Skip to main content
  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
  • Impact Factor 13.965
  • Facebook
  • Twitter

  • My alerts
  • Sign In
  • Join

  • Advanced search

Header Publisher Menu

  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

Circulation Research

  • My alerts
  • Sign In
  • Join

  • Impact Factor 13.965
  • Facebook
  • Twitter
  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
Cellular Biology

Characterization of a Resident Population of Adventitial Macrophage Progenitor Cells in Postnatal VasculatureNovelty and Significance

Peter J. Psaltis, Amrutesh S. Puranik, Daniel B. Spoon, Colin D. Chue, Scott J. Hoffman, Tyra A. Witt, Sinny Delacroix, Laurel S. Kleppe, Cheryl S. Mueske, Shuchong Pan, Rajiv Gulati, Robert D. Simari
Download PDF
https://doi.org/10.1161/CIRCRESAHA.115.303299
Circulation Research. 2014;115:364-375
Originally published June 6, 2014
Peter J. Psaltis
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Amrutesh S. Puranik
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Daniel B. Spoon
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Colin D. Chue
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Scott J. Hoffman
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tyra A. Witt
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sinny Delacroix
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Laurel S. Kleppe
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Cheryl S. Mueske
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shuchong Pan
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rajiv Gulati
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert D. Simari
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (P.J.P., A.S.P., D.B.S., C.D.C., S.J.H., T.A.W., S.D., L.S.K., C.S.M., S.P., R.G., R.D.S.); Monash Cardiovascular Research Centre, Monash University, Clayton, Victoria, Australia (P.J.P.); Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia (P.J.P., S.D.); and Kansas University Medical Center, The University of Kansas, Kansas City (R.D.S).
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Tables
  • Supplemental Materials
  • Info & Metrics

Jump to

  • Article
    • Abstract
    • Introduction
    • Methods
    • Results
    • Discussion
    • Acknowledgments
    • Sources of Funding
    • Disclosures
    • Footnotes
    • References
  • Figures & Tables
  • Supplemental Materials
  • Info & Metrics
  • eLetters
Loading

Abstract

Rationale: Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal.

Objective: We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate.

Methods and Results: Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1+CD45+ cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67+) Lin−c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b− subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1+CD45+ adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE−/− and LDL-R−/− mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries.

Conclusions: The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage progenitors in other tissues.

  • atherosclerosis
  • cells
  • leukocytes
  • macrophages
  • monocyte–macrophage precursor cells
  • progenitor cells

Introduction

Members of the mononuclear phagocyte system (MPS), including macrophages and dendritic cells, mediate innate immunity and coordinate the regulation of inflammation. In the vasculature, cells of the MPS regulate vascular form and function in health and disease, highlighted by their role in atherosclerosis. While intimal macrophages are considered as a source of foam cells in atheroma,1 adventitial macrophages also initiate plaque formation via stimulation of angiogenesis2 and participate in the pathogenesis of inflammatory vasculopathies and aneurysm formation.3 Although the existing paradigm of macrophage origins in vascular disease emphasizes recruitment of bone marrow (BM)– or splenic-derived monocytes via the circulation,1 recent data suggest that in established atheroma, macrophage burden may be maintained predominantly by local proliferation rather than circulatory monocyte renewal.4 This new observation fits within an emerging understanding that diversity of tissue macrophage subsets may be aligned with different developmental origins.5,6

Classically activated, inflammatory macrophages are generally considered to derive from circulating monocytes, which descend from hematopoietic stem and progenitor cells (HPCs). In adult murine BM, Lin−Sca-1−c- Kit+/IntCD135+CD115+CX3CR1+ macrophage/dendritic cell progenitors (MDPs) have been identified as the common precursor for circulating monocytes and their macrophage and dendritic cell progeny,7 with recent evidence placing Lin−c-Kit+CD135−CD115+Ly6C+CD11b− common monocyte progenitors (cMoPs) as the link between MDPs and monocytes.8 Despite this hierarchy of BM monocyte–macrophage ancestry, the origin of some tissue macrophages, including those belonging to the alternatively activated pathway, is less clearly defined. During prenatal development, F4/80Bright macrophages arise in different organs through a process that originates in the yolk sac before the onset of definitive hematopoiesis.9 Postnatally, the renewal, maintenance, and proliferation of certain tissue-resident macrophages occur locally, largely autonomous of BM hematopoiesis and circulatory monocyte recruitment.6,10–12 To date, this has been attributed to a remarkable capacity for mature tissue macrophages to undergo self-renewal, enabling them to proliferate indefinitely without loss of functional differentiation, through uncoupling of differentiation and cell cycle withdrawal by the inactivation of specific transcription factors.13 However, another explanation for the local proliferation and turnover of postnatal tissue macrophages, previously untested, is the existence of tissue-resident macrophage progenitor cells. With increasing recognition that postnatal blood vessels contain resident populations of diverse progenitor cell populations,14 we recently identified that murine arteries contain clonogenic cells of hematopoietic significance.15 Here, we phenotypically, transcriptionally, and functionally characterize a population of resident adventitial macrophage progenitor cells (AMPCs) within healthy and atherosclerotic vasculature and determine their noncirculatory origins, as well as their capacity to generate macrophage and dendritic cell progeny.

Methods

Detailed methods are provided in the Online Data Supplement. Single-cell tissue disaggregates were prepared from mice belonging to C57BL/6, ApoE−/−, LDL-R−/−, green fluorescent protein (GFP), and op/op (C3Fe a/a-Csf1op/CSF1op) strains under age and dietary conditions specified below. Hematopoietic colony-forming unit (CFU) assays, flow cytometry analysis, BM and splenic cell adoptive transfer experiments, and tissue immunostaining were performed using techniques described previously.15 Magnetic-activated cell sorting was used to fractionate freshly isolated aortic disaggregates into subpopulations based on expression of stem cell antigen-1 (Sca-1 or Ly6A) and the pan-leukocyte marker, CD45. These different aortic cell subsets were compared for (1) clonogenicity and cell cycle activity, (2) transcriptional profiling by RNA microarray and quantitative reverse transcription polymerase chain reaction validation, (3) expression of surface markers associated with macrophage ancestry in BM, and (4) long-term in vivo fate after carotid artery adventitial transfer to atheroprone recipients. All experiments complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD) and were approved by the Mayo Clinic Institutional Animal Care and Use Committee.

Statistical Analysis

Comparisons were performed with parametric or nonparametric unpaired or paired 2-sample t tests or ANOVA (with post-test comparisons), as appropriate. Results are expressed as mean±SEM of multiple experiments, unless otherwise specified. In all cases, statistical significance was established at 2-tailed P<0.05.

