Commentary on Cutting Edge Science Macrophage Proliferation in Atherosclerosis An Historical Perspective Michael E. Rosenfeld

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t seems that every decade seminal observations related to the pathogenesis of atherosclerosis are revisited especially when new technology, animal models, or reagents are developed. This is the case with this study by Robbins et al1 from the Massachusetts General Hospital. These investigators have used a variety of powerful techniques to address the question of what roles macrophage proliferation and monocyte recruitment play in accounting for the numbers of macrophages within the intima of atherosclerotic plaques in mice at different stages of lesion development. This study makes an important new contribution because it provides quantitative evidence that monocyte recruitment followed by differentiation into macrophages predominates at early stages of lesion development in mice, whereas macrophage proliferation is predominant at later stages. The authors started by continuously infusing the thymidine analogue, bromodeoxyuridine (BrDU) into 4-month-old apolipoprotein E–deficient (ApoE−/−) mice on a high cholesterol diet. After enzyme digestion of the aorta, they measured the BrDU incorporation into macrophages (Lin− CD11b+ CD11c−/low F4/80high cells) by flow cytometry and found that 92% of the macrophages were labeled. They corroborated these finding by also analyzing 4′,6-diamidino-2-phenylindole staining for S/G2/M phase cells, H3 histone phosphorylation, and positive immunostaining for another proliferation marker Ki67 in tissue sections. Depletion of circulating monocytes had no effect on BrDU incorporation, suggesting that monocyte recruitment was not playing a role in replenishing the pool of proliferating macrophages in the established lesions. They analyzed this further using the classical technique of parabiosis. They joined the circulations of diet-fed 4-month-old CD45.1+ ApoE−/− mice with established lesions with CD45.2+ ApoE−/− mice for 5 weeks. They found low levels (5%) of macrophage chimerism in the aortic lesions. Separation of the parabionts also had no effect on the number of macrophage chimeras in the established plaques. In contrast, when they joined 8-week-old mice that had not been fed the high cholesterol diet before parabiosis and measured chimerism 4 weeks later, the percent chimerism was similar for circulating monocytes and the macrophages within the early lesions indicating that monocyte recruitment was the likely source of the macrophages in the early lesions. As it was From the Departments of Pathology and Environmental and Occupational Health Sciences, University of Washington, Seattle. Correspondence to Michael E. Rosenfeld, PhD, Departments of Pathology and Environmental and Occupational Health Sciences, Box 358050, University of Washington, Seattle, WA. E-mail [email protected] (Arterioscler Thromb Vasc Biol. 2014;34:e21-e22.) © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.114.303379

not clear to what extent circulating monocytes replenish the plaque macrophages during the long term, they transplanted the bone marrow from CD45.1+ ApoE−/− mice into 4-month-old CD45.2+ ApoE−/− mice. By 5 months later, the CD45.1+ cells had replenished many of the macrophages within the plaques. Intriguingly, they also provided evidence that the type 1 scavenger receptor A (Msr1) seems to play a role in mediating macrophage proliferation in the mouse lesions. They transplanted irradiated 8-week-old Ldlr−/− (low-density lipoprotein receptor deficient) mice with a mixture of WT CD45.1+ and Msr1−/− CD45.2+ bone marrow cells and after BrdU infusion they compared the proliferation of Msr1+/+ and Msr1−/− macrophages at 26 weeks of age. Surprisingly, there were considerably fewer BrdU+ Msr1−/− macrophages in the lesions. Macrophage proliferation in atherosclerotic lesions is not a new concept. In 1948, McMillan and Duff2 reported on mitotic activity in foam cells in atherosclerotic lesions in cholesterolfed rabbits. In the 1960s, Spraragen et al3 and McMillan and Stary4 went on to apply the technique of 3H-thymidine autoradiography to provide more definitive data that cellular proliferation was an active process within the rabbit lesions. Shortly thereafter, thymidine incorporation was further documented in lesions from rabbits,5,6 swine,7 Rhesus monkeys,8 and humans.9 In the 1980s, several of us at the University of Washington applied newly developed cell type–specific monoclonal antibodies (RAM-11 and HAM-56) to definitively demonstrate that the labeled cells were both macrophages and smooth muscle cells.10–12 In studies of Watanabe Heritable Hyperlipemic (WHHL) and comparably hypercholesterolemic fat–fed rabbits, we combined the techniques of immunocytochemistry and thymidine autoradiography on single sections to identify which cell types were proliferating.10 We found that 30% of the labeled cells were definitively macrophages. In keeping with the observations of Robbins et al for a possible role for the type A scavenger receptor and lipid accumulation in mediating the macrophage proliferation, we also found that many of the labeled macrophages in the rabbit lesions were foam cells and that lipid accumulation regardless of whether it was from endogenous (WHHL) or exogenous (fat-fed) sources did not compromise macrophage proliferation. However, in contrast to the conclusions of Robbins et al, we observed that the early lesions in the rabbits had more macrophage proliferation (percentage of total cells) than more advanced lesions. Concurrently, Gordon et al11 and Katsuda et al12 simultaneously applied the cell type–specific antibodies along with a monoclonal antibody to the proliferating cell nuclear antigen to demonstrate macrophage proliferation in human coronary and aortic lesions. These results were later corroborated by additional studies.13–17 Of particular note was a study by Lutgens et al17 on autopsy specimens of the descending aorta. These investigators used the proliferation marker Ki67 and the macrophage

