Abstract 4139230: Mapping the Molecular Progression of Human Coronary Atherosclerosis Confirms Key Role of Smooth Muscle Cell Phenotype and Highlights Novel Regulators of Phenotypic Fate

Background: clarity in the molecular mechanisms contributing to pre-clinical stages of coronary atherosclerosis is needed to develop novel early diagnostic tools and more effective preventive interventions. Methods: We completed untargeted proteomic and RNA expression analysis on proximal coronary arterial (CA) tissues from young adults who died of trauma with no antemortem suspicion of coronary disease (n=322, mean age (range): 34.1 yrs. (15-59); sex: M-239, F-83; race: W-218, B-88, other-16), including n=180 (56%) with morphologic evidence of pre-clinical atherosclerosis. Proteomic data (n=1900 proteins) were analyzed using manifold learning and unsupervised latent feature (LF) detection methods to define the pseudo-temporal progression of disease from normal to advanced atherosclerosis and to identify molecular drivers of disease initiation and progression. Correlation and network mapping of the mRNA data (n=16,707 transcripts, including n=1707 annotated transcription factors [TFs]) from the same arterial samples was used to define the transcriptional regulatory networks (TRNs) associated with the observed proteomic LF dynamics. Results: Four of seven identified LFs had a significant (FDR <p.01) association with plaque progression. Among these, a standout LF clearly represented quiescent, contractile smooth muscle cells (SMC) with progressive and then poignantly reduced abundance in late-stage lesions. This LF was enriched with proteins related to cell adhesion and SMC contractility, including 14 previously implicated in the pathogenesis of atherosclerosis via GWAS analyses. Analysis of TRNs revealed 36 regulatory TFs with (1) a predicted binding site in the promotor of SMC LF marker proteins, (2) observed expression that was strongly associated with this SMC LF and (3) significant correlation between predicted activity and observed abundance, including the established SMC phenotype regulator SRF. Expression of regulatory TFs and SMC LF markers in public single-cell RNAseq data from normal and atherosclerotic human coronary tissues suggested both SMC intrinsic and cell-cell crosstalk TRNs. Conclusions: The pseudotemporal pattern of the SMC LF was consistent with SMC phenotype changes as early initiating events in atherosclerosis of the CA. Therapies to promote or inhibit activity of these identified TFs may prevent against early lesion formation and atherosclerosis progression by supporting SMC phenotype stability.