76 Complex stem cell models reveal causality and disease mechanisms of aortic disease genetic variants from the 100,000 Genomes Project

Abstract Introduction Induced pluripotent stem cell (iPSC) derived aortic vascular smooth muscle cells (VSMC) recapitulate key disease features leading to wall failure. We hypothesise iPSC models of thoracic aortic diseases (TAD) can determine causality of genetic variants from the 100,000 Genomes Project. Methods Variants in LOX (122070160 T>TA), MYLK (123401069 ACT>A) and SMAD4 (51076665 C>T) were selected, and genetic editing used to create mutant iPSC lines. The mutant lines were differentiated into VSMCs, analysed with bulk mRNAseq and findings validated in vitro. Results Mutant VSMCs demonstrated TAD transcriptomic signatures, with common & distinct pathways mediating disease pathogenesis in different variants. EdU incorporation in LOX and SMAD4 mutants confirmed enrichment of pro-proliferative GO terms. An active and basal contraction defect in MYLK and LOX was demonstrated in both 2D and 3D models. LOX mutant showed elevated proteo-/glycosaminoglycans and soluble collagen verifying ECM remodelling GO terms. Increased mitochondrial basal respiration confirmed metabolic defect in SMAD4 and MYLK. Perturbed calcium signalling in MYLK, or an inflammatory phenotype strongly associated with SMAD4 were highlighted. Differential expression of osteochondrogenic/fibroblastic markers in MYLK, and senescent/osteoblast markers in LOX were suggestive of VSMC phenotypic switching. Several targetable signalling pathways were identified. Conclusion Despite distinct initiators, there is a convergence on abnormal ECM turnover and contractility which in combination are likely the driving factor behind reduced aortic wall integrity. iPSC TAD models offer a precision medicine tool through determining causality of genetic variants, identifying disease mechanisms and providing a blueprint for therapeutic targeting.