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e22   Arterioscler Thromb Vasc Biol   October 2014 marker CD68 and in keeping with our studies of rabbits they reported that early American Heart Association type II lesions had the highest frequency of macrophage proliferation. A major limitation of the study by Robbins et al is that it is difficult to extrapolate their observations to human atherosclerosis. Normal human muscular arteries have an intima (often referred to as diffuse intimal thickening) that contains resident macrophages.18 Normal mouse arteries do not have an intima. Thus in the mouse, monocyte recruitment leads to the formation of the intima and must predominate at early stages. In the human, it may be that resident macrophages are also induced to proliferate as part of the early inflammatory response and that macrophage proliferation contributes more to expansion of early lesions in humans than it would in mice. In fact, cells in human diffuse intimal thickenings have been shown to express proliferation markers.16 Additional unanswered questions are to what degree the macrophage proliferation actually contributes to lesion progression rather than just replenishment of the macrophage population and to what degree the scavenger receptor status regulates the proliferation. This is underscored by the controversy that exists as to whether the Msr1 plays a role in lesion development as there has been contradictory evidence that knockout or overexpression of the Msr1 affects lesion area and composition in several different mouse models.19–25 Nevertheless, the major strength of the studies of Robbins et al is that based on several different and simultaneously used quantitative approaches, they have conclusively demonstrated that macrophage proliferation contributes to maintaining the macrophage population of mouse lesions. Furthermore, this study has refocused our attention on the importance of macrophage proliferation in atherosclerosis and has for the first time provided quantitative evidence that at certain stages of the disease, macrophage proliferation may be a predominant mechanism supporting the chronic inflammatory response that is characteristic of atherosclerosis.

Disclosures None.

References 1. Robbins CS, Hilgendorf I, Weber GF, et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat Med. 2013;19:1166–1172. 2. McMillan GC, Duff GL. Mitotic activity in the aortic lesions of experimental atherosclerosis in rabbits. Arch Pathol 1948;46:179–182. 3. Spraragen SC, Bond VP, Dahl LK. Role of hyperplasia in vascular lesions of cholesterol-fed rabbits studied with thymidine-H3 autoradiography. Circ Res. 1962;11:329–336. 4. McMillan GC, Stary HC. Preliminary experience with mitotic activity of cellular elements in the atherosclerotic plaques of cholesterol-fed rabbits studied by labeling with tritiated thymidine. Ann N Y Acad Sci. 1968;149:699–709. 5. Cavallero C, Turolla E, Ricevuti G. Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits. 1. Colchicine and (3H)thymidine studies. Atherosclerosis. 1971;13:9–20. 6. Stary HC, McMillan GC. Kinetics of cellular proliferation in experimental atherosclerosis. Radioautography with grain counts in cholesterol-fed rabbits. Arch Pathol. 1970;89:173–183.

