Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade Ying Wanga,b, Vivek Nandaa,b,c, Daniel Direnzoa, Jianqin Yea, Sophia Xiaoa, Yoko Kojimaa, Kathryn L. Howea, Kai-Uwe Jarra, Alyssa M. Floresa, Pavlos Tsantilasa, Noah Tsaoa, Abhiram Raob,d, Alexandra A. C. Newmane, Anne V. Eberharda, James R. Priestf, Arno Ruusaleppg, Gerard Pasterkamph,i, Lars Maegdefesselj,k, Clint L. Millerl,m,n, Lars Lindo, Simon Koplevp, Johan L. M. Björkegrenp, Gary K. Owense, Erik Ingelssonb,o,q, Irving L. Weissmanr,1, and Nicholas J. Leepera,b,1 aDivision of Vascular Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305; bStanford Cardiovascular Institute, Stanford University, Stanford, CA 94305; cDepartment of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, AL 35294; dDepartment of Bioengineering, Stanford University, Stanford, CA 94305; eRobert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22904; fDivision of Pediatric Cardiology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; gDepartment of Cardiac Surgery, Tartu University Hospital, Tartu, Estonia 50406; hDepartment of Cardiology, University Medical Center Utrecht, 3584CX Utrecht, the Netherlands; iLaboratory of Clinical Chemistry, University Medical Center Utrecht, 3584CX Utrecht, the Netherlands; jDepartment for Vascular and Endovascular Surgery, Klinikum Rechts der Isar, Technical University Munich, 80333 Munich, Germany; kGerman Center for Cardiovascular Research (DZHK partner site), 10785 Munich, Germany; lCenter for Public Health Genomics, Department of Public Health Sciences, University of Virginia, Charlottesville, VA 22904; mDepartment of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22904; nDepartment of Biomedical Engineering, University of Virginia, Charlottesville, VA 22904; oDepartment of Medical Sciences, Uppsala University, SE-751 05 Uppsala, Sweden; pDepartment of Genetics and Genomic Sciences, Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029-6574; qDivision of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; and rInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Irving L. Weissman, April 20, 2020 (sent for review April 8, 2020; reviewed by Joshua Beckman and Yogen Kanthi) Atherosclerosis is the process underlying heart attack and stroke. This is one of the reasons why all currently approved therapies Despite decades of research, its pathogenesis remains unclear. target risk factors for disease (e.g., hypertension, diabetes, and Dogma suggests that atherosclerotic plaques expand primarily via hyperlipidemia) rather than the cells that actually constitute the accumulation of cholesterol and inflammatory cells. However, the lesion. recent evidence suggests that a substantial portion of the plaque While there is little doubt that bone marrow-derived in- “ ” may arise from a subset of dedifferentiated vascular smooth flammatory cells home to the developing lesion (3, 5), emerging muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the ma- ture SMC can give rise to a hyperproliferative cell which appears to Significance promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with pro- Cardiovascular disease remains the world’s leading killer, de- phagocytic complement fragments, we find that this cell also es- spite the widespread use of cholesterol-lowering medicines. capes immune surveillance by neighboring macrophages, thereby Recent studies suggest a portion of this residual risk may result exacerbating its relative survival advantage. Mechanistic studies from the clonal expansion of cells within the atherosclerotic indicate this phenomenon results from a generalized opsonin- plaques of diseased blood vessels. How these cells promote sensing defect acquired by macrophages during polarization. This inflammation and whether they can be therapeutically tar- defect coincides with the noncanonical up-regulation of so-called geted remain unclear. The current study suggests that clonally don’t eat me molecules on inflamed phagocytes, which reduces expanding cells may cause disease by triggering the comple- their capacity for programmed cell removal (PrCR). Knockdown ment cascade while evading immune surveillance. However, or knockout of the key antiphagocytic molecule CD47 restores these cells also appear to be susceptible to therapies that the ability of macrophages to sense and clear opsonized targets reactivate phagocytic clearance pathways, suggesting a po- in vitro, allowing for potent and targeted suppression of clonal tential treatment approach for heart disease similar to current SMC expansion in the plaque in vivo. Because integrated clinical oncology efforts directed against the cancer stem cell. and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that Author contributions: Y.W., D.D., Y.K., J.R.P., L.M., G.K.O., E.I., and N.J.L. designed re- the clonally expanding SMC may represent a translational target search; I.L.W. guided experimental design; Y.W., V.N., D.D., J.Y., S.X., Y.K., K.L.H., K.