Regulatory mechanisms in multiple vascular diseases locus LRP1 involve repression by SNAIL and extracellular matrix remodeling

Abstract

BackgroundGenome-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. MethodsFine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells (iPSC) 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. 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. ResultsMulti-trait co-localization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure and pulmonary function trait. We found rs11172113-T allele to associate with higher LRP1 expression. 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 and lung development. LRP1 KO showed potentiated canonical TGF-{beta} signaling through enhanced phosphorylation of SMAD2/3. Analyses of protein content of decellularized extracts indicated partial extracellular matrix (ECM) remodeling involving enhanced secretion of CYR61, a known LRP1 ligand involved in vascular integrity and TIMP3, implicated in extracellular matrix maintenance and also known to interact with LRP1. ConclusionsOur 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/539992v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@11926a8org.highwire.dtl.DTLVardef@390a37org.highwire.dtl.DTLVardef@cdad4dorg.highwire.dtl.DTLVardef@12cbf92_HPS_FORMAT_FIGEXP M_FIG C_FIG