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

Abstract

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 (SMC), 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 with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMC 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 potentially unstable atherosclerotic plaques.

Keywords: Adult stem cells; Atherosclerosis; Stem cells; Vascular Biology.

Figure 1. AdvSca1-SM cell–derived YFP⁺ cells are distributed throughout the vascular wall and atherosclerotic plaque.

(A) Schematic of experimental approach. (B) A subset of animals were injected i.v. with fluorescently labeled Griffonia simplicifolia lectin I (GSL I) isolectin B4 5 minutes prior to sacrifice to label functional vasculature in vivo. Representative immunofluorescence of aortic root sections from 24-week plaques (n = 6) stained for YFP (AdvSca1-SM and AdvSca1-SM–derived cells; green); lectin (red); DAPI for all cell nuclei (blue). Arrows indicate functional adventitial microvasculature (lectin labeled) surrounded by YFP⁺ cells; arrowheads indicate intraplaque YFP⁺ cells. (C) Representative immunofluorescence image of 28-week aortic root plaque (n = 5) stained for αSMA (red) and YFP (green). Arrows indicate YFP⁺ medial cells. (D and E) Aortic root slides from 24-week plaques (n = 10) stained for YFP (green), αSMA (red), Ter-119 (magenta), and DAPI (blue). Low-power and high-power insert (D) show YFP⁺ cells in the cap of the plaque. (E) YFP⁺ cells in the plaque cap (arrows), YFP⁺/αSMA⁺ cells in the plaque cap (asterisk), and YFP⁺ cells in the body of the plaque (arrowheads). Scale bars: 50 μm, 100 μm (low-power D).

Figure 2. scRNA-Seq analysis demonstrates major shifts in vascular cell types in the setting of atherosclerosis.

The aortic sinus, aortic arch, brachiocephalic artery, and carotid arteries were harvested at baseline and after 16 weeks of normal or high fat chow and processed for scRNA-Seq; 3 mice per condition were pooled for analysis. (A) UMAP of all cells that passed quality control. Cluster identity was assigned using representative gene expression profiles. (B) Dot plot showing the top 5 unique genes that define each cluster. (C) UMAP of all cells from baseline, 16-week control, and 16-week atherogenic samples. (D) Stacked bar plot of YFP⁺ and YFP^– cells from 16-week control and atherogenic samples. Arrows indicate increases (red) or decreases (blue) in the cell population as a result of atherosclerosis. (E) RNA velocity analysis of all cells after 16 weeks of atherogenic diet.

Figure 3. AdvSca1-SM cells differentiate into fibroblasts or SMCs or remain in a stem-like state in atherosclerosis.

(A) Feature plots showing distribution of YFP⁺ and YFP^– cells on the UMAP. (B) Feature plots of major fibroblast (Col1a1, Col1a2, Col3a1, Dcn, Lum, and Tcf21) and stem cell (Ly6a/Sca1, Cd34, Scara5, Pi16) markers in YFP⁺ cells. (C) Representative aortic root image from 24-week plaques (n = 10) stained for YFP (green), Sca1 (red), and DAPI (blue). Arrows indicate YFP⁺ Sca1⁺ cells in the adventitia; arrowheads indicate YFP⁺ Sca1^– cells. * = cardiomyocyte autofluorescence. (D) Aortic sinus, aortic arch, brachiocephalic artery, and carotid arteries from 16-week control (n = 5) or atherosclerotic (n = 11) mice were processed for flow. Representative image of a YFP and SCA1 density plot from one atherogenic animal. (E) Double RNAscope and immunofluorescence image of an aortic root from 24-week atherogenic animals showing lumican (Lum; fibroblast cell marker; red) mRNA and YFP (green). Arrows indicate YFP⁺/lumican⁺ cells; arrowheads indicate YFP⁺/lumican^– cells; n = 3. (F) Feature plots of SMC genes (Acta2, Cnn1, Mhy11, Tagln) in YFP⁺ cells. (G) 24-week aortic root plaques (n = 10) stained for YFP (green), αSMA (red), and DAPI (blue). Arrows indicate YFP⁺ αSMA⁺ cells forming the fibrous cap of the plaque or contributing to the media. Arrowheads indicate YFP⁺ αSMA^– cells. PA = pulmonary artery; n = 11. (H) Stacked bar plot showing phenotypic shifts in YFP⁺ cells between 16-week control and atherogenic samples. Arrows indicate increases (red) or decreases (blue) in the cell population with atherosclerosis. (I) GO Biological Process between the Fib_2 cluster and all other cell clusters. Arrows indicate the top 4 processes positively associated with Fib_2. (J) Violin plots demonstrating the stronger collagen gene signature (Col1a1, Col1a2, and Col3a1) in Fib_2 compared with Fib_3. All scale bars are 50 μm.

