Université Paris Cité, Inserm, PARCC, Paris, France.
Circ Res. 2024 Nov 8;135(11):1084-1097. doi: 10.1161/CIRCRESAHA.124.325269. Epub 2024 Oct 2.
Genome-wide association studies implicate common genetic variations in the (low-density lipoprotein receptor-related protein 1 gene) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown.
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 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.
Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in for fibromuscular dysplasia, migraine, pulse pressure, and spontaneous coronary artery dissection. We found the rs11172113-T allele to associate with higher expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL (Zinc Finger Protein SNAI1) to repress expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. 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. 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.
Our findings support allele-specific expression repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased 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.
全基因组关联研究表明，（低密度脂蛋白受体相关蛋白 1 基因）基因座中的常见遗传变异与多种血管疾病和特征有关。然而，潜在的生物学机制尚不清楚。
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（成簇规律间隔短回文重复序列-CRISPR 相关蛋白 9）对人类诱导多能干细胞进行基因组编辑，以删除或修饰候选增强子区域并生成 基因敲除细胞系。通过中胚层谱系将细胞分化为平滑肌细胞。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、批量 RNA 测序和质谱法进行细胞表型变化。
多性状共定位分析表明，rs11172113 是纤维肌性发育不良、偏头痛、脉搏压和自发性冠状动脉夹层的 基因中最可能的因果变异。我们发现 rs11172113-T 等位基因与 表达升高相关。诱导多能干细胞衍生的平滑肌细胞中的基因组缺失支持 rs11172113 位于调节 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）通过涉及疾病风险等位基因的 SNAIL 相互作用的等位基因特异性机制抑制 表达。基因敲除减少了诱导多能干细胞衍生的平滑肌细胞的增殖和迁移。差异表达的基因富含含胶原的细胞外基质和结缔组织发育。基因敲除和 rs11172113 增强子缺失显示通过增强 SMAD2/3（Mothers against decapentaplegic homolog 2/3）的磷酸化增强了经典 TGF-β（转化生长因子-β）信号传导。脱细胞提取物的蛋白质含量分析表明，涉及血管完整性的已知 LRP1 配体 CYR61（富含半胱氨酸的血管生成蛋白 61）和细胞外基质维持所涉及的 TIMP3（金属蛋白酶抑制剂 3）的部分细胞外基质重塑，也已知与 LRP1 相互作用。
我们的研究结果支持内皮到间充质转化调节剂 SNAIL 的等位基因特异性 表达抑制。我们提出平滑肌细胞中 表达的减少可能通过 TGF-β重塑细胞外基质，这是该血管疾病多效性基因座的潜在机制。