Results

Adventitial Sca-1+CD45+ Cells Contain Macrophage Progenitors

To understand the context and specificity of the hematopoietic potential of aorta, short-term CFU assays were performed on freshly isolated, single-cell preparations of various hematopoietic and nonhematopoietic adult C57BL/6 murine tissues. As described previously,15 great care was taken to avoid peripheral blood contamination of aortic extracts, and this was verified in numerous ways, including the use of flow cytometry (Online Figure I).16 Enzymatic digestion of full-length aorta from 12-week-old C57BL/6 mice yielded 1.5 to 2×106 cells. Although all tissues tested generated CFUs to a varying degree, aortic cells were distinguished by a robust hematopoietic CFU capacity, which was notable for their distinct predisposition to generate lineage-specific macrophage colonies (CFU-M) at a mean frequency of 13.7±1.4 per 105 aortic cells plated (n=11) or ≈200 to 300 per aorta (Figure 1A). In addition to fulfilling conventional morphological criteria, the MPS-enriched content of aortic CFU assays was supported by their substantial upregulation of macrophage and dendritic cell marker expression after 14 days of culture in methylcellulose, with absence of granulocytes (Figure 1B). Unlike aorta, CFUs generated from BM, peripheral blood, and spleen comprised much higher proportions of multilineage CFU-GM (granulocyte–macrophage) and -GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte), along with CFU-G (granulocyte) and BFU-E (burst forming units-erythroid).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

Enrichment of macrophage colonies within the aorta’s adventitial Sca-1+CD45+ compartment. A, Total and macrophage (M) colony-forming unit (CFU) counts from different tissues of 12-week-old C57BL/6 mice (n=8–13). B, Representative flow cytometry density plots of aortic CFU progeny after 14 days of methylcellulose culture showing absence of Gr-1 expression and abundance of CD11b+F4/80Hi (macrophage) and CD11b+CD11c+ (dendritic cell) staining. C, Representative flow cytometry plots of aortic disaggregates for stem cell antigen-1 (Sca-1) and CD45 expression with IgG isotype control staining also shown. D, Immunofluorescent detection of Sca-1+CD45+ coexpression in the adventitia of 12-week-old C57BL/6 aortic root. The low magnification image is stained with Hoechst for nuclei, with the yellow dotted line indicating the external elastic lamina and the inset box corresponding to the higher magnification images. Adv indicates adventitia. Scale bars: 20 μm (white) and 5 μm (yellow). Pie chart shows mean expression of the different Sca-1(S)/CD45(45) subsets in C57BL/6 aortic root adventitia from tissue immunostaining of 30 sections, 5 mice. E, CFU frequency from different aortic Sca-1/CD45 fractions. Unf indicates unfractionated cells. n=4 to 8 different experiments per fraction, each performed from ≥6 C57BL/6 mice. *P<0.05, †P<0.01, ‡P<0.001 for comparison with Sca-1+CD45+ cells. Also see Online Figure III. F, Representative images of small (<100 cells) and large (>100 cells) CFU-M, with quantification of the proportion of large CFU-M from unfractionated and Sca-1+CD45+ cultures (n=7–10). G, Content of monocytes within the aortic Sca-1+CD45+ and Sca-1−CD45+ fractions as assessed by flow cytometry (n=11). H, Nuclear Ki67 expression determined for different aortic cell subsets by flow cytometry. Total indicates all aortic cells. n=8 C57BL/6 mice. §P<0.0001 for comparison of Sca-1+CD45+ cells vs each other population. I, Proportion of cells in different aortic subpopulations that were in S or G2/M phases of cell cycle, assessed by flow cytometry of bromodeoxyuridine incorporation. n=8. P<0.0001. Also see Online Figure VI. For all graphical data, mean±SEM is shown, and *P<0.05, †P<0.01, ‡P<0.001, §P<0.0001.

Previously, we established that arterial capacity for CFU formation is predominantly contained within the adventitial subpopulation of cells expressing Sca-1.15 As others have reported that adventitial Sca-1+Lineage−CD45− cells possess smooth muscle differentiation potential but lack hematopoietic clonogenicity,17,18 we focused on the previously unstudied population of adventitial Sca-1+CD45+ cells. By flow cytometry, CD45 expression was present on 35.9±4.7% of Sca-1+ cells from C57BL/6 aortas (n=15; Figure 1C). Coexpression of Sca-1 and CD45 in healthy murine aorta was independently confirmed by tissue immunofluorescence and shown to be predominantly contained within the adventitia (Figure 1D, Online Figure II). Magnetic separation of the Sca-1+CD45+ subset of aortic cells resulted in 4-fold enrichment in hematopoietic CFU yield (Figure 1E, Online Figure III). These CFUs were exclusively CFU-M, which were more commonly of larger size than those from unfractionated cultures (Figure 1F). Exposure of Sca-1+CD45+ cells to either macrophage colony-stimulating factor (M-CSF) or interleukin-4 and GM colony-stimulating factor augmented CFU-M recovery (Online Figure IVA) and facilitated in vitro differentiation toward macrophage (CD115+F4/80+; Online Figure IVB) or dendritic cell (CD11c+) phenotypes (Online Figure IVC), respectively.

By comparison to aortic Sca-1+CD45+ cells, hematopoietic colony formation was nearly absent from the Sca-1+CD45− fraction, consistent with previous depictions that these cells harbor smooth muscle and not hematopoietic progenitor capacity.17,18 Critically, colony formation was also 16-fold less from resident Sca-1−CD45+ cells, with 25% of these colonies comprising CFU-GM and -GEMM. The much lower yield of CFU-M from Sca-1−CD45+ cells was in spite of this population containing a higher frequency of Lin−CD11bHiF4/80Lo monocytes than the Sca-1+CD45+ subset (Figure 1G). Along with the fact that CFU-M were generated at low frequency from peripheral blood (Figure 1A), this provided initial evidence that the cells responsible for arterial macrophage colonies are not monocytes, consistent with recent data showing the nonmonocyte origins of tissue-resident macrophages.9,11,12 This was supported in separate experiments in which freshly disaggregated aortic cells were subjected to magnetic sorting on the basis of their expression of a panel of lineage markers, which included the monocyte/macrophage antigen CD11b, along with CD5, CD19, CD45R, Gr-1, Ter119. CFU-M were almost exclusively retrieved from the lineage-depleted fraction, with negligible CFU-M recovery from the lineage-positive cells which included CD11b+ monocytes and macrophages (Online Figure V).

Together with augmented CFU-forming ability, the adventitial Sca-1+CD45+ compartment also contained the highest proportion of actively cycling cells in aorta, as corroborated by Ki67 staining analyzed by flow cytometry of aortic disaggregates (Figure 1H) and tissue immunostaining (Online Figure VI), and bromodeoxyuridine incorporation (Figure 1I, Online Figure VI). The mean rate of apoptosis, as determined by annexin V and 7-amino-actinomycin D (7AAD) uptake, was ≈10% for aortic Sca-1+CD45+ cells and did not differ significantly with aging (Online Figure VII). Thus, aortic adventitia is enriched in macrophage progenitors that reside in the population of proliferative Sca-1+CD45+ cells.

