Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients.
Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients.
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DOI：
10.1016/j.jvs.2018.05.223
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发表时间：
2018-12
影响因子：
4.3
通讯作者：
Brewster LP
中科院分区：
文献类型：
--
作者：
Chadid T;Morris A;Surowiec A;Robinson S;Sasaki M;Galipeau J;Pollack BP;Brewster LP
再生医学致力于阻止或逆转慢性疾病的病理后果。许多糖尿病患者患有外周动脉疾病（PAD），这增加了他们本来就很高的截肢风险。细胞疗法是一种很有前途的 PAD 再生医学方法，可用于将再生细胞集中注射到濒危组织床中。已知间充质干细胞 (MSC) 通过基质支持和旁分泌刺激新血管（血管生成）来促进组织再生。虽然人们对人类糖尿病 MSC (dMSC)，特别是来自 PAD 患者的 MSC 知之甚少，但 dMSC 的扩增能力有限，但可以通过补充人血小板裂解物 (PL) 来改善。 PL 富含许多生长因子，包括表皮生长因子 (EGF)，众所周知，它对细胞增殖和生存信号通路非常重要。我们假设 dMSC 的 EGF 受体通路存在可逆缺陷。这项工作的目的是使用 PAD 患者的 dMSC 来检验这一假设。对来自 5 名接受大截肢的患者（6 条肢体）的骨髓来源 (BM) 和脂肪组织来源 (ATD) dMSC 的 EGF 和重要血管生成素的分泌组表达进行了表征。使用蛋白质印迹来表征标准培养物 (5% FBS + FGF-2)、5% 人血小板裂解物 (PL) 或 5% FBS + EGF 下 dMSC 中 AKT 和 Erk1/2 的表达。健康供体 MSC 为对照细胞。在人脐静脉内皮细胞 (EC) 上测试了 BM 和 ATD-dMSC 的血管生成活性。酌情使用配对 t 检验、方差分析和 Kruskal-Wallis 检验。 BM 和 ATD-dMSC 均具有典型的 MSC 表面标志物表达、相似的扩增曲线，并且在其分泌组中不表达 EGF。 dMSCs 的 PL 补充改善了 AKT 信号传导，但对 Erk1/2 的 FGF-2 激活具有抵抗力。补充 EGF 导致与 PL 相似的 AKT 表达，但 PL 在 30 分钟和 60 分钟时 AKT 磷酸化程度更高。来自 BM 和 ATD-dMSC 的条件培养基 (CM) 具有高水平的显着血管生成素（VEGF、MCP-1、HGF），可刺激 EC 增殖和迁移，并且 dMSC 与 EC 共培养导致 3D 凝胶中的 EC 芽比单独的 EC 颗粒显着更长。与 FGF-2 相比，补充 PL 和 EGF 可改善 dMSC 中的 AKT 表达，但与 EGF 相比，PL 可改善 pAKT。因此，PL 补充策略可能会改善 AKT 信号传导，这对于细胞治疗中 MSC 的存活很重要。此外，BM 和 ATD-dMSC 具有相似的分泌组和强大的体外血管生成活性，这支持在再生医学策略中从两个储存库中寻找 dMSC。
Regenerative medicine seeks to stall or reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral artery disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). While little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. The secretome expression of EGF and prominent angiogens were characterized from bone marrow-derived (BM) and adipose tissue-derived (ATD) dMSCs from 5 patients (6 limbs) undergoing major amputation. Western blot was used to characterize the AKT and Erk1/2 expression in dMSCs under standard culture (5% FBS + FGF-2), 5% human platelet lysate (PL), or 5% FBS + EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, ANOVA and Kruskal-Wallis tests were used as appropriate. Both BM and ATD-dMSCs had typical MSC surface marker expression, similar expansion profiles, and did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling but were resistant to FGF-2 activation of Erk1/2. EGF supplementation led to similar AKT expression as PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media (CM) from both BM and ATD-dMSCs had robust levels of prominent angiogens (VEGF, MCP-1, HGF), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in 3D gel than EC-alone pellets. PL and EGF supplementation improves AKT expression in dMSCs over that of FGF-2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Further, BM and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.
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影响因子：
23.9
作者：
Guimarães-Camboa N;Cattaneo P;Sun Y;Moore-Morris T;Gu Y;Dalton ND;Rockenstein E;Masliah E;Peterson KL;Stallcup WB;Chen J;Evans SM
通讯作者：
Evans SM
影响因子：
4.3
作者：
Krawiec JT;Liao HT;Kwan LL;D'Amore A;Weinbaum JS;Rubin JP;Wagner WR;Vorp DA
通讯作者：
Vorp DA
影响因子：
4.3
作者：
Dick, Florian;Diehm, Nicolas;Baumgartner, Iris
通讯作者：
Baumgartner, Iris
影响因子：
3.8
作者：
Morris, Andrew D.;Dalal, Sidd;Brewster, Luke P.
通讯作者：
Brewster, Luke P.
DOI：
10.1016/s0020-1383(14)70009-9
发表时间：
2014-10-01
影响因子：
2.5
作者：
Narbona-Carceles, Javier;Vaquero, Javier;Eugenia Fernandez-Santos, Maria
通讯作者：
Eugenia Fernandez-Santos, Maria