Atherosclerotic plaque fibroblasts derive from adventitial and medial Pdgfra-lineage-positive cells and predominantly maintain fibroblast identity.
AIMS: Fibroblasts are mesenchymal cells in the healthy vascular adventitia. In atherosclerosis, single-cell sequencing datasets suggest fibroblasts are abundant in plaques. However, their identity, origin, and fate during plaque progression remain unclear, which we aim to unravel here. APPROACH AND RESULTS: To robustly define fibroblast identity, origin, and fate, we employed meta-analyses of 54 single-cell RNA sequencing libraries, including murine smooth muscle cell (Myh11) and endothelial cell (EC) (Cdh5) lineage reporter mice with and without atherosclerosis; human control and atherosclerotic arteries; and murine adventitia and atherosclerotic plaques processed separately from low-density lipoprotein (LDL) receptor knockout (Ldlr-/-) mice. These meta-analyses showed that murine and human plaque fibroblast identity was robustly defined by Pdgfra, Pi16, Cygb, and Serpinf1 mRNA. Ninety-five percent of plaque fibroblasts do not derive from the Myh11 lineage, while no Cdh5-lineage-positive cells were present in the fibroblast cluster. We identified five murine arterial fibroblast subsets in atherosclerotic murine aorta: progenitor fibroblasts, matrix fibroblasts, inflammatory fibroblasts, an EC-like fibroblast subset, detected in both adventitia and plaques, and Col5a3+ fibroblasts, unique to the adventitia. We next studied fibroblast identity, origin, and fate using pseudotime analysis and Pdgfra-CreERT2/tdTomato lineage reporter mice (Pdgfra Lin+). Healthy Pdgfra Lin+ reporter mice showed predominant adventitial tdTomato expression, and infrequent medial and intimal Pdgfra Lin+ cells co-expressing MYH11 and PECAM1, respectively. The Pdgfra Lin+ plaque area increased with diet duration. Pdgfra Lin+ cells largely maintain fibroblast identity in the plaque, while <10% co-express SMC markers (MYH11, SM22α), or contribute to ACTA2+ cap cells. ECs gaining mesenchymal markers are transcriptionally distinct from Cdh5-lineage-negative fibroblasts gaining EC markers. Plaque-resident EC-like fibroblasts displayed a mesenchymal-to-endothelial transition transcriptome, which was induced in human primary fibroblasts in vitro by starvation, and dampened or reversed by IL1B, TGFB1, TGFB3, and oxidized LDL. Cross-species integration showed that all murine plaque fibroblasts were conserved in human atherosclerosis, with one additional subset partially resembling murine subsets, and three human-specific subsets. Importantly, human fibroblast subsets differentially correlated to human plaque traits, with EC-like fibroblasts correlating to plaque instability. CONCLUSION: Our results indicate that 95% of plaque-residing fibroblasts are Myh11 Lin- Plaque fibroblasts have a dual origin, predominantly adventitial Pdgfra Lin+ progenitor fibroblasts, with a minor contribution from medial Pdgfra Lin+  Myh11+ SMCs. Most plaque fibroblasts maintain fibroblast identity. Murine plaque fibroblast subsets were conserved in human atherosclerosis. EC-like fibroblasts are linked to human plaque instability. Intervening in progenitor-to-specific fibroblast transitions could present a new avenue to promote plaque stability in atherosclerosis.