Genomic and Phenotypic Characterization of Aortic Sca-1+CD45+ Cells

To further understand the innate differences among aortic cell populations according to their Sca-1 and CD45 expression, we performed, analyzed, and validated nonbiased transcriptional profiles of disaggregated aortic cells. Unsupervised clustering analysis of genome-wide expression arrays indicated that the combined presence of Sca-1 and CD45 was associated with extensive differences in the transcriptional profile of aortic cells (Figure 2A–2C, Online Table I). There were 44 genes that were upregulated in Sca-1+CD45+ cells compared with both the Sca-1−CD45+ (resident leukocytes) and Sca-1+CD45− (smooth muscle progenitor) fractions, including those that encode Csf2 (granulocyte–macrophage colony-stimulating factor), Il5 (interleukin-5), Ccl1 (chemokine [C-C motif] ligand 1), Cxcl12 (chemokine [C–X–C motif] ligand 12), and Gata3. Many other genes were differentially regulated between Sca-1+CD45+ cells and each of the other 2 populations, with 879 and 552 genes upregulated by a factor of ≥2-fold compared with Sca-1−CD45+ and Sca-1+CD45− cells, respectively (Figure 2C). These covered a broad range of biological pathways, including hematopoiesis, inflammation, innate and acquired immunity, and extracellular matrix metabolism, all of which are central to vascular maintenance and disease (Figure 2D and 2E). Quantitative reverse transcription polymerase chain reaction validation of 12 of these gene products provided a Spearman r coefficient of 0.712 (P<0.01) with the microarray (Online Table II).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

RNA microarray profiling of different stem cell antigen-1 (Sca-1)/CD45 fractions of aortic cells. Volcano plots (A) and heat map (B) from RNA microarray showing differential gene expression between Sca-1+CD45+, Sca-1−CD45+ and Sca-1+CD45− aortic cells. C, Unsupervised clustering analysis of genome-wide expression arrays indicated that the combined presence of stem cell antigen-1 (Sca-1) and CD45 was associated with extensive differences in the genomic imprint of aortic cells compared with Sca-1−CD45+ and Sca-1+CD45− cells, respectively. D and E, Histograms from Ingenuity analysis depict the top 10 biological pathways that had differential gene regulation between aortic Sca-1+CD45+ cells and Sca-1−CD45+ cells (D) and between aortic Sca-1+CD45+ cells and Sca-1+CD45− cells (E).

We used flow cytometry to characterize the hematopoietic surface markers of these subpopulations. Compared with the other Sca-1/CD45 subpopulations in adult C57BL/6 aorta, a higher percentage of Sca-1+CD45+ cells expressed CX3CR1, CD115 (c-fms), and c-Kit (CD117), which are established markers of MDPs and cMoPs in BM7,8 (Figure 3A, Online Figure VIII). Further immunophenotypic characterization was then performed to examine for MDP-like (Lin−c-Kit+CD135+CD115+CX3CR1+)7 and cMoP-like (Lin−c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b−)8 surface marker profiles within the aortic Sca-1+CD45+ fraction, where the panel of lineage markers used was CD31, NK1.1, CD3, CD19, CD11c, Ly6G, Ter119, and a live-dead marker. Although expression of the MDP-like markers accounted for <5 per 100 000 aortic cells, the aortic Sca-1+CD45+ gate was found to contain a well-delineated subpopulation of cMoP-like cells (Figure 3B). The frequency of these Sca-1+CD45+Lin−c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b− cells in C57BL/6 aorta was in the order of 15 to 30 per 100 000 cells, which approximated the frequency of CFU-M from unfractionated aortic cell culture (see Figure 1A). This subpopulation contained a high proportion of Ki67+ cells (69.6±3.2%, n=9 mice), consistent with a proliferative progenitor (as distinct from quiescent stem cell) phenotype (Figure 3B).

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

Immunophenotypic profiling of aortic Sca-1+CD45+ cells for macrophage ancestral markers. Flow cytometry gating strategies used on C57BL/6 aortic disaggregates first involved selecting single cells and excluding cell debris. A, Representative flow cytometry density plots show the expression of c-Kit, CX3CR1, and CD115 on gated Sca-1+CD45+ cells. Accompanying graphs compare expression of these markers on different stem cell antigen-1 (Sca-1)/CD45 gates from C57BL/6 aorta. n=6 mice. §P<0.0001. B, C57BL/6 aorta contained a subset of Lin−Sca-1+CD45+ cells with the c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b− phenotype recently used to identify common monocyte progenitors (cMoPs) in bone marrow.8 This subfraction contained a high percentage of proliferating Ki67+ cells (density plots are representative of n=9 mice). FSC-A indicates forward scatter-area; FSC-H, forward scatter-height; MDP, macrophage/dendritic cell progenitor; and SSC-A, side scatter-area. For other IgG isotype controls, see Online Figure VIII.

Collectively, the above data indicate the distinctive genotypic and phenotypic profiles portended by the dual expression of Sca-1 and CD45 on aortic cells. The CFU-M–enriched Sca-1+CD45+ fraction expresses immunophenotypic markers consistent with macrophage ancestry and includes a small subfraction of proliferative Lin−c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b− cells that has been identified as cMoP-like.

Maintenance of Aortic CFU-M Progenitors Is Not From Circulating Cells

Our data suggest that a key difference between macrophage ancestors in BM and spleen and CFU-M progenitors in aorta is that the latter express Sca-1, as this marker is known to be downregulated and ultimately lost with progression of BM hematopoiesis to lineage specification.7 This prompted us to investigate whether postnatal aortic Sca-1+CD45+ cells and their CFU-M progeny are maintained by circulatory trafficking from BM or spleen. In the first instance, we noted that isolation of Sca-1+CD45+ cells from BM and spleen yielded completely different CFU profiles that lacked the CFU-M specificity of the Sca-1+CD45+ fraction in aorta (Online Figure IX). Then BM or splenic cells from GFP donors were transferred intravenously to lethally irradiated C57BL/6 mice. In both sets of adoptive transfer experiments, there was rapid reconstitution of blood with GFP+ cells (85.8±2.8% at 2 weeks and 94.4±0.6% at 12 weeks; n=8). By 16 weeks, chimerism of GFP+ cells in whole aorta was 15.9±3.8%, contributing to 42.9±6.9% of the aorta’s Sca-1+CD45+ compartment but to few CD45− cells (Figure 4A, Online Figure X). As consistent with our previous experience,15 recipient aortas demonstrated diminished CFU yield (total CFU at 16 weeks, 1.9±0.4 per 105 unfractionated aortic cells) compared with the nonirradiated state (Figure 1A), indicating radiosensitivity. These aortic colonies remained predominantly CFU-M (920/982; 93.7%), with the majority still recovered from Sca-1+CD45+ cells (796/920; 86.5%). Despite nearly complete GFP reconstitution of blood monocytes, donor GFP+ cells contributed to only 3.3% (30/920) of all aortic CFU-M and 1.0% (8/796) of CFU-M generated from aortic Sca-1+CD45+ cells (n=8 experiments; Figure 4A, Online Figure XC). Therefore, although circulating cells contributed to the aorta’s Sca-1+CD45+ population, those Sca-1+CD45+ cells responsible for CFU-M capacity were of host origin and not the result of recruitment to the vessel wall from BM- or splenic-derived cells. In contrast, 90.3% (56/62) of nonmacrophage colonies, including CFU-GM and -GEMM that were mostly generated from aortic Sca-1− cells, were of donor GFP+ source. These results indicate mixed sources of hematopoietic CFUs in aorta. Circulatory surveillance of BM and splenic hematopoietic stem cells/HPCs is largely responsible for the aorta’s rare content of CFU-granulocyte, -GM, -GEMM, and burst-forming units–erythroid, consistent with the previous model proposed by Massberg et al.19 In contrast, Sca-1+CD45+ CFU-M progenitors, which are much more prevalent in aorta, are inefficiently reconstituted from BM and spleen, suggesting that they may be maintained locally.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

