Epigenetic regulation at LRP1 risk locus for cardiovascular diseases and assessment of cellular function in hiPSC derived smooth muscle cells
Authors: Liu L , Jouve C , Sebastien Hulot J , Georges A , Bouatia-Naji N 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC)

Background/Introduction
Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of myocardial infarction in young and middle-aged women. A common genetic variant, rs11172113, located in LRP1 (low density lipoprotein receptor-related protein 1) first intron, was associated with several vascular diseases, including SCAD, coronary artery disease and migraine. However, the biological mechanisms through which rs11172113 influence LRP1 function in the context of non-atherosclerotic arterial lesions is not fully understood.

Purpose
We aim at defining the specific mechanisms of rs11172113 genotype affecting LRP1 expression in contractile smooth muscle cells (SMCs), leading to alterations of their physiological function.

Methods
We applied in silico functional annotation to select variants and measured their enhancer activity using luciferase reporter assay in rat primary SMCs (A7r5). We performed siRNA knockdown of LRP1 and 4 transcription factors (TFs) predicted to interact with rs11172113 in human induced pluripotent stem cells (iPSCs) derived SMCs, harboring either synthetic or contractile phenotype. We edited iPSCs prior to differentiation using CRISPR-Cas9 to generate 100 bp deletion of the enhancer region containing rs11172113. We also created frame-shift indels in exons 2 or 5 of LRP1 in iPSCs to create SMCs knockouts and differentiated into SMCs. We then performed a proteomic and transcriptomic characterization of LRP1 knockout effect in these iPSC derived SMCs. We used a computational method of cell tracking and relative cell surface area change to characterize the contractility of LRP1 knockout effect in these iPSC derived SMCs. We performed the assessment of fluo-4 labeled Ca2+ mobilization to characterize the ability of calcium release in iPSC derived SMCs after LPR1 knockout.

Results
Seven variants in LRP1 locus co-located with enhancer (histone marks) and open chromatin regions (ATAC-Seq peaks) in SMCs and arterial tissues. Reporter assay in rat SMCs confirmed that rs11172113 belongs to a genomic region showing enhancer activity in vitro. iPSCs with homozygous deletion of rs11172113 enhancer region presented the same pluripotency compared with wild type, and iPSC derived SMCs showed positive expression of specific markers for each phenotype (ACTA2, TAGLN for both, MYH11 for contractile SMCs and CALM2 for synthetic SMCs). We found that the deletion of rs11172113 enhancer region decreased the expression of LRP1 while LRP1 knockdown increased cell migration capacity in SMCs. Preliminary results in LRP1-knockout iPSC-derived SMCs suggest LRP1 to enhance the expression of cell contraction markers and decrease capacity of cell proliferation.
Conclusions We confirmed rs11172113 to regulate LRP1 expression in iPSCs derived synthetic and contractile SMCs. Our results support LRP1 effect on SMCs cellular function alteration as a potential mechanism in genetic susceptibility for vascular disease.