Remember me
Research ArticleStem cellsVascular biology
Open Access | 
10.1172/jci.insight.174639
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Dubner, A.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Lu, S.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Jolly, A.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Strand, K.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Mutryn, M.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by Hinthorn, T. in: JCI | PubMed | Google Scholar
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by Noble, T. in: JCI | PubMed | Google Scholar
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Nemenoff, R.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Moulton, K.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Majesky, M.
in:
JCI
|
PubMed
|
Google Scholar
|
1Department of Medicine, Division of Renal Diseases and Hypertension,
2Integrated Physiology PhD Program,
3School of Medicine, Consortium for Fibrosis Research and Translation,
4Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
5Biomedical Sciences and Biotechnology MS program, University of Colorado Graduate School, Anschutz Medical Campus, Aurora, Colorado, USA.
6Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA.
7Center for Developmental Biology & Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
8Departments of Pediatrics, Laboratory Medicine & and Pathology, University of Washington, Seattle, Washington, USA.
9Cardiovascular Pulmonary Research Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Address correspondence to: Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, C281, Research Complex 2, Room 7002, Aurora, Colorado 80045, USA. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. Or to: Mark W. Majesky, Department of Pediatrics, Division of Cardiology, University of Washington, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-5, Seattle, Washington 98101, USA. Phone: 206.884.3661; Email: mwm84@uw.edu.
Find articles by
Weiser-Evans, M.
in:
JCI
|
PubMed
|
Google Scholar
|
Published November 22, 2023 - More info
Published in Volume 8, Issue 22 on November 22, 2023
AbstractWe 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.
Graphical Abstract
Introduction
Atherosclerosis is a complex inflammatory condition and the major driver of cardiovascular disease, a spectrum of diseases resulting in approximately 32% of global deaths (1, 2). Approaches to management of atherosclerosis include lipid-lowering and antiinflammatory medications. Unfortunately, statins fail to fully resolve CVD risk, and despite intense lipid lowering, many patients have residual risks. Additionally, antiinflammatory treatments have limitations due to cost and the need for chronic use, which carries increased risk of infection and sepsis. Thus, additional therapeutic approaches are needed to directly target the vascular wall cells in atherosclerotic lesions (3–8). Surprisingly, few if any therapies focus on the pathological mechanisms of resident vascular cells, and this may provide insights for future therapy beyond current care.
Historically, research in the field of atherosclerosis has centered on the role of the innermost layer of the blood vessel, the intima, in driving atherosclerosis progression through endothelial dysfunction, lipid accumulation/oxidation, and macrophage infiltration (1, 3–5). More recently, lineage-tracing studies in murine models and human atherosclerotic tissues have defined the important role vascular smooth muscle cells (SMC) play in disease progression. However, while less frequently studied, it has been well recognized that cells in the outermost layer of the vessel, the adventitia, are critical contributors to early stages in the pathogenesis of atherosclerosis. Furthermore, the “outside-in” hypothesis posits that expansion of adventitial microvessels, the vasa vasorum (VV), acts as an early and potent driver of plaque progression by facilitating inflammatory cell infiltration and tertiary lymphoid organogenesis and by supplying the oxygen/nutrient needs of the growing plaque (6–10).
Recent research has demonstrated that the adventitia is highly dynamic and home to a wide variety of cells, including fibroblasts, leukocytes, and resident vascular progenitor cells (7, 11–13). Our group discovered a subpopulation of these adventitial progenitor cells (AdvSca1-SM cells; adventitial location, Sca1 expression, SMC origin) derived from mature, contractile SMC that undergo KLF4-dependent reprogramming, thus transitioning into a multipotent progenitor (14). Subsequent research demonstrated that maintenance of the AdvSca1-SM stemlike phenotype is dependent on continuous KLF4 activity and that AdvSca1-SM cells preferentially differentiate into myofibroblasts to contribute to perivascular fibrosis in response to acute vascular injury (15, 16). However, the contribution of AdvSca1-SM cells to chronic diseases such as atherosclerosis has not been established.