Maintenance of aortic Sca-1+CD45+ macrophage colony-forming unit (CFU-M) progenitors occurs independently of bone marrow (BM) hematopoietic progenitor cells (HPCs), blood monocytes, and aortic macrophages. A, top left, Adoptive transfer of BM cells was performed from green fluorescent protein (GFP) donors to irradiated C57BL/6 recipients with aortic harvest at 16 weeks. Bottom left, Graphs show percentage of GFP+ chimerism in different aortic stem cell antigen-1 (Sca-1)(S)/CD45(45) fractions (left) and the total yield of CFUs from different aortic Sca-1/CD45 subpopulations (right). n=4 experiments, each performed using 6 recipient aortas. Top right, Table shows the numbers of total macrophage (M) and nonmacrophage (non-M) CFUs from different aortic Sca-1/CD45 fractions in these experiments, along with the number and percentage of GFP+ colonies for each Sca-1/CD45 fraction and CFU subtype. Bottom right, Representative flow cytometry density plot of CD11b and F4/80 staining in aorta 16 weeks after BM cell transfer, with CD11b+F4/80Hi macrophages gated by red circle. Adjacent density plot depicts GFP+ (donor) chimerism for the gated macrophage population, with mean±SEM % donor chimerism shown from n=6. B, C57BL/6 mice were treated for 30 days with liposomal clodronate or vehicle control (n=6 each group). Despite resulting in marked reduction of circulating Lin−CD11b+ monocytes (density plots), this was accompanied by a small increase in aortic CFU-M. C and D, Aortas were harvested from osteopetrotic (op/op) mice and their wild-type (WT) littermates (age, 16–21 days). Graphs compare (C) their respective frequencies of monocytes, macrophages, and Sca-1+CD45+ cells (n=9 per group) and (D) their yield of total hematopoietic CFUs and different CFU subtypes (n=6 per group). Graphs summarize mean±SEM values. *P<0.05, †P<0.01, ‡P<0.001, §P<0.0001 (compared with Sca-1+CD45+ fraction in [A]). BFU-E indicates burst-forming units–erythroid; CFU-G, CFU-granulocyte; FSC, forward scatter; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM, granulocyte–macrophage; and SSC, side scatter.

Importantly, in these experiments we also observed that aortic CD11b+F4/80Hi macrophages were equally of both donor and recipient origin 16 weeks after irradiation and adoptive transfer (49.4±5.5% GFP+; Figure 4A). This suggested the contribution of a local source to aortic macrophages, as has been shown recently for resident macrophages in other organs.12 At the same time, it also provided additional evidence to dismiss the possibility that macrophages in the vessel wall were responsible for generating CFU-M, because 96.7% of these colonies were GFP− (Figure 4A, Online Figure XC).

To further corroborate that aortic Sca-1+CD45+ CFU-M progenitors cannot be attributed to the recruitment of circulating monocytes or the presence of proliferative macrophages in the vessel wall, we studied 2 different models of monocyte/macrophage depletion: (1) liposomal clodronate treatment4 (Figure 4B) and (2) osteopetrotic (op/op) mice that are genetically deficient in M-CSF20 (Figure 4C and 4D, Online Figure XI). Previously, clodronate treatment has been shown to result in virtual disappearance of circulating monocytes within 24 hours.4 We repeatedly administered liposomal clodronate to adult C57BL/6 mice for 30 days, because this time period exceeds the interval thought to be required for macrophage turnover in postnatal murine tissues, including atherosclerotic aorta.4,12 Despite resulting in marked reduction of circulating monocytes, clodronate treatment was in fact associated with a small but significant increase in the frequency of CFU-M generated from aortic cell cultures (Figure 4B), providing more evidence that blood-derived monocytes are not the source of aortic CFU-M progenitors.

In comparison with wild-type littermates, aortas from weaning-age M-CSF–deficient op/op mice had preserved numbers of Sca-1+CD45+ cells (Figure 4C) and generated similar frequencies of CFU-M under standard colony outgrowth conditions (ie, without addition of M-CSF; Figure 4D), despite the expected reduction in their frequency of circulating monocytes, as well as monocytes and macrophages in spleen (Online Figure XI) and aorta (Figure 4C). These results affirm that the aorta’s capacity to produce CFU-M is independent of cells that are reduced in op/op mice, namely circulating monocytes and monocytes and macrophages that are resident in the aortic wall. Op/op mice also had markedly diminished CFU yield from BM attributable to osteopetrosis, but enhanced hematopoiesis in spleen, resulting from displacement of hematopoietic stem cells and HPCs from BM (Online Figure XI). Interestingly, yield of nonmacrophage colonies, especially CFU-GM and -GEMM, was increased from op/op aortas (Figure 4D), demonstrating that extramedullary hematopoiesis impacted the frequency of multilineage HPCs in aorta but not macrophage-specific progenitors.

Aortic Sca-1+CD45+ Macrophage Progenitors Are Upregulated in Atherosclerosis

Hyperlipidemia has been shown to have a stimulatory effect on BM hematopoietic stem cells/HPCs, providing potential mechanistic insight into leukocyte upregulation in atherosclerotic vessels.21 However, recent evidence has suggested a switch in the predominant source of macrophages in atheroma of ApoE−/− mice from circulating monocytes in early lesion development to local macrophage proliferation in established intimal lesions.4 By comparison with age-matched C57BL/6 mice, we observed an increase in the frequency of Sca-1+CD45+ cells from disaggregates of whole aorta from 5-month-old ApoE−/− mice, especially in those fed 12 weeks of Western diet (WD; Figure 5A, Online Figure XII). The aortas of these hyperlipidemic mice also contained increased adventitial levels of M-CSF, as determined by quantitative ELISA (Online Figure XIIB), along with increases in the generation of CFU-M (1.8-fold for chow diet ApoE−/− relative to C57BL/6; 2.3-fold for WD ApoE−/− relative to C57BL/6; Figure 5B), but not other CFU subtypes (data not shown). Similar results were observed for LDL-R−/− mice (Online Figure XIII).

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5.

Sca-1+CD45+ macrophage progenitor cells in atherosclerosis. Frequencies of (A) Sca-1+CD45+ cells determined by flow cytometry and (B) macrophage colony-forming units (CFU-M) from disaggregates of whole aorta from 5-month-old C57BL/6, chow diet (CD)– and Western diet (WD)–fed ApoE−/− mice, 12 weeks after splenectomy or sham surgery (n≥6 per group). C, Relative content of different leukocyte subsets in aortas of splenectomized WD ApoE−/− mice (n=8) compared with sham counterparts (n=6). DCs indicates dendritic cells; Monos, monocytes; and Mϕs, macrophages. D, Immunostaining of WD ApoE−/− aortic root showing at low magnification the presence of Sca-1+ cells within the media and atheroma plaque (Pl). Yellow dotted lines indicate the external and internal elastic lamina. Inset box corresponds to the adjacent high magnification images showing an Sca-1+CD45+ cell within plaque. Graph depicts quantitative summary of the prevalence of stem cell antigen-1 (Sca-1) and CD45 coexpression in atheroma, adventitia (Adv), and the combination of both from 22 aortic root sections, 5 mice. E, CFU-M yield from disaggregates of Adv or remaining vessel wall (non-Adv) after dissection of WD ApoE−/− aortas (n=7). F, Low- and high-power images show predominantly adventitial distribution of cycling Ki67+ cells from WD ApoE−/− aortic root, with (G) quantitative summary from n=14 sections, 3 mice. H, Percentage frequency of Ki67+ expression among all cells counted in atheroma and adventitia of these sections. I, Pie charts summarize the distribution of Ki67 staining among the different Sca-1/CD45 subsets in atheroma, adventitia, and both. Also see Online Movie I. J, Confirmation of the cycling nature of Sca-1+CD45+ cells in WD ApoE−/− aorta by flow cytometry assessment of bromodeoxyuridine incorporation (n=10). For tissue immunostaining, nuclei are counterstained blue with Hoechst. Scale bars: 20 μm (white) and 5 μm (yellow). Graphs summarize mean±SEM values. *P<0.05, †P<0.01, ‡P<0.001, §P<0.0001.

