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.