BACKGROUND Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1 gene) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown. METHODS Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry. RESULTS Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and spontaneous coronary artery dissection. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL (Zinc Finger Protein SNAI1) to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix and connective tissue development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3 (Mothers against decapentaplegic homolog 2/3). Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61 (cystein rich angiogenic protein 61), a known LRP1 ligand involved in vascular integrity and TIMP3 (Metalloproteinase inhibitor 3), implicated in extracellular matrix maintenance and also known to interact with LRP1. CONCLUSIONS Our findings support allele-specific LRP1 expression repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

中文翻译：

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SNAIL 抑制 LRP1 导致血管疾病遗传风险中的 ECM 重塑。


背景 全基因组关联研究表明 LRP1 （低密度脂蛋白受体相关蛋白 1 基因） 基因座的常见遗传变异具有多种血管疾病和性状的风险。然而，潜在的生物学机制尚不清楚。方法 精细映射分析包括贝叶斯共定位，以确定最可能的因果变异。使用 CRISPR-Cas9 （成簇规则间隔短回文重复序列-CRISPR 相关蛋白 9） 对人诱导多能干细胞进行基因组编辑，以删除或修改候选增强子区域并生成 LRP1 敲除细胞系。细胞通过中胚层谱系分化为平滑肌细胞。使用荧光素酶报告基因测定、转录因子敲低和染色质免疫沉淀评估转录调控。使用细胞测定、大量 RNA 测序和质谱法进行细胞表型变化。结果 多性状共定位分析指出 rs11172113 是 LRP1 中纤维肌发育不良、偏头痛、脉压和自发性冠状动脉夹层最可能的致病变异。我们发现 rs11172113-T 等位基因与较高的 LRP1 表达相关。诱导多能干细胞来源的平滑肌细胞中的基因组缺失支持 rs11172113 位于调节 LRP1 表达的增强子区域。我们发现转录因子 MECP2 （甲基 CpG 结合蛋白 2） 和 SNAIL （锌指蛋白 SNAI1） 通过等位基因特异性机制抑制 LRP1 表达，涉及 SNAIL 与疾病风险等位基因的相互作用。LRP1 敲除减少了诱导多能干细胞衍生的平滑肌细胞增殖和迁移。 差异表达基因富集用于含胶原的细胞外基质和结缔组织发育。rs11172113 增强子的 LRP1 敲除和缺失显示通过增强 SMAD2/3 的磷酸化（母亲对抗 decapentaplegic 同源物 2/3）增强经典 TGF-β （转化生长因子 β） 信号传导。对脱细胞提取物蛋白质含量的分析表明，部分细胞外基质重塑涉及 CYR61（富含半胱氨酸的血管生成蛋白 61）的分泌增强，CYR61 （富含半胱氨酸的血管生成蛋白 61） 是一种参与血管完整性的已知 LRP1 配体和 TIMP3 （金属蛋白酶抑制剂 3），与细胞外基质维持有关，也已知与 LRP1 相互作用。结论 我们的研究结果支持内皮-间充质转化调节因子 SNAIL 抑制等位基因特异性 LRP1 表达。我们提出平滑肌细胞中 LRP1 表达降低以重塑 TGF-β 增强的细胞外基质，这是血管疾病多效性位点的潜在机制。