-MSCs can support angiogenesis. Our strategy also illustrates the usefulness of comparative targeted proteomic approach to decipher molecular pathways underlying rTMAT-MSCs properties.
了解干细胞/祖细胞治疗效果的机制，其中包括脂肪组织来源的间充质基质细胞（AT-MSCs），对于临床应用具有重要意义。由于大多数此类细胞在移植后数天或数周内死亡，并且不会在移植的器官或组织中持续存在，因此它们的作用似乎主要是通过旁分泌信号通路介导的，并且通过过表达抗衰老蛋白端粒酶逆转录酶（TERT）和抗细胞凋亡转录因子心肌营养素（MYOCD）得到增强。
通过结合二维凝胶电泳（2DE）和基质辅助激光解吸/电离飞行时间（MALDI-TOF/TOF）质谱的蛋白质组学方法，我们旨在分析衰老的鼠 AT-MSCs 的可溶性和囊泡分泌组以及它们的血管生成功能如何被 TERT 和 MYOCD 的过表达所调节。
我们培养了来自 1 岁雄性 C57BL/6 小鼠的 mock 转导的 AT-MSCs 和过表达 TERT 和 MYOCD 的“ rejuvenated ”AT-MSCs（rTMAT-MSCs）。我们在无血清条件下从 3 个 mock 转导的 AT-MSCs 和 rTMAT-MSCs 培养物以及它们相应的条件培养基（CM）和外泌体富集部分（Exo+）中建立了蛋白质组。
蛋白质组学分析显示，与 mock 转导的 AT-MSCs 相比，rTMAT-MSCs 的 CM 中基质金属蛋白酶 2（MMP-2）及其抑制剂金属蛋白酶抑制剂 2（TIMP2）增加了 2 倍，但在 Exo+中没有增加。在功能水平上，rTMAT-MSCs-CM，并且在较小程度上，其 Exo+部分，增加了人静脉内皮细胞（HUVECs）的管形成，这可以被抗 MMP2 抗体阻断，并被抗 TIMP2 抗体增强。总之，我们的结果确定 MMP2 和其抑制剂 TIMP2 为 rTMAT-MSCs 支持血管生成的新候选物。我们的策略还说明了比较靶向蛋白质组学方法在破译 rTMAT-MSCs 特性背后的分子途径方面的有用性。