In this study, we characterized the multifaceted contributions of AdvSca1-SM cells to atherosclerosis. Immunofluorescence microscopy demonstrated that AdvSca1-SM–derived cells are found throughout the adventitia, media, and atherosclerotic plaque. Using single-cell RNA sequencing (scRNA-Seq), we established the major differentiation trajectories of AdvSca1-SM–derived cells as well as their ability to communicate with other cells in the plaque microenvironment. Surprisingly, we found that AdvSca1-SM cell–specific KO of Klf4 altered both the differentiation trajectories of AdvSca1-SM cells and the transcriptomic profiles of non-AdvSca1-SM cell–derived SMC and macrophages, leading to increased fibrous cap thickness and intraplaque collagen deposition with corresponding reductions in necrotic core area, cholesterol crystal accumulation, and macrophage recruitment. These findings indicate that AdvSca1-SM cells are major regulators of atherosclerotic plaque progression and that KLF4-dependent phenotypic transitions of both AdvSca1-SM cells and other vascular cells are critical to plaque complexity, thus highlighting a potential future therapeutic target.
ResultsInduction of atherosclerosis in AdvSca1-SM cell lineage mice. Our previous research indicated a preferential differentiation of AdvSca1-SM cells into myofibroblasts in the setting of unilateral carotid ligation, a well-established model for acute vascular injury, neointima formation, and adventitial remodeling (15, 16). In these experiments, AdvSca1-SM cells were integral to adventitial remodeling and arterial fibrosis. However, the role of AdvSca1-SM cells in chronic vascular diseases, specifically atherosclerosis, has not been established. To elucidate the role of these adventitial progenitor cells in atherosclerosis, we utilized the AdvSca1-SM cell lineage tracing mouse model we previously developed and validated (Gli1-CreERT/Rosa26-YFP) (15). Compared with other cells in the vasculature, AdvSca1-SM cells express high levels of Gli1. Upon tamoxifen injections, the tamoxifen-inducible Gli1-Cre recombinase is activated, and AdvSca1-SM cells and AdvSca1-SM–derived cells are selectively and permanently labeled with the YFP reporter; a complete lack of medial SMC or intimal cell labeling confirms the specificity of this system. After cell labeling, AdvSca1-SM lineage mice were injected with a gain-of-function mutant PCSK9-AAV to knock down LDL receptors and were placed on a high-fat/high-cholesterol diet for 8–28 weeks to induce hypercholesterolemia and atherosclerotic plaque formation, as previously described (Figure 1A and Supplemental Figure 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.174639DS1) (17, 18).
Figure 1AdvSca1-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).
AdvSca1-SM–derived cells are found throughout the vessel wall and plaque in both early- and late-stage atherosclerosis. We first sought to determine the spatial distribution of AdvSca1-SM cells and their progeny within the vessel wall and plaque. The majority of published studies of atherosclerosis progression primarily focused on lipid accumulation and monocyte recruitment on the intimal surface of the vessel (1, 3, 5). However, additional work has demonstrated the integral role of the VV in driving plaque progression (1, 3–9). Moreover, using an in vivo Matrigel plug assay, we previously demonstrated the ability of AdvSca1-SM cells to form microvasculature, raising the question of whether AdvSca1-SM cells could be involved in the expansion of the adventitial microvasculature in the setting of atherosclerosis (14). To investigate this possibility, a subset of mice after 16 or 24 weeks of atherogenic diet were i.v. injected with fluorescently conjugated isolectin B4 to label functional vasculature. Using this approach, we detected microvessels in the adventitial area of the aortic root. Similar to our findings in acute vascular injury, there was an expansion of YFP+ AdvSca1-SM cell–derived cells in the adventitia, with many frequently associated with the adventitial microvasculature, suggesting a neovascularization role for AdvSca1-SM cells in atherosclerosis and supporting our previous Matrigel plug experiments (Figure 1B). While the majority of YFP+ cells were found in the adventitia, they were also observed in the medial layer of the aortic root (Figure 1C). Finally, we observed YFP+ cells localized to the plaque itself, both via formation of the fibrous cap (Figure 1, D and E) as well as within the core of the plaque. These data indicate roles for AdvSca1-SM cells throughout the vascular wall in the context of chronic atherosclerosis and suggest multifaceted contributions to atherogenesis.