Because the spleen has been implicated as an important reservoir from which proinflammatory monocytes are recruited to generate macrophages in atherosclerosis22,23 and myocardial infarction,24 we also interrogated the frequency of CFU-M progenitor cells in aortas of splenectomized mice. Neither CFU-M, Sca-1+CD45+, macrophage nor dendritic cell frequencies were found to be diminished in healthy, atheroprone or atherosclerotic aortas, 3 months after splenectomy, despite the anticipated reduction that splenectomy caused to aortic monocyte content (Figure 5A–5C). These data are consistent with those from our splenic cell adoptive transfer studies (Online Figure X) in verifying that the spleen is not a significant source of aortic macrophage progenitors via supply of circulating HPCs or monocytes.

We next set out to define the distribution of local macrophage progenitor cells during murine atherosclerosis development. In aortas from C57BL/6 and prelesional chow diet ApoE−/−mice, Sca-1+CD45+ cells were almost exclusively contained within the adventitia. After 12 weeks of WD, ApoE−/− and LDL-R−/− aortas displayed transmural distribution of Sca-1+CD45+ cells, also involving media and lesional neointima (Figure 5D, Online Figures XII and XIII). Despite the presence of Sca-1+CD45+ cells and macrophages in neointimal plaque, the great majority of CFU-M from WD ApoE−/− aortas were still derived from adventitia, as determined when the adventitia was separated by microscopic dissection from the remaining vessel wall before digestion and culture (Figure 5E). Quantification of Ki67 nuclear expression in sections of atheroma-containing aortic root revealed that almost 75% of proliferating (Ki67+) cells were located within the adventitia, the confines of which we defined as being within 5 cell layers from the external elastic lamina (Figure 5F and 5G). Of the 11.0±1.0% of adventitial cells that were Ki67+ (Figure 5H), a majority were Sca-1+CD45+ (63.7±5.4% from 14 sections, 3 mice; Figure 5I, Online Movie I). In contrast, only 5.3±1.3% of cells in atheroma itself were Ki67+ (P<0.001 versus adventitia), with 65.2±11.8% of these being Sca-1−CD45+. Overall, microscopic quantification of Ki67 staining for aortic root revealed that the highest proportion of actively cycling cells in WD ApoE−/− aorta was contained within the Sca-1+CD45+ population, followed by Sca-1−CD45+ cells (Figure 5I). In keeping with the predominantly adventitial distribution of CFU-M progenitors in WD aorta, a higher percentage of adventitial Sca-1+CD45+ cells were proliferative than was the case for Sca-1+CD45+ cells in plaque (15.4±1.5% versus 1.8±0.6%; P<0.0001). By flow cytometry assessment of bromodeoxyuridine incorporation in disaggregates of whole aorta from WD ApoE−/− mice, 19.8±1.2% of Sca-1+CD45+ cells were in the S or G2/M phases of cycle (n=10; Figure 5J). Taken together, these observations reveal spatial diversity and heterogeneity of Sca-1+CD45+ cells within atherosclerotic aorta, highlighting that the majority of clonogenic and proliferative Sca-1+CD45+ macrophage progenitors do in fact remain in the adventitia.

Sca-1+CD45+ AMPCs Have Durable MPS Cell Fate

Finally, to interrogate the in vivo fate of Sca-1+CD45+ cells within atherosclerotic arteries, we isolated cells by Sca-1 and CD45 expression from aortas of GFP mice and injected each fraction into carotid artery adventitia of recipient 8-week-old ApoE−/− mice (Figure 6A). WD was then administered for 16 weeks, during and after which GFP+ donor cells were not detectable systemically (Figure 6B). At study end, donor-derived cells were identified in the injected artery of all recipients of Sca-1+CD45+ cells (n=7 mice; Figure 6C) to a greater extent than was the case 48 hours after their administration (Figure 6A). From all carotid artery sections examined from GFP+Sca-1+CD45+ recipients, 89.4% (583/652) of GFP+ cells were located in adventitia and 10.6% (69/652) in plaque (Figure 6D), although the latter was increased to 18.6% (69/371) in sections that actually contained atheroma. Of the cells enumerated in atheroma, 7.5±3.5% of cells were GFP+, and 22.6±4.4% of all cells in adventitia were GFP+ (Figure 6E). Phenotypic analysis revealed active cycling (Ki67+) of some donor-derived cells (Online Figure XIV), as well as expression of macrophage (F4/80, monocyte/macrophage antibody-2 [MOMA-2]) and dendritic cell (CD11c) markers (Figure 6F). By comparison, GFP was absent after injection of cell-free matrigel and was rarely identified after transfer of Sca-1−CD45+ or Sca-1+CD45− cells (Figure 6C). Although this in vivo model revealed the striking capacity of Sca-1+CD45+ cells to engraft after allogeneic transfer and provide long-term MPS progeny in their new environment, our findings suggest that the major contribution of these progenitors is to the maintenance of the adventitial, rather than neointimal, macrophage compartment, which is known to expand in atherosclerosis and play an important regulatory role in pathogenic processes, such as vasa vasorum formation.2

Figure 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 6.

Fate of Sca-1+CD45+ cells in hyperlipidemic arteries. A, Confocal images show initial adventitial retention of GFP+Sca-1+CD45+ cells 2 days after injection to left carotid artery of 8-week-old ApoE−/− mouse. Ad indicates adventitia. B, Flow cytometry histograms indicate absence of GFP+ chimerism in blood or bone marrow (BM) 12 weeks after adventitial delivery of GFP+Sca-1+CD45+ cells. Dotted histograms correspond to GFP− controls. C, Immunostaining for green fluorescent protein (GFP) expression in carotid artery sections 16 weeks after Western diet and delivery of matrigel only, GFP+Sca-1−CD45+, GFP+Sca-1+CD45−, or GFP+Sca-1+CD45+ cells. In the 2 sections from GFP+Sca-1+CD45+ cell transfer, higher magnification images of the inset boxes show donor-derived cells in adventitia (Adv) in No. 1 and atheroma plaque (Pl) in No. 2. c/l indicates contralateral (right) carotid artery; and L, lumen. D, Pie chart depicts the percentage distribution of GFP+Sca-1+CD45+–derived cells between adventitia and atheroma in recipient carotid arteries (28 sections, 7 mice). E, Quantification of the percentage of cells in atheroma and adventitia that were donor-derived (GFP+). More than 3500 nuclei assessed in all. *P<0.05. F, Costaining for GFP with F4/80, MOMA-2, and CD11c after delivery of GFP+Sca-1+CD45+ cells, showing that these cells made a durable contribution to macrophage and dendritic cell progeny in recipient carotid adventitia. Representative IgG control staining is also shown. Nuclei are counterstained blue with Hoechst. Scale bars: 50 μm (orange), 20 μm (white), and 10 μm (yellow).

