To date, many attempts are employed to increase the regenerative potential of stem cells. In this study, we evaluated the hypothesis of whether an autophagy modulation could alter differentiation potency of CD146+ cells into mature pericyte, endothelial, and cardiomyocyte lineage. In this study, CD146+cells were enriched from the human bone marrow aspirates and trans-differentiated into mature endothelial cells, pericytes, and cardiomyocytes after exposure to autophagy stimulator (50-μM Met)/inhibitor (15-μM HCQ). The protein levels of autophagy proteins were monitored by western blotting. NO content was measured using the Griess assay. Using real-time PCR assay and western blotting, we monitored the lineage protein and gene levels. Pro-inflammatory cytokine and angiocrine factors were measured by ELISA. The fatty acid change was determined by gas chromatography. We also measured exosome secretion capacity by measuring AChE activity and real-time PCR assay. Data revealed the modulation of autophagy factors, Beclin-1, P62, and LC3 II/I ratio in differentiating CD146+ cells after exposure to Met and HCQ (p < 0.05). The inhibition of autophagy increased NO content compared to the Met-treated cells (p < 0.05). Real-time PCR analysis showed that the treatment of CD146+ cells with autophagy modulators altered the expression of VE-cadherin, cTnI, and α-SMA (p < 0.05). Met increased the expression of VE-cadherin, α-SMA, and cTnI compared to the HCQ-treated cells (p < 0.05) while western blotting revealed the protein synthesis of all lineage-specific proteins under the stimulation and inhibition of autophagy. None statistically significant differences were found in the levels of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 after autophagy modulation. Fatty acid profile analysis revealed the increase of unsaturated fatty acids after exposure to HCQ (p < 0.05). The treatment of cells with HCQ increased the levels of TNF-α and IL-6 compared to the Met-treated cells. Data revealed the increase of exosome biogenesis and secretion to the supernatant in cells treated with HCQ compared to the Met groups (p < 0.05). In summary, autophagy modulation could alter differentiation potency of CD146+cells which is important in cardiac regeneration.

中文翻译：

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自噬调节改变了 CD146+ 细胞向内皮细胞、周细胞和心肌细胞的分化能力。

迄今为止，人们进行了许多尝试来提高干细胞的再生潜力。在这项研究中，我们评估了自噬调节是否可以改变 CD146+ 细胞分化为成熟周细胞、内皮细胞和心肌细胞谱系的假设。在这项研究中，CD146+ 细胞从人骨髓抽吸物中富集，并在暴露于自噬刺激剂 (50-μM Met)/抑制剂 (15-μM HCQ) 后转分化为成熟的内皮细胞、周细胞和心肌细胞。通过蛋白质印迹监测自噬蛋白的蛋白质水平。使用 Griess 测定法测量 NO 含量。使用实时 PCR 测定和蛋白质印迹，我们监测谱系蛋白和基因水平。通过ELISA测量促炎细胞因子和血管分泌因子。通过气相色谱法测定脂肪酸变化。我们还通过测量 AChE 活性和实时 PCR 测定来测量外泌体分泌能力。数据显示，暴露于 Met 和 HCQ 后，自噬因子、Beclin-1、P62 和 LC3 II/I 比率在分化 CD146+ 细胞中的调节作用（p < 0.05）。与 Met 处理的细胞相比，自噬的抑制增加了 NO 含量（p < 0.05）。实时 PCR 分析表明，用自噬调节剂处理 CD146+ 细胞改变了 VE-cadherin、cTnI 和 α-SMA 的表达（p < 0.05）。与 HCQ 处理的细胞相比，Met 增加了 VE-cadherin、α-SMA 和 cTnI 的表达（p < 0.05），而蛋白质印迹显示在自噬的刺激和抑制下所有谱系特异性蛋白质的蛋白质合成。自噬调节后 Tie-1、Tie-2、VEGFR-1 和 VEGFR-2 水平无统计学差异。脂肪酸谱分析显示接触 HCQ 后不饱和脂肪酸增加（p < 0.05）。与 Met 处理的细胞相比，用 HCQ 处理细胞增加了 TNF-α 和 IL-6 的水平。数据显示，与 Met 组相比，HCQ 处理的细胞中外泌体生物合成和分泌到上清液中的增加（p < 0.05）。总之，自噬调节可以改变CD146+细胞的分化能力，这对心脏再生很重要。与 Met 处理的细胞相比，用 HCQ 处理细胞增加了 TNF-α 和 IL-6 的水平。数据显示，与 Met 组相比，HCQ 处理的细胞中外泌体生物合成和分泌到上清液中的增加（p < 0.05）。总之，自噬调节可以改变CD146+细胞的分化能力，这对心脏再生很重要。与 Met 处理的细胞相比，用 HCQ 处理细胞增加了 TNF-α 和 IL-6 的水平。数据显示，与 Met 组相比，HCQ 处理的细胞中外泌体生物合成和分泌到上清液中的增加（p < 0.05）。总之，自噬调节可以改变CD146+细胞的分化能力，这对心脏再生很重要。