scRNA-Seq defines shifts in vascular cell phenotypes in atherosclerosis. Given the wide distribution of AdvSca1-SM cell–derived YFP+ cells in the vascular adventitia, media, and atherosclerotic plaque, we used unbiased scRNA-Seq to define the possible functions of AdvSca1-SM cells in atherosclerosis. Arteries from AdvSca1-SM lineage tracing mice at both baseline (after tamoxifen treatment) and after 16 weeks of either control or atherogenic conditions were harvested, processed into single-cell suspensions, and sorted by FACS based on YFP expression; YFP+ and YFP– cells were subjected to scRNA-Seq. It is important to note that entire aortae, BCA, carotid arteries, and aortic root with both plaque and nonplaque regions were combined to obtain sufficient cells for sequencing. Data sets from all samples were combined, passed through quality control measures, and processed using the Seurat pipeline, generating a total of 26 cell clusters (Figure 2A and Supplemental Figure 4). Gene expression profiles were used to assign identities to the different clusters, and the top 5 differentially expressed genes per cluster are shown in Figure 2B. As expected, we detected shifts in cell populations between baseline and 16 weeks of control or atherogenic treatment (Figure 2C). We then examined the changes in cell populations between mice on control or atherogenic diet after 16 weeks of treatment. The clusters that showed the most variability as a result of atherosclerosis were the fibroblast clusters (Fib_1, Fib_2, Fib_3, Fib_4). Specifically, we observed increases in the size of Fib_1, Fib_3, and Fib_4 clusters, along with a decrease in Fib_2 (Figure 2D). We also observed a slight decrease in the AdvSca1-SM cell cluster, suggesting an increased pattern of differentiation into other cell types in the setting of atherosclerosis. Since AdvSca1-SM cells are multipotent progenitor cells and don’t represent a static cell population, RNA velocity analysis was used to predict future cell transition patterns, as described previously (19). Validating our previous findings, the streamplots indicate marked differentiation away from the most stemlike state, the AdvSca1-SM cell cluster, and into other cell types (Figure 2E). These data are in agreement with previous scRNA-Seq data showing shifts in cell phenotype with atherosclerosis progression and confirm a transition of AdvSca1-SM cells away from a stemlike phenotype (20–24).
Figure 2scRNA-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.
AdvSca1-SM cells preferentially differentiate into fibroblasts or remain in a stem-like state in atherosclerosis, with minor contribution to SMC populations. Leveraging our AdvSca1-SM lineage tracing mouse model, we investigated specific changes in the YFP+ AdvSca1-SM and AdvSca1-SM–derived cell populations in the setting of atherosclerosis. YFP+ cells were primarily found in the stemlike AdvSca1-SM, and fibroblast-like Fib_1, Fib_2, and Fib_3 clusters (Figure 3A). These clusters expressed high levels of myofibroblast (Tcf21) and extracellular matrix (ECM) markers (Col1a1, Col1a2, Col3a1, Lum, Dcn), and stem cell markers (Ly6a, Cd34, Scara5, Pi16) (Figure 3B). However, the distribution of these markers varied between the clusters, with the AdvSca1-SM cell cluster representing the most stem-like phenotype, Fib_2 having elevated collagen gene expression compared with the other fibroblast clusters, and Fib_1 having the highest expression of myofibroblast marker Tcf21.
Figure 3AdvSca1-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.
As noted in the scRNA-Seq data, a substantial population of YFP+ cells do not differentiate into other cell types but rather retain expression of a stemlike phenotype, as shown by expression of the stem cell marker Ly6a (SCA1). Confirming these data, aortic roots stained for YFP and SCA1 exhibited a large population of YFP+ SCA1+ cells in the adventitia (Figure 3C). Flow cytometry analysis of animals after 16 weeks of atherogenic diet also supported these findings, demonstrating a considerable proportion of YFP+ SCA1+ stemlike AdvSca1-SM cells (Figure 3D). As noted above, both scRNA-Seq and flow experiments were performed on whole arterial tissue, as compared with immunofluorescence analysis, which focused specifically on plaque-affected regions of the vasculature. Since atherosclerosis is a focal disease, many of the stemlike AdvSca1-SM cells detected in scRNA-Seq and flow cytometry are likely from regions of uninvolved tissue.