-U.J., for treating atherosclerosis. A.M.F., P.T., N.T., A. Rao, A.A.C.N., A.V.E., J.R.P., G.P., L.M., J.L.M.B., E.I., and N.J.L. per- formed research; Y.W., A.A.C.N., J.R.P., A. Ruusalepp, G.P., L.M., C.L.M., L.L., S.K., J.L.M.B., G.K.O., E.I., I.L.W., and N.J.L. contributed new reagents/analytic tools; Y.W., V.N., D.D., atherosclerosis | clonality | smooth muscle cells | efferocytosis | CD47 J.Y., Y.K., K.L.H., K.-U.J., A.M.F., J.R.P., G.P., L.M., C.L.M., L.L., S.K., J.L.M.B., G.K.O., E.I., and N.J.L. analyzed data; I.L.W. provided feedback on data; and Y.W., K.-U.J., G.K.O., E.I., and N.J.L. wrote the paper. espite being the world’s leading killer (1), the root causes of Reviewers: J.B., Vanderbilt University Medical Center; and Y.K., University of Michigan. Datherosclerotic cardiovascular disease remain elusive (2, 3). Competing interest statement: I.L.W. and N.J.L. are cofounders of Forty Seven, Inc., an This is due in part to the fact that cells which contribute to immunooncology company. This company was recently acquired by Gilead Sciences; the growing atherosclerotic plaque frequently undergo extensive purchase did not include stock in Gilead. I.L.W. and N.J.L. do not currently have any phenotypic modulation, wherein they down-regulate the very consulting agreement with Gilead Sciences. “cell-specific” markers needed for their definitive identification Published under the PNAS license. 1 (4). This process is commonly referred to as “dedifferentiation” To whom correspondence may be addressed.Email:[email protected] or nleeper@ stanford.edu. in the field of vascular biology. As a result, there are major This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ambiguities regarding the origins of the individual components doi:10.1073/pnas.2006348117/-/DCSupplemental. of the plaque and how they contribute to disease pathogenesis. First published June 15, 2020. 15818–15826 | PNAS | July 7, 2020 | vol. 117 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.2006348117 Downloaded by guest on September 25, 2021 data suggest that it is a phenotypically-modulated (“dediffer- whether systemic C3 expression is associated with risk for coronary entiated”) cell in the vascular smooth muscle cell (SMC) lineage artery disease (CAD); 2) if the dedifferentiated SMC is a source of which may actually give rise to the majority of cells within the C3 in human plaque; and 3) if CAD patients harbor a C3- atherosclerotic plaque (6, 7). Sophisticated lineage-tracing studies producing cell that resembles the Sca1+ SMC identified above. revealed that the progeny of these cells have remarkable plasticity First, we measured circulating complement levels in 1,003 el- and can assume a wide variety of phenotypes. These range from derly individuals enrolled in the Prospective Investigation of the cells which resemble cap-stabilizing myofibroblasts (which are Vasculature in Uppsala Seniors (PIVUS) study (SI Appendix, likely beneficial), to cells previously assumed to represent bone Fig. S3A) (17). Consistent with prior reports (18), we found that marrow-derived macrophages (which are likely detrimental), and age- and sex-adjusted C3 levels were associated with endothelial- even to cells which express stem cell markers (whose role is un- dependent vasodilation (a marker of vascular function), carotid known) (6, 8–13). This final point is important because Benditt and intimal–medial thickness (CIMT; a measure of atherosclerotic colleagues reported a clonal origin of human atherosclerosis nearly plaque burden), and major adverse cardiovascular event rate 50 y ago (14), and recent studies revealed that murine SMCs can (MACE; a composite end point which includes incident heart expand in a clonal fashion under periods of stress (7, 8, 10). attack and stroke) (Fig. 2A). These associations were attenuated These data indicate that there may be a cell which resembles after adjusting for inflammatory comorbidities, such as body an “atherosclerotic