Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVD). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolemia and aging. Murine aorta scRNA-seq analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, while traditional markers such as cluster of differentiation (CD)90 and vimentin also marked transgelin + vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+) and lysyl oxidase (Lox+), were reproduced in an independent RNAseq dataset. Gene ontology analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in adventitia of aged C57BL/6J mice, and mildly hypercholesterolemic LDLR-KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14 + and LOX + fibroblasts in aged and hypercholesterolemic aortic adventitia respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14 + and LOX + fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets differentially correlated to human atherosclerotic plaque traits. We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and cardiovascular disease in humans and mice. Biological relevance is evident from regulation of fibroblast clusters by age and hypercholesterolemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.

Platelet-derived growth factor B (PDGF-B) is a mitogenic, migratory and survival factor. Cell-associated PDGF-B recruits stabilizing pericytes towards blood vessels through retention in extracellular matrix. We hypothesized that the genetic ablation of cell-associated PDGF-B by retention motif deletion would reduce the local availability of PDGF-B, resulting in microvascular pericyte loss, microvascular permeability and exacerbated atherosclerosis. Therefore, Ldlr^-/-Pdgfb^ret/ret mice were fed a high cholesterol diet. Although plaque size was increased in the aortic root of Pdgfb^ret/ret mice, microvessel density and intraplaque hemorrhage were unexpectedly unaffected. Plaque macrophage content was reduced, which is likely attributable to increased apoptosis, as judged by increased TUNEL+ cells in Pdgfb^ret/ret plaques (2.1-fold) and increased Pdgfb^ret/ret macrophage apoptosis upon 7-ketocholesterol or oxidized LDL incubation in vitro. Moreover, Pdgfb^ret/ret plaque collagen content increased independent of mesenchymal cell density. The decreased macrophage matrix metalloproteinase activity could partly explain Pdgfb^ret/ret collagen content. In addition to the beneficial vascular effects, we observed reduced body weight gain related to smaller fat deposition in Pdgfb^ret/ret liver and adipose tissue. While dampening plaque inflammation, Pdgfb^ret/ret paradoxically induced systemic leukocytosis. The increased incorporation of 5-ethynyl-2′-deoxyuridine indicated increased extramedullary hematopoiesis and the increased proliferation of circulating leukocytes. We concluded that Pdgfb^ret/ret confers vascular and metabolic effects, which appeared to be protective against diet-induced cardiovascular burden. These effects were unrelated to arterial mesenchymal cell content or adventitial microvessel density and leakage. In contrast, the deletion drives splenic hematopoiesis and subsequent leukocytosis in hypercholesterolemia.

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.