Figure 4. Dynamic, bidirectional differentiation between AdvSca1-SM cells and SMC in the setting of atherosclerosis.

(A) The transitional cluster consists of YFP⁺ and YFP^– cells and exhibits features of both SMC and AdvSCa1-SM cells, roughly divided in half (bottom). Feature plots show that the upper portion of the transitional cluster expresses more contractile SMC genes (Acta2, Myh11, Cnn1), whereas the lower portion expresses more mesenchymal stem cell markers (Ly6a, Cd34, Pdgfra). (B) KEGG pathway differences between the transitional cluster and the AdvSca1-SM/fibroblast clusters or SMC clusters. (C) Representative image of 14-week atherogenic brachiocephalic artery (n = 14) from SMC lineage tracing mice (Myh11-CreERT^+/–/Rosa26-YFP^+/+) stained for YFP (green; expression indicates SMC, not AdvSca1-SM). Arrows indicate adventitial YFP⁺ Sca1⁺ cells; arrowheads indicate adventitial progenitors not expressing YFP (YFP^–/Sca1⁺); orange arrows indicate DAPI⁺ cells in the vessel media that are negative for YFP. A, adventitia. Scale bar = 50 μm.

Figure 5. Depletion of KLF4 in AdvSca1-SM cells modifies the fate of YFP⁺ cells in the setting of atherosclerosis.

Arteries from 16-week WT and Klf4 KO AdvSca1-SM lineage atherogenic mice were processed for scRNA-Seq. (A) Left: UMAP projection of YFP⁺ and YFP^- cells from WT and Klf4 KO AdvSca1-SM cell lineage tracing mice. Right: UMAP projection showing the different fates of YFP⁺ cells in atherosclerosis as a consequence of depleting KLF4 in AdvSca1-SM cells. Arrows indicate major phenotypic shifts in KO mice. (B) Stacked bar graph of YFP⁺ cells from WT and Klf4 KO mice after 16-week atherogenic diet. Arrows indicate increases (red) or decreases (blue) in cell populations as a function of Klf4 depletion. (C) Violin plots showing higher expression of ECM related genes (Col1a1, Col1a2) in YFP⁺ Fib_2 cluster from Klf4 KO mice compared to WT mice after 16 weeks of atherogenic diet. Col1a1 is also higher in YFP⁺ Fib_3 cluster cells from Klf4 KO mice compared to WT mice, but there is no difference with Col1a2 in this cluster. (D) Violin plots showing higher expression of SMC contractile genes (Acta2, Myh11, Cnn1) in YFP⁺ Transitional cluster cells from Klf4 KO mice compared to WT mice after 16 weeks of atherogenic diet.

Figure 6. KLF4 depletion in AdvSca1-SM cells alters the fate of YFP^– non–AdvSca1-SM–derived cells.