Discussion

Prevailing concepts of macrophage development recognize that there are diverse origins for different cellular subtypes of the MPS.5 In particular, growing evidence indicates that circulating monocytes, and by extension BM MDPs, are not the sole source of resident macrophages in many postnatal tissues.6,9,11,12 Although recent emphasis has focused on the capacity of mature macrophages to undergo self-renewal and local proliferation,4,13 an alternative inference of these observations is that there may exist local niches of tissue-resident, nonmedullary macrophage progenitor cells that persist through adult life. To address this, the current study used murine aorta as a template for the interrogation of tissue macrophage progenitor cells.

Using hematopoietic CFU assays, along with a combination of genetic, phenotypic, and functional analyses, we discovered the existence of a distinct population of clonogenic adventitial progenitors that coexpress Sca-1 and CD45, are strongly predisposed to generate MPS cells, and lack granulocyte potential. A significant proportion of these cells are actively cycling in situ at any given time, under both healthy and atherosclerotic conditions. Consistent with recent evidence on the local origins of postnatal tissue macrophages, these AMPCs are not the result of circulatory trafficking of HPCs. Just as importantly, multiple lines of evidence suggest that their existence cannot be interpreted as the proliferation of monocytes recruited from the peripheral blood or the self-renewal and rapid turnover of resident mature macrophages. First, others have shown that proliferative monocytes8 and mature macrophages from wild-type mice are not capable of forming CFU-M.25 Second, although of variable size, the CFU-M generated by aortic Sca-1+CD45+ cells frequently comprise hundreds to thousands of cells and in prior adoptive transfer experiments have displayed remarkable long-lasting and self-renewing properties, months after primary and secondary transplantation.15 Third and fundamentally, in the present study, lineage-positive aortic cells, including those that expressed CD11b, showed minimal capacity to generate CFU-M unlike their Lin− counterparts, while the frequency of CFU-M was 16 times lower from Sca-1−CD45+ cells than Sca-1+CD45+ cells, despite higher monocyte content in the former. Fourth, neither clodronate-treated C57BL/6 mice nor M-CSF–deficient mice displayed diminished aortic CFU-M yield. Fifth, in adoptive BM and splenic transfer experiments, recipient mice had ≈95% chimeric reconstitution of circulating monocytes with an almost equal mixture of donor- and recipient-derived macrophages in aorta, yet produced only 3.3% donor-derived aortic CFU-M. Sixth, aortic CFU-M were not diminished in splenectomized mice despite their reduced vessel wall content of monocytes and Ly6CHi monocytes. Finally, despite the presence of macrophages in atherosclerotic neointima, the CFU-M capacity of aortas from WD ApoE−/− mice was predominantly contained within the adventitia.

Unlike previous work reporting the presence of arterial Sca-1+CD45− smooth muscle progenitor cells,17 our data draw attention to the fact that a considerable proportion of adventitial Sca-1+ cells express CD45 and it is within this highly heterogeneous fraction that macrophage clonogenicity is primarily contained. These cells are transcriptionally and functionally distinct from their neighboring CD45-negative counterparts. As is the case for proliferative, self-renewing microglia in the central nervous system (CNS),10 our results in the op/op mice indicate that the presence of Sca-1+CD45+ AMPCs is not dependent on M-CSF, although we have shown that this factor does facilitate their ability to generate CFU-M and differentiate to macrophages. Intriguingly, the presence of Sca-1 on the surface of vascular AMPCs directly contravenes classical hierarchical depictions of murine BM hematopoiesis, where Sca-1 is absent from lineage-committed myeloid precursor cells, including granulocyte–macrophage progenitors and MDPs.7 However, in keeping with the profiles ascribed to Sca-1− BM MDPs and cMoPs,8 this study demonstrates that the adventitial Sca-1+CD45+ population does contain cells that express c-Kit, CX3CR1, and CD115 and moreover a small subset of cMoP-like Lin−c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b− cells, which are highly proliferative and whose frequency approximates the yield of CFU-M from healthy aortas. Isolation of these rare cells from fresh aortic disaggregates by multicolor fluorescence-activated cell sorting to test their clonogenicity has thus far proven extremely challenging because of difficulties in preserving cell viability. However, it remains an important objective to further delineate the specific identify of the clonogenic AMPC subpopulation moving forward.

Although our adoptive transfer model may have been confounded by the effects of irradiation, it demonstrated that BM and splenic HPCs contribute minimally to the presence of CFU-M progenitors in postnatal aorta. Even with compromised clonal capacity after irradiation, 96.7% CFU-M and 50% of aortic macrophages were still host-derived 16 weeks after BM or splenic cell transfer, suggesting substantial local contribution. It is presumed that these macrophages were generated in the vessel wall after irradiation and were not already pre-existent there, because recent profiling of tissue and vascular macrophage kinetics has shown substantial turnover within 21 to 28 days.4,12 Future studies dedicated to investigating the BM independence of AMPCs and macrophage responses in healthy and diseased vasculature will require molecular fate-mapping strategies, similar to those used to investigate the origins of other tissue macrophages.6,9,12

Circulating monocytes and their BM-derived progenitor cells have long been considered the source of intimal macrophages and foam cells in atheroma,1 whereas the spleen has also been recognized as an intermediate reservoir of monocytes.22 The present study reveals that in both mild and severe hyperlipidemia, aortic frequency of Sca-1+CD45+ expression and CFU-M yield progressively increase and this is not affected by surgical removal of the spleen and its accompanying reduction in vessel wall monocyte content. In lesional ApoE−/− and LDL-R−/− sections, we observed substantial proliferation along the adventitial aspect of the external elastic lamina, especially within the Sca-1+CD45+ population. Interestingly, although Sca-1/CD45 costaining breaches the adventitia in the setting of atheroma to involve the other mural layers, the progenitors responsible for CFU-M are still mostly adventitial. Taken together with the findings from our BM transplant studies, it is apparent that there is great diversity in the biological properties and origins of Sca-1+CD45+ cells in the vessel wall. We conclude that the vast majority of Sca-1+CD45+ cells in atheroma are neither AMPCs nor the result of adventitial-to-neointimal translocation of AMPC progeny, but are more likely to be leukocytes and lymphocytes that have originated from the circulation. The results of our carotid adventitial transfer model show that donor Sca-1+CD45+ cells, but not the other aortic subsets, generated macrophages and dendritic cells 16 weeks after delivery, although these were predominantly adventitial. This fits with the proliferative, clonogenic nature of Sca-1+CD45+ AMPCs, which are located to be able to contribute to the presence of MPS populations found in the adventitia of healthy arteries,16 as well as the rapid expansion of adventitial macrophages that occurs during the development of atherosclerosis and aneurysms, where these cells also help regulate growth of pathogenic vasa vasorum.2,26,27 We are currently focusing on the proangiogenic properties of Sca-1+CD45+ AMPCs, which we hypothesize may underlie one of their major pathophysiological functions during arterial wall remodeling. Although our results indicate that adventitial progenitors may contribute to only a small proportion of neointimal leukocytes, alternative model systems, such as molecular fate-mapping and arterial graft interposition, will be required before definitively excluding a greater role for AMPCs as a source of locally maintained macrophages in atheroma.4

In summary, we have identified a population of Sca-1+CD45+ macrophage progenitor cells that reside in postnatal murine vascular adventitia. The definition of these resident progenitors provides a new context to consider the role of the adventitia in vascular disease, including atherosclerosis. Moreover, it provides a novel alternative paradigm to account for the local proliferation and turnover of tissue macrophages, advancing our understanding of the vessel wall as a source of tissue-resident macrophages and their progenitors more broadly.

