Abstract Body: Introduction: Adventitial fibroblasts play an important role in maintaining vascular homeostasis but their functions in atherogenesis remain elusive. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been demonstrated to have complex function in atherogenesis. However, it is not clear whether IKKβ signaling in fibroblasts can also affect atherogenesis. This study aims to investigate the role of fibroblast IKKβ in the regulation of atherosclerosis development in LDL receptor-deficient (LDLR^-/-) mice.

Hypothesis: IKKβ signaling regulates fibroblast functions to affect diet-induced atherosclerosis development.

Methods: To define the role of fibroblast IKKβ in atherogenesis, we generated a novel inducible fibroblast-specific IKKβ-deficient LDLR^-/- (IKKβ^ΔFibLDLR^-/-) mouse model. IKKβ^ΔFibLDLR^-/- and control littermates (IKKβ^F/FLDLR^-/-) were fed a high-fat Western diet for 12 weeks to induce atherosclerosis development. Atherosclerotic lesion sizes and plaque vulnerability were analyzed. Total RNAs were isolated from aortas for gene expression analysis. Aortas were collected for single-cell RNA sequencing (scRNA-seq) analysis.

Results and Conclusions: Deficiency of fibroblast IKKβ did not affect diet-induced obesity and atherosclerotic lesion areas in LDLR^-/- mice. IKKβ^ΔFibLDLR^-/- mice, however, had increased plaque vulnerability characterized by a thin fibrous cap, increased necrotic core, and decreased extracellular matrix contents. Aortic gene expression analysis demonstrated that IKKβ^ΔFibLDLR^-/- mice had decreased expression of genes that regulate fibroblast activation, myofibroblast differentiation, cell survival, and apoptosis. scRNA-seq analysis of the whole aorta confirmed that deficiency of IKKβ led to dysregulated genes and pathways mediating fibroblast activation and differentiation in the fibroblast clusters. In addition, scRNA-seq results revealed a decreased overall smooth muscle cell population as well as smooth muscle-like cell subpopulation derived from fibroblast origin. These results demonstrate that IKKβ functions in fibroblasts to regulate atherosclerotic plaque vulnerability, likely through regulating fibroblast differentiation and apoptosis. This study suggests IKKβ signaling in atherogenesis is complex, and IKKβ in different cell types has different effects on atherosclerosis development.