Oscillatory and elevated flow distinctly regulate gene expression in human coronary artery endothelial cells

Background

Atherosclerosis develops at arterial sites exposed to disturbed flow, while plaque rupture and plaque erosion predominantly occur in regions subjected to elevated flow. The impact of elevated flow on regulation of endothelial gene expression is less well studied; therefore, we undertook a comprehensive analysis of primary human coronary artery endothelial cell (HCAEC) gene expression under elevated flow, comparing it to gene expression induced by normal physiological and oscillatory flow.

Methods

Analysis of HCAEC mRNA, microRNA and protein expression cultured under oscillatory shear stress (OSS), physiological laminar shear stress (LSS), and elevated shear stress (ESS) for 72 hours. Identification of changes in RNA isoform expression and proximity of flow-responsive genes to established coronary artery disease (CAD) risk loci were also performed.

Results

2,175 shear-regulated genes were identified, with 665 uniquely responsive to ESS. Both ESS and OSS induced significant changes in RNA isoform selection, predicted to affect 848 and 580 genes respectively. Signalling pathways regulating CAD pathogenesis including HIPPO, TGFβ/BMP, and IRF, showed altered RNA isoform selection which may influence plaque development and plaque erosion. 65% of linkage disequilibrium (LD)-filtered CAD-associated genetic variants contained at least one OSS or ESS-regulated gene within 250Kb. Proteomic analysis identified 289 proteins differentially expressed under OSS and 171 under ESS, with notable discordance between mRNA and protein changes observed in 28.7% (OSS vs LSS) and 16.6% (ESS vs LSS) genes. Additionally, 40 shear-responsive microRNAs were identified.

Conclusion

Elevated flow elicits a distinct gene expression programme in HCAECs, modulating pathways central to CAD pathogenesis.

Research Perspective

What New Question Does This Study Raise?

• Plaque erosion predominantly occurs on the upstream surface of the plaque, where the endothelium is exposed to elevated flow, which we show within this study to evoke a significant and largely unique regulation of the transcriptome, miRome and proteome within primary human coronary artery endothelial cells.

• Identify shear stress as a significant regulator of alternative splicing, with elevated flow causing the greatest shift in alternative transcript selection.

• Identify 135 oscillatory, and 101 elevated differentially expressed genes in proximity to CAD risk loci.

What Question Should be Addressed Next?

• Study of the response of the coronary endothelium directly in patients to understand how the risk factors involved in plaque erosion change endothelial function.

• Creation of physiological 3D arterial models replicating in vivo observations to study the precipitating factors involved in plaque erosion.