Transcriptomic characterization of iPSC-derived SMCs edited for LRP1 full knockout or enhancer deletion

Genome-wide association studies (GWAS) implicate common genetic variations in the low-density lipoprotein receptor-related protein 1 locus (LRP1) in risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown.To tackle this issue, human induced pluripotent stem cells (iPSCs) were genome-edited using CRISPR-Cas9 to delete or modify candidate enhancer regions, and generate LRP1 knockout cell lines (KO). Cells were differentiated into smooth muscle cells (SMCs) through a mesodermal lineage. Phenotype changes in cells were conducted using cellular assays, bulk RNA-sequencing and mass spectrometry.Genomic deletion in iPSC-derived SMCs supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 and SNAIL to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 KO decreased iPSC-derived SMCs proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix, connective tissue development and lung development. LRP1 KO and deletion of rs11172113 enhancer showed potentiated canonical TGF-beta signaling through enhanced phosphorylation of SMAD2/3. Altogether, our findings support allele specific LRP1 gene repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in SMCs to remodel the ECM enhanced by TGF-beta as a potential mechanism of this pleiotropic locus for vascular diseases.

全基因组关联研究（Genome-wide Association Studies, GWAS）揭示，低密度脂蛋白受体相关蛋白1基因座（low-density lipoprotein receptor-related protein 1, LRP1）上的常见遗传变异与多种血管疾病及性状的发病风险相关。然而，其潜在的生物学机制尚不明确。为解决这一问题，本研究利用CRISPR-Cas9技术对人类诱导多能干细胞（induced pluripotent stem cells, iPSCs）进行基因组编辑，以敲除或修饰候选增强子区域，并构建LRP1敲除细胞系（knockout, KO）。通过中胚层谱系分化，将上述细胞诱导为平滑肌细胞（smooth muscle cells, SMCs）。随后通过细胞实验、批量RNA测序（bulk RNA-sequencing）与质谱分析法对细胞表型变化进行检测。对iPSC来源的SMCs进行基因组缺失实验证实，rs11172113位于调控LRP1表达的增强子区域内。本研究发现，转录因子MECP2与SNAIL可通过等位基因特异性机制抑制LRP1表达，其中SNAIL可与疾病风险等位基因发生相互作用。LRP1敲除可降低iPSC来源的SMCs的增殖与迁移能力。差异表达基因显著富集于含胶原的细胞外基质、结缔组织发育及肺发育相关通路。LRP1敲除与rs11172113增强子缺失均可通过增强SMAD2/3的磷酸化水平，激活经典转化生长因子-β（transforming growth factor-β, TGF-β）信号通路。综上，本研究证实内皮-间质转化调控因子SNAIL可通过等位基因特异性机制抑制LRP1基因表达。我们提出，SMCs中LRP1表达下调可重塑TGF-β介导的细胞外基质（extracellular matrix, ECM），这可能是该多效性基因座导致血管疾病的潜在分子机制。