Smooth muscle-derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner

We previously established that vascular smooth muscle-derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM-derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMCs), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared to WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMCs and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potential unstable atherosclerotic plaques. Overall design: AdvSca1-SM lineage tracing mice (Klf4 WT or KO) were treated with tamoxifen to knock in YFP reporter and flox out Klf4 (in the case of KO mice). Athero mice were given PCSK9 AAV injections at 0 and 2 weeks, then put on high fat/high cholesterol diet to induce atherosclerosis; control mice did not receive PCSK9 and were left on standard chow. 3 mice per condition were pooled, and aortic sinus, aortic arch, BCA, and carotid arteries were processed into a single cell digest. Samples were FACS sorted for YFP expression, then each sample was sequenced separately.