We also confirmed the differentiation of YFP+ cells into fibroblasts using immunofluorescence and RNAscope microscopy. Investigation of aortic roots revealed YFP+ cells in both the adventitia and plaque that coexpress the fibroblast marker, lumican (Lum) (Figure 3E). Please note that not all Lum+ cells coexpressed YFP; however, these data further support that AdvSca1-SM have the potential for differentiation toward a fibroblast phenotype in the setting of atherosclerosis. In addition to differentiation toward a fibroblast phenotype or remaining as a stem cell, we detected a subset of YFP+ AdvSca1-SM cells differentiating toward a SMC fate. YFP+ cells found in the SMC clusters were found to express genes specific to mature vascular SMC, including Acta2, Myh11, Cnn1, and Tagln (Figure 3F). This differentiation profile suggested by scRNA-Seq was then confirmed using immunofluorescence microscopy, in which we identified YFP+/αSMA+ cells in the plaque fibrous cap and aortic media (Figure 3G). Finally, we identified extremely rare differentiation of AdvSca1-SM cells into other cell types, including macrophages, adipocytes, and endothelial cells (Supplemental Figure 6).
As expected, the proportion of YFP+ cells in each of the cell clusters was altered in mice on an atherogenic diet compared with controls. Specifically, there were increased percentages of YFP+ cells in Fib_1 and Fib_3 clusters with a corresponding decrease in Fib_2 and AdvSca1-SM cell clusters in the setting of atherosclerosis (Figure 3H). To elucidate the functional importance of these cell shifts, we further characterized the phenotypes of the different fibroblast clusters using GO Biological Process pathway analysis. We determined that, compared with all other cell clusters, Fib_2 was enriched for pathways related to ECM and collagen organization (Figure 3I). These findings were confirmed by comparing average expression of collagen genes between the various fibroblast clusters, with Fib_2 showing the highest expression of these genes (Figure 3J). Collectively, these data indicate that chronic hyperlipidemia–induced atherosclerosis promotes activation and a shift in the phenotype of AdvSca1-SM cells predominantly toward a variety of fibroblast-like cells as well as the contribution of AdvSca1-SM cells to adventitial remodeling and plaque formation.
Dynamic exchange between AdvSca1-SM cells and SMC in the setting of atherosclerosis. RNA velocity analysis of our data (Figure 2E) revealed that, not only do AdvSca1-SM cells differentiate away from their most stem-like state and into other cell types, but a variety of cell transitions occur in the fibroblast and SMC clusters. Of particular interest was the transitional cluster, which occupies Uniform Manifold Approximation and Projection (UMAP) space between SMC and AdvSca1-SM/fibroblast clusters and shows a bidirectional differentiation trajectory. The first trajectory demonstrated a shift from AdvSca1-SM/fibroblast clusters toward a SMC phenotype, a trajectory that we confirmed using immunofluorescence imaging for αSMA (Figure 3, F and G). However, the RNA velocity analysis also suggested a reverse trajectory from non–AdvSca1-SM–derived mature SMC (YFP–) toward the AdvSca1-SM/fibroblast–like clusters. Further characterization of this transitional cluster revealed 2 unique subpopulations, (a) an upper cluster, which expresses more SMC genes and is largely YFP–, and (b) a lower cluster, which expresses more stem/fibroblast genes and is largely YFP+ (Figure 4A). Further supporting the identity as an intermediate or transitional cell population, KEGG pathway analysis of the transitional cluster showed it to be less contractile than mature SMC clusters but more contractile than the fibroblast/AdvSca1-SM clusters (Figure 4B). These data support the reprogramming of YFP– mature SMC toward a stemlike AdvSca1-SM phenotype, consistent with our original discovery of AdvSca1-SM cells (14). To confirm this, we exposed SMC lineage tracing mice (Myh11-CreERT/Rosa26-YFP) to tamoxifen to label SMC, and we then put them on the same atherogenic regimen as the AdvSca1-SM lineage tracing mice. We found that SMC-derived YFP+ cells were found in the vascular adventitia, where they coexpressed SCA1, indicating the transition of a mature, contractile SMC into multipotent progenitor cells (Figure 4C). Interestingly, we also observed YFP– cells (i.e., non-SMC–derived) within the media of the vessel (
Comments (0)