Context-dependent regulation of endothelial inflammation and atherosclerosis by endothelial microRNA-33

Atherosclerosis arises through the metabolic and inflammatory perturbation of numerous cells, including immune cells and endothelial cells (ECs). microRNA-33 (miR-33) regulates lipid metabolism and inflammatory responses of immune cells, but the impact of miR-33 on atherosclerosis progression has mixed effects, pointing to context- and cell-type-specific functions. Notably, the role of EC miR-33 in atherosclerosis remains unexplored, despite the central involvement of metabolic and inflammatory pathways in EC function. We sought to determine the definitive role of EC miR-33 in atherosclerosis progression.

We generated mice with an inducible EC-specific miR-33 knockout (iECKO), followed by PCSK9-AAV8 injection and western diet feeding. Detailed plaque analyses and scRNAseq were performed. For acute inflammation, we analyzed TNFα-mediated leukocyte recruitment in the air pouch model. In vitro approaches included the culture of human aortic ECs to analyze gene expression under inflammatory and lipid-laden conditions with miR-33 mimicry.

iECKO mice showed accelerated lesion initiation, but this effect did not persist in advanced atherosclerosis, which is likely due to chronic hypercholesterolemia-driven downregulation of miR-33 that masks its deletion at later stages. Transcriptomic analyses revealed that cholesterol loading alters EC responses to inflammation, which can be partially rescued by miR-33 mimicry. Accordingly, iECKO mice exhibited heightened sensitivity to acute, normocholesterolemic inflammation, which is paralleled with regulation of E-selectin levels.

Our work underscores the nuanced effects of miR-33 manipulation and highlights how well-described regulators of atherosclerosis progression may have unique cell type- and disease stage-dependent roles. Additionally, our work further implicates miR-33 as a regulator of EC function and identifies a new potential role in acute inflammation via E-selectin regulation.