Arteries from 16-week WT and Klf4-KO AdvSca1-SM lineage atherogenic mice were processed for scRNA-Seq. (A) Left: UMAP projection of YFP⁺ and YFP^– cells from WT and Klf4 KO AdvSca1-SM cell lineage tracing mice. Right: UMAP projection showing the different fates of YFP^– cells in atherosclerosis as a consequence of depleting KLF4 in AdvSca1-SM cells. Arrows indicate major phenotypic shifts in KO mice, including changes to the SMC and macrophage clusters. (B) Stacked bar graph of YFP^– cells from WT and Klf4-KO mice after 16-week atherogenic diet. Arrows indicate increases (red) or decreases (blue) in the cell population as a result of KLF4 depletion. (C) Violin plot showing expression of SMC contractile genes (Acta2, Tagln, Myh11) in YFP^– cells in the major SMC cluster from Klf4-KO mice compared with WT mice. (D) Violin plot showing expression of Ccl2 in YFP^– cells of the major macrophage cluster from Klf4-KO mice compared with WT mice. (E) Flow cytometry analysis of single-cell arterial digests from both WT and Klf4-KO mice after 24 weeks of atherogenic diet. (F) CellChat analysis was performed on the fibroblast/AdvSca1-SM and Mac_1 clusters. Bubble plot showing elevated levels of COL1A1/COL1A2 from the fibroblast clusters signaling to SDC4 in Mac_1 in the setting of atherosclerosis. (G) Violin plots show expression of Sdc4 and Abcg1 in Mac_1 from Klf4-KO mice in the setting of atherosclerosis. Abca1 is not significantly different between the genotypes. Statistical analysis done with 2-tailed Student’s t test.

Figure 7. Depletion of KLF4 in AdvSca1-SM cells does not alter overall plaque burden but reduces late-stage plaque complexity.

(A) The aortic arch and descending aorta from 24-week atherogenic WT (n = 6) and Klf4-KO (n = 4) mice were stained using Sudan IV to assess overall atherosclerotic plaque burden. Two-tailed Student’s t test analysis of staining is shown to the right. (B) Representative H&E images of aortic root cross sections from WT (n = 6) and Klf4-KO mice (n = 6). Insets show higher magnification of the plaque structure with significantly smaller necrotic cores in Klf4-KO mice compared with WT mice. Two-tailed Student’s t test analyses of plaque percent coverage and necrotic core area (P = 0.0221) are shown below. (C) Representative H&E images of stained aortic root cross sections from WT (n = 6) and Klf4-KO (n = 6) mice. Arrows indicate cholesterol clefts. (D) Representative immunofluorescence images of aortic root cross sections from WT (n = 5) and Klf4-KO (n = 6) mice for CD68 (red) and DAPI (blue). Two-tailed Student’s t test analysis of staining is shown to the right; P = 0.0117. (E) Representative immunofluorescence images of aortic root cross sections from WT (n = 5) and Klf4-KO (n = 6) mice for YFP (green), αSMA (red), and DAPI (blue). Two-tailed Student’s t test analysis of staining is shown to the right; P = 0.026. (F) Representative Masson’s trichrome images from WT (n = 6) and Klf4-KO (n = 6) mice aortic roots with collagen (blue), cytoplasm (pink), and cell nuclei (brown). Two-tailed Student’s t test analysis of staining is shown to the right; P = 0.0165. Scale bars: 50 μm, 200 μm (E).

Figure 8. Schematic of the multifaceted roles of AdvSca1-SM cells in atherosclerosis.

In the setting of atherosclerosis, AdvSca1-SM cells are capable of differentiating into a variety of cell types (primarily fibroblasts and SMC) and migrating throughout the vascular wall and the atherosclerotic plaque. In addition to directly differentiating into other cell types, AdvSca1-SM cells can signal to other cell types, including macrophages, fibroblasts, and SMC to alter their phenotypes. Genetic modulation of AdvSca1-SM cells drastically alters plaque composition complexity, indicating a central role for AdvSca1-SM cells in atherosclerosis.