From the Department of Cardiac Surgery, North Campus Research Complex, University of Michigan, Ann Arbor (J.G., D.Z., L.J., P.Q., Y.E.C., B.Y.).
The Second Xiangya Hospital, Central South University, Changsha, People's Republic of China (J.G., D.Z., L.J.).
Arterioscler Thromb Vasc Biol. 2020 Jul;40(7):1651-1663. doi: 10.1161/ATVBAHA.120.313033. Epub 2020 May 14.
SMAD3 pathogenic variants are associated with the development of thoracic aortic aneurysms. We sought to determine the role of SMAD3 in lineage-specific vascular smooth muscle cells (VSMCs) differentiation and function. Approach and Results: c.652delA, a frameshift mutation and nonsense-mediated decay, was introduced in human-induced pluripotent stem cells using CRISPR-Cas9. The wild-type and (c.652delA) human-induced pluripotent stem cells were differentiated into cardiovascular progenitor cells or neural crest stem cells and then to lineage-specific VSMCs. Differentiation, contractility, extracellular matrix synthesis, and TGF-β (transforming growth factor-β) signaling of the differentiated VSMCs were analyzed. The homozygous frameshift mutation resulted in SMAD3 deficiency and was confirmed in human-induced pluripotent stem cells by Sanger sequencing and immunoblot analysis. In cardiovascular progenitor cell-VSMCs, SMAD3 deletion significantly disrupted canonical TGF-β signaling and decreased gene expression of VSMC markers, including SM α-actin, myosin heavy chain 11, calponin-1, SM22α, and key controlling factors, SRF and myocardin, but increased collagen expression. The loss of SMAD3 significantly decreased VSMC contractility. In neural crest stem cells-VSMCs, SMAD3 deficiency did not significantly affect the VSMC differentiation but decreased ELN (elastin) expression and increased phosphorylated SMAD2. Expression of mir-29 was increased in VSMCs, and inhibition of mir-29 partially rescued ELN expression.
SMAD3-dependent TGF-β signaling was essential for the differentiation of cardiovascular progenitor cell-VSMCs but not for the differentiation of neural crest stem cell-VSMCs. The lineage-specific TGF-β responses in human VSMCs may potentially contribute to the development of aortic root aneurysms in patients with mutations.
SMAD3 致病变体与胸主动脉瘤的发展有关。我们试图确定 SMAD3 在血管平滑肌细胞（VSMC）分化和功能中的作用。
使用 CRISPR-Cas9 在人诱导多能干细胞中引入 c.652delA 移码突变和无义介导的衰变。野生型和（c.652delA）人诱导多能干细胞分化为心血管祖细胞或神经嵴干细胞，然后分化为特定谱系的 VSMC。分析分化的 VSMC 的分化、收缩性、细胞外基质合成和 TGF-β（转化生长因子-β）信号传导。纯合移码突变导致 SMAD3 缺乏，并通过 Sanger 测序和免疫印迹分析在人诱导多能干细胞中得到证实。在心血管祖细胞-VSMC 中，SMAD3 缺失显著破坏了经典的 TGF-β 信号通路，并降低了 VSMC 标志物的基因表达，包括 SM α-肌动蛋白、肌球蛋白重链 11、钙调蛋白 1、SM22α 和关键调控因子 SRF 和心肌调节蛋白，但增加了胶原蛋白的表达。SMAD3 的缺失显著降低了 VSMC 的收缩性。在神经嵴干细胞-VSMC 中，SMAD3 缺乏对 VSMC 分化没有显著影响，但降低了 ELN（弹性蛋白）的表达，增加了磷酸化 SMAD2 的表达。在 VSMC 中 mir-29 的表达增加，mir-29 的抑制部分挽救了 ELN 的表达。
SMAD3 依赖性 TGF-β 信号对心血管祖细胞-VSMC 的分化是必需的，但对神经嵴干细胞-VSMC 的分化不是必需的。人类 VSMC 中的谱系特异性 TGF-β 反应可能有助于突变患者主动脉根部动脉瘤的发展。
