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BackgroundThe protein 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a key stimulator of glycolytic flux. Systemic, partial PFKFB3 inhibition previously decreased total plaque burden and increased plaque stability. However, it is unclear which cell type conferred these positive effects. Myeloid cells play an important role in atherogenesis, and mainly rely on glycolysis for energy supply. Thus, we studied whether myeloid inhibition of PFKFB3-mediated glycolysis in Ldlr–/–LysMCre+/–Pfkfb3fl/fl (Pfkfb3fl/fl) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr–/–LysMCre+/–Pfkfb3wt/wt control mice (Pfkfb3wt/wt).Methods and ResultsAnalysis of atherosclerotic human and murine single-cell populations confirmed PFKFB3/Pfkfb3 expression in myeloid cells, but also in lymphocytes, endothelial cells, fibroblasts and smooth muscle cells. Pfkfb3wt/wt and Pfkfb3fl/fl mice were fed a 0.25% cholesterol diet for 12 weeks. Pfkfb3fl/fl bone marrow-derived macrophages (BMDMs) showed 50% knockdown of Pfkfb3 mRNA. As expected based on partial glycolysis inhibition, extracellular acidification rate as a measure of glycolysis was partially reduced in Pfkfb3fl/fl compared to Pfkfb3wt/wt BMDMs. Unexpectedly, plaque and necrotic core size, as well as macrophage (MAC3), neutrophil (Ly6G) and collagen (Sirius Red) content were unchanged in advanced Pfkfb3fl/fl lesions. Similarly, early lesion plaque and necrotic core size and total plaque burden were unaffected.ConclusionPartial myeloid knockdown of PFKFB3 did not affect atherosclerosis development in advanced or early lesions. Previously reported positive effects of systemic, partial PFKFB3 inhibition on lesion stabilization, do not seem conferred by monocytes, macrophages or neutrophils. Instead, other Pfkfb3-expressing cells in atherosclerosis might be responsible, such as DCs, smooth muscle cells or fibroblasts.

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-/-Pdgfbret/ret mice were fed a high cholesterol diet. Although plaque size was increased in the aortic root of Pdgfbret/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 Pdgfbret/ret plaques (2.1-fold) and increased Pdgfbret/ret macrophage apoptosis upon 7-ketocholesterol or oxidized LDL incubation in vitro. Moreover, Pdgfbret/ret plaque collagen content increased independent of mesenchymal cell density. The decreased macrophage matrix metalloproteinase activity could partly explain Pdgfbret/ret collagen content. In addition to the beneficial vascular effects, we observed reduced body weight gain related to smaller fat deposition in Pdgfbret/ret liver and adipose tissue. While dampening plaque inflammation, Pdgfbret/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 Pdgfbret/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 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. Methods & Results 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. Conclusion 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.