Acknowledgments

We thank Megan Crouch, James Tarara, and Holly Lamb and staff of the Flow Cytometry Core Facility, Mayo Clinic, Rochester, MN.

Sources of Funding

P.J. Psaltis is funded by a Post-doctoral Overseas Biomedical Fellowship from the National Health and Medical Research Council of Australia. A.S. Puranik is funded by the James Nutter Family & Maria Long Family Fellowship in Cardiology at the Mayo Clinic.

Disclosures

None.

Footnotes

  • In May 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.87 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.303299/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    AMPC
    adventitial macrophage progenitor cell
    BM
    bone marrow
    CFU
    colony-forming unit
    CFU-M
    macrophage colony-forming unit
    cMoP
    common monocyte progenitor
    GEMM
    granulocyte, erythrocyte, monocyte, megakaryocyte
    GM
    granulocyte–macrophage
    HPC
    hematopoietic progenitor cell
    M-CSF
    macrophage colony-stimulating factor
    MDP
    macrophage/dendritic cell progenitor
    MPS
    mononuclear phagocyte system
    WD
    Western diet

  • Received December 20, 2013.
  • Revision received May 18, 2014.
  • Accepted June 6, 2014.
  • © 2014 American Heart Association, Inc.

References

  1. 1.↵
    1. Moore KJ,
    2. Tabas I
    . Macrophages in the pathogenesis of atherosclerosis. Cell. 2011;145:341–355.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Moulton KS,
    2. Vakili K,
    3. Zurakowski D,
    4. Soliman M,
    5. Butterfield C,
    6. Sylvin E,
    7. Lo KM,
    8. Gillies S,
    9. Javaherian K,
    10. Folkman J
    . Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A. 2003;100:4736–4741.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Daugherty A,
    2. Manning MW,
    3. Cassis LA
    . Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest. 2000;105:1605–1612.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Robbins CS,
    2. Hilgendorf I,
    3. Weber GF,
    4. et al
    . Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat Med. 2013;19:1166–1172.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Geissmann F,
    2. Manz MG,
    3. Jung S,
    4. Sieweke MH,
    5. Merad M,
    6. Ley K
    . Development of monocytes, macrophages, and dendritic cells. Science. 2010;327:656–661.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Yona S,
    2. Kim KW,
    3. Wolf Y,
    4. Mildner A,
    5. Varol D,
    6. Breker M,
    7. Strauss-Ayali D,
    8. Viukov S,
    9. Guilliams M,
    10. Misharin A,
    11. Hume DA,
    12. Perlman H,
    13. Malissen B,
    14. Zelzer E,
    15. Jung S
    . Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38:79–91.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Auffray C,
    2. Fogg DK,
    3. Narni-Mancinelli E,
    4. et al
    . CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation. J Exp Med. 2009;206:595–606.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Hettinger J,
    2. Richards DM,
    3. Hansson J,
    4. Barra MM,
    5. Joschko AC,
    6. Krijgsveld J,
    7. Feuerer M
    . Origin of monocytes and macrophages in a committed progenitor. Nat Immunol. 2013;14:821–830.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Schulz C,
    2. Gomez Perdiguero E,
    3. Chorro L,
    4. Szabo-Rogers H,
    5. Cagnard N,
    6. Kierdorf K,
    7. Prinz M,
    8. Wu B,
    9. Jacobsen SE,
    10. Pollard JW,
    11. Frampton J,
    12. Liu KJ,
    13. Geissmann F
    . A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science. 2012;336:86–90.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Ginhoux F,
    2. Greter M,
    3. Leboeuf M,
    4. Nandi S,
    5. See P,
    6. Gokhan S,
    7. Mehler MF,
    8. Conway SJ,
    9. Ng LG,
    10. Stanley ER,
    11. Samokhvalov IM,
    12. Merad M
    . Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–845.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Jenkins SJ,
    2. Ruckerl D,
    3. Cook PC,
    4. Jones LH,
    5. Finkelman FD,
    6. van Rooijen N,
    7. MacDonald AS,
    8. Allen JE
    . Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science. 2011;332:1284–1288.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Hashimoto D,
    2. Chow A,
    3. Noizat C,
    4. et al
    . Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity. 2013;38:792–804.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Sieweke MH,
    2. Allen JE
    . Beyond stem cells: self-renewal of differentiated macrophages. Science. 2013;342:1242974.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Psaltis PJ,
    2. Harbuzariu A,
    3. Delacroix S,
    4. Holroyd EW,
    5. Simari RD
    . Resident vascular progenitor cells–diverse origins, phenotype, and function. J Cardiovasc Transl Res. 2011;4:161–176.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Psaltis PJ,
    2. Harbuzariu A,
    3. Delacroix S,
    4. Witt TA,
    5. Holroyd EW,
    6. Spoon DB,
    7. Hoffman SJ,
    8. Pan S,
    9. Kleppe LS,
    10. Mueske CS,
    11. Gulati R,
    12. Sandhu GS,
    13. Simari RD
    . Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta. Circulation. 2012;125:592–603.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Galkina E,
    2. Kadl A,
    3. Sanders J,
    4. Varughese D,
    5. Sarembock IJ,
    6. Ley K
    . Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med. 2006;203:1273–1282.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Hu Y,
    2. Zhang Z,
    3. Torsney E,
    4. Afzal AR,
    5. Davison F,
    6. Metzler B,
    7. Xu Q
    . Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest. 2004;113:1258–1265.
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Passman JN,
    2. Dong XR,
    3. Wu SP,
    4. Maguire CT,
    5. Hogan KA,
    6. Bautch VL,
    7. Majesky MW
    . A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1+ smooth muscle progenitor cells. Proc Natl Acad Sci U S A. 2008;105:9349–9354.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Massberg S,
    2. Schaerli P,
    3. Knezevic-Maramica I,
    4. Köllnberger M,
    5. Tubo N,
    6. Moseman EA,
    7. Huff IV,
    8. Junt T,
    9. Wagers AJ,
    10. Mazo IB,
    11. von Andrian UH
    . Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues. Cell. 2007;131:994–1008.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Cecchini MG,
    2. Dominguez MG,
    3. Mocci S,
    4. Wetterwald A,
    5. Felix R,
    6. Fleisch H,
    7. Chisholm O,
    8. Hofstetter W,
    9. Pollard JW,
    10. Stanley ER
    . Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development. 1994;120:1357–1372.
    OpenUrlAbstract
  21. 21.↵
    1. Murphy AJ,
    2. Akhtari M,
    3. Tolani S,
    4. Pagler T,
    5. Bijl N,
    6. Kuo CL,
    7. Wang M,
    8. Sanson M,
    9. Abramowicz S,
    10. Welch C,
    11. Bochem AE,
    12. Kuivenhoven JA,
    13. Yvan-Charvet L,
    14. Tall AR
    . ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice. J Clin Invest. 2011;121:4138–4149.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Robbins CS,
    2. Chudnovskiy A,
    3. Rauch PJ,
    4. et al
    . Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions. Circulation. 2012;125:364–374.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Swirski FK,
    2. Libby P,
    3. Aikawa E,
    4. Alcaide P,
    5. Luscinskas FW,
    6. Weissleder R,
    7. Pittet MJ
    . Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata. J Clin Invest. 2007;117:195–205.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Dutta P,
    2. Courties G,
    3. Wei Y,
    4. et al
    . Myocardial infarction accelerates atherosclerosis. Nature. 2012;487:325–329.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Aziz A,
    2. Soucie E,
    3. Sarrazin S,
    4. Sieweke MH
    . MafB/c-Maf deficiency enables self-renewal of differentiated functional macrophages. Science. 2009;326:867–871.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Rateri DL,
    2. Howatt DA,
    3. Moorleghen JJ,
    4. Charnigo R,
    5. Cassis LA,
    6. Daugherty A
    . Prolonged infusion of angiotensin II in apoE(-/-) mice promotes macrophage recruitment with continued expansion of abdominal aortic aneurysm. Am J Pathol. 2011;179:1542–1548.
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Blomkalns AL,
    2. Gavrila D,
    3. Thomas M,
    4. et al
    . CD14 directs adventitial macrophage precursor recruitment: role in early abdominal aortic aneurysm formation. J Am Heart Assoc. 2013;2:e000065.
    OpenUrlAbstract/FREE Full Text

