In Vitro Lineage-Specific Differentiation of Vascular Smooth Muscle Cells in Response to SMAD3 Deficiency: Implications for SMAD3-Related Thoracic Aortic Aneurysm.

响应 SMAD3 缺乏的血管平滑肌细胞的体外谱系特异性分化 ： 对 SMAD3 相关胸主动脉瘤的影响。

• 影响指数: 10.514
• 发表时间: 07 2020
• 来源期刊: Arterioscler Thromb Vasc Biol
• DOI: 10.1161/ATVBAHA.120.313033
• PMID: 32404006
• 文章类型: Journal Article
• 中科院分区: Q1
• 方向: 医学
• 作者列表: Gong J,Zhou D,Jiang L,Qiu P,Milewicz DM,Chen YE,Yang B

关键词: aortic aneurysm, thoracic; calponin; elastin; myocardin; neural crest

Mesh: Aortic Aneurysm, Thoracic / genetics metabolism pathology physiopathology; Cell Differentiation; Cell Lineage; Cells, Cultured; Elastin / genetics metabolism; Extracellular Matrix / metabolism pathology; Frameshift Mutation; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells / metabolism; MicroRNAs / genetics metabolism; Muscle, Smooth, Vascular / metabolism pathology; Myocytes, Smooth Muscle / metabolism pathology; Phosphorylation; Signal Transduction; Smad2 Protein / genetics metabolism; Smad3 Protein / deficiency genetics; Transforming Growth Factor beta / metabolism; Vascular Remodeling; Vasoconstriction

Abstract

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: SMAD3 c.652delA, a frameshift mutation and nonsense-mediated decay, was introduced in human-induced pluripotent stem cells using CRISPR-Cas9. The wild-type and SMAD3-/- (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 SMAD3-/- 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 SMAD3 mutations.