7. Florentin RA, Nam SC, Lee KT, Lee KJ, Thomas WA. Increased mitotic activity in aortas of swine after three days of cholesterol feeding. Arch Pathol. 1969;88:463–469. 8. Stary HC. Proliferation of arterial cells in atherosclerosis. Adv Exp Med Biol. 1974;43:59–81. 9. Villaschi S, Spagnoli LG. Autoradiographic and ultrastructural studies on the human fibro-atheromatous plaque. Atherosclerosis. 1983;48:95–100. 10. Rosenfeld ME, Ross R. Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1990;10:680–687. 11. Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci USA. 1990;87:4600–4604. 12. Katsuda S, Coltrera MD, Ross R, Gown AM. Human atherosclerosis. IV. Immunocytochemical analysis of cell activation and proliferation in lesions of young adults. Am J Pathol. 1993;142:1787–1793. 13. Gown AM. Cell type and cell state specific antibodies in the analysis of early lesions of human atherosclerosis. Am J Hypertens. 1992;5(6 Pt 2):114S–117S. 14. Rekhter MD, Gordon D. Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques. Am J Pathol. 1995;147:668–677. 15. Brandl R, Richter T, Haug K, Wilhelm MG, Maurer PC, Nathrath W. Topographic analysis of proliferative activity in carotid endarterectomy specimens by immunocytochemical detection of the cell cycle-related antigen Ki-67. Circulation. 1997;96:3360–3368. 16. Orekhov AN, Andreeva ER, Mikhailova IA, Gordon D. Cell proliferation in normal and atherosclerotic human aorta: proliferative splash in lipidrich lesions. Atherosclerosis. 1998;139:41–48. 17. Lutgens E, de Muinck ED, Kitslaar PJ, Tordoir JH, Wellens HJ, Daemen MJ. Biphasic pattern of cell turnover characterizes the progression from fatty streaks to ruptured human atherosclerotic plaques. Cardiovasc Res. 1999;41:473–479. 18. Gown AM, Tsukada T, Ross R. Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191–207. 19. Suzuki H, Kurihara Y, Takeya M, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997;386:292–296. 20. Sakaguchi H, Takeya M, Suzuki H, Hakamata H, Kodama T, Horiuchi S, Gordon S, van der Laan LJ, Kraal G, Ishibashi S, Kitamura N, Takahashi K. Role of macrophage scavenger receptors in diet-induced atherosclerosis in mice. Lab Invest. 1998;78:423–434. 21. de Winther MP, Gijbels MJ, van Dijk KW, van Gorp PJ, suzuki H, Kodama T, Frants RR, Havekes LM, Hofker MH. Scavenger receptor deficiency leads to more complex atherosclerotic lesions in APOE3Leiden transgenic mice. Atherosclerosis. 1999;144:315–321. 22. Herijgers N, de Winther MP, Van Eck M, Havekes LM, Hofker MH, Hoogerbrugge PM, Van Berkel TJ. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knockout mice. J Lipid Res. 2000;41:1402–1409. 23. Van Eck M, De Winther MP, Herijgers N, Havekes LM, Hofker MH, Groot PH, Van Berkel TJ. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on cholesterol levels and atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 2000;20:2600–2606. 24. Kamada N, Kodama T, Suzuki H. Macrophage scavenger receptor (SR-A I/II) deficiency reduced diet-induced atherosclerosis in C57BL/6J mice. J Atheroscler Thromb. 2001;8:1–6. 25. Jalkanen J, Leppänen P, Närvänen O, Greaves DR, Ylä-Herttuala S. Adenovirus-mediated gene transfer of a secreted decoy human macrophage scavenger receptor (SR-AI) in LDL receptor knock-out mice. Atherosclerosis. 2003;169:95–103.

Key Words: Editorials ◼ atherosclerosis ◼ inflammation ◼ macrophages ◼ monocytes

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Macrophage Proliferation in Atherosclerosis: An Historical Perspective Michael E. Rosenfeld Arterioscler Thromb Vasc Biol. 2014;34:e21-e22; originally published online August 28, 2014; doi: 10.1161/ATVBAHA.114.303379 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

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