Novelty and Significance

What Is Known?

  • In some adult tissues, resident macrophages seem to be maintained locally, without contribution from circulating monocytes.

  • Recent evidence suggests that this may also apply for macrophages in established atheroma.

  • The adventitia of arteries is a niche for different progenitor cell populations.

What New Information Does This Article Contribute?

  • The adventitia of postnatal murine aorta contains clonogenic macrophage progenitor cells that are Sca-1+CD45+.

  • These adventitial macrophage progenitor cells (AMPCs) are independent of circulating monocytes and are not replenished by bone marrow or spleen-derived hematopoietic progenitor cells.

  • AMPCs have increased prevalence in atherosclerotic aorta, where they remain predominantly adventitial, contributing macrophages to adventitia and to a lesser extent to atheroma.

Recent evidence suggests that some macrophages are not derived from bone marrow hematopoietic progenitor cells or circulating monocytes; instead, they are maintained locally, supposedly by self-renewal. Our study identifies that among different murine tissues, aorta has a robust and augmented capacity for forming macrophage colonies that arise from an adventitial population of proliferative Sca-1+CD45+ cells. These clonogenic cells are neither monocytes nor adventitial macrophages, nor are they reconstituted by circulating hematopoietic progenitor cells. AMPCs are therefore a distinct, local progenitor cell population, resident in the vessel wall. AMPCs are more abundant in the setting of atherosclerosis, providing macrophage progeny to the adventitia of atherosclerotic arteries, while also contributing to a smaller extent to atheroma cell burden. These results identify AMPCS as a new cellular participant in arterial remodeling, building on the important but underappreciated role of adventitia in vascular homeostasis and disease. Our findings suggest novel mechanisms of disease and potentially identify a new target for therapy.

View Abstract
Back to top
Previous ArticleNext Article

This Issue

Circulation Research
July 18, 2014, Volume 115, Issue 3
  • Table of Contents
Previous ArticleNext Article

Jump to

  • Article
    • Abstract
    • Introduction
    • Methods
    • Results
    • Discussion
    • Acknowledgments
    • Sources of Funding
    • Disclosures
    • Footnotes
    • References
  • Figures & Tables
  • Supplemental Materials
  • Info & Metrics

Article Tools

  • Print
  • Citation Tools
    Characterization of a Resident Population of Adventitial Macrophage Progenitor Cells in Postnatal VasculatureNovelty and Significance
    Peter J. Psaltis, Amrutesh S. Puranik, Daniel B. Spoon, Colin D. Chue, Scott J. Hoffman, Tyra A. Witt, Sinny Delacroix, Laurel S. Kleppe, Cheryl S. Mueske, Shuchong Pan, Rajiv Gulati and Robert D. Simari
    Circulation Research. 2014;115:364-375, originally published June 6, 2014
    https://doi.org/10.1161/CIRCRESAHA.115.303299

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
  •  Download Powerpoint
  • Article Alerts
    Log in to Email Alerts with your email address.
  • Save to my folders

Share this Article

  • Email

    Thank you for your interest in spreading the word on Circulation Research.

    NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

    Enter multiple addresses on separate lines or separate them with commas.
    Characterization of a Resident Population of Adventitial Macrophage Progenitor Cells in Postnatal VasculatureNovelty and Significance
    (Your Name) has sent you a message from Circulation Research
    (Your Name) thought you would like to see the Circulation Research web site.
  • Share on Social Media
    Characterization of a Resident Population of Adventitial Macrophage Progenitor Cells in Postnatal VasculatureNovelty and Significance
    Peter J. Psaltis, Amrutesh S. Puranik, Daniel B. Spoon, Colin D. Chue, Scott J. Hoffman, Tyra A. Witt, Sinny Delacroix, Laurel S. Kleppe, Cheryl S. Mueske, Shuchong Pan, Rajiv Gulati and Robert D. Simari
    Circulation Research. 2014;115:364-375, originally published June 6, 2014
    https://doi.org/10.1161/CIRCRESAHA.115.303299
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects

  • Basic, Translational, and Clinical Research
    • Mechanisms
    • Pathophysiology
    • Cell Biology/Structural Biology

Circulation Research

  • About Circulation Research
  • Editorial Board
  • Instructions for Authors
  • Abstract Supplements
  • AHA Statements and Guidelines
  • Permissions
  • Reprints
  • Email Alerts
  • Open Access Information
  • AHA Journals RSS
  • AHA Newsroom

Editorial Office Address:
3355 Keswick Rd
Main Bldg 103
Baltimore, MD 21211
CircRes@circresearch.org

Information for:
  • Advertisers
  • Subscribers
  • Subscriber Help
  • Institutions / Librarians
  • Institutional Subscriptions FAQ
  • International Users
American Heart Association Learn and Live
National Center
7272 Greenville Ave.
Dallas, TX 75231

Customer Service

  • 1-800-AHA-USA-1
  • 1-800-242-8721
  • Local Info
  • Contact Us

About Us

Our mission is to build healthier lives, free of cardiovascular diseases and stroke. That single purpose drives all we do. The need for our work is beyond question. Find Out More about the American Heart Association

  • Careers
  • SHOP
  • Latest Heart and Stroke News
  • AHA/ASA Media Newsroom

Our Sites

  • American Heart Association
  • American Stroke Association
  • For Professionals
  • More Sites

Take Action

  • Advocate
  • Donate
  • Planned Giving
  • Volunteer

Online Communities

  • AFib Support
  • Garden Community
  • Patient Support Network
  • Professional Online Network

Follow Us:

  • Follow Circulation on Twitter
  • Visit Circulation on Facebook
  • Follow Circulation on Google Plus
  • Follow Circulation on Instagram
  • Follow Circulation on Pinterest
  • Follow Circulation on YouTube
  • Rss Feeds
  • Privacy Policy
  • Copyright
  • Ethics Policy
  • Conflict of Interest Policy
  • Linking Policy
  • Diversity
  • Careers

©2018 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. The American Heart Association is a qualified 501(c)(3) tax-exempt organization.
*Red Dress™ DHHS, Go Red™ AHA; National Wear Red Day ® is a registered trademark.

  • PUTTING PATIENTS FIRST National Health Council Standards of Excellence Certification Program
  • BBB Accredited Charity
  • Comodo Secured