Local proliferation dominates lesional macrophage accumulation in atherosclerosis

Abstract

During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall. The observation that circulating monocytes give rise to lesional macrophages has reinforced the concept that monocyte infiltration dictates macrophage buildup. Recent work has indicated, however, that macrophage accumulation does not depend on monocyte recruitment in some inflammatory contexts. We therefore revisited the mechanism underlying macrophage accumulation in atherosclerosis. In murine atherosclerotic lesions, we found that macrophages turn over rapidly, after 4 weeks. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation through the involvement of scavenger receptor A (SR-A). Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.

Figure 1. Macrophages turn over rapidly in lesions

(a) Identification of aortic macrophages in established atherosclerosis. Contour plots demonstrate gating scheme for aortic macrophages in Apoe^−/− mice consuming HCD for 8 weeks. (b) BrdU-containing osmotic pumps were implanted in Apoe^−/− mice consuming HCD for 8 weeks. Data depict BrdU incorporation in aortic macrophages 1 and 4 weeks following implantation of osmotic pumps. (c) Percentage of aortic macrophages that incorporate BrdU. Shown is quantification of data in b as well as BrdU⁺ macrophages in mice where pumps were removed for 4 weeks (mean ± SEM, n = 8 (week 1), n = 3 (week 4), n = 2 (week 4 removal)). (d) Immunofluorescence (IF) shows BrdU⁺ Mac3⁺ macrophages in aortic root lesions. (e) IF showing co-localization of BrdU and DAPI in intimal and adventitial macrophages of aortic root sections following 4 weeks of BrdU administration; 10x magnification. (f) Enumeration of BrdU⁺ Mac3⁺ macrophages by IF in aortic root sections (mean ± SEM, n = 2–3). * P < 0.05. (g) Lesion growth during BrdU labeling period. (h) Lesional macrophage content during BrdU labeling period. Data show area staining for Mac-3.

Figure 2. Lesional macrophage accumulation occurs largely independent of monocyte recruitment

(a) Effect of repeated clodronate liposome administration on peripheral blood monocytes (mean ± SEM, n = 4). (b) Effect of clodronate liposome treatment on BrdU incorporation by aortic macrophages. Data show percentage of BrdU⁺ macrophages in aortic tissue following 5 d clodronate administration (mean ± SEM, n = 6–9). (c) Data show number of BrdU⁺ macrophages in aortic tissue following 5 d clodronate treatment (mean ± SEM, n = 6–9). (d) Data show total number of macrophages in aortic tissue following 5 d clodronate treatment (mean ± SEM, n = 6–9). (e) CD45.1⁺ and CD45.2⁺ Apoe^−/− HCD mice were joined in parabiosis for 5 weeks. Data show Ly-6C^high monocyte chimerism in the blood, spleen, and aorta and macrophage chimerism in the aortic tissue (mean ± SEM; n = 6–8). (f) Representative contour plots demonstrate monocyte and macrophage chimerism in aortic tissue. (g) Immunohistochemistry (IHC) of aortic root sections showing CD45.1 and CD45.2 staining in CD45.1⁺ parabionts. (h) CD45.1⁺ and CD45.2⁺ Apoe^−/− HCD mice were joined in parabiosis for 5 weeks, separated and assessed for chimerism 2 weeks later. Shown is chimerism for monocytes in the blood, spleen, and aorta as well as macrophages in the aorta (mean ± SEM; n = 4–8).* P < 0.05.

Figure 3. In situ proliferation dominates macrophage accumulation in atherosclerosis

(a) Data show colony-forming units–granulocytes and macrophages (CFU-GM) in spleen, bone marrow and aortic tissue of Apoe^−/− HCD mice (mean ± SEM, n = 3). * P < 0.05. (b) BrdU incorporation following adoptive transfer of GFP⁺ Ly6C^high monocytes. One representative experiment is shown. (c) BrdU pulse labeling of aortic macrophages (mean ± SEM, n = 7). (d) Cell cycle analysis of aortic tissue macrophages. Histograms depict DAPI staining (mean ± SEM, n = 7). * P < 0.05. (e) Percentage of aortic tissue macrophages in S and G2/M phases of the cell cycle. (mean ± SEM, n = 3–7). * P < 0.05. (f) Phospho-histone H3 staining of aortic macrophages in G2/M phases of the cell cycle. (g) Analysis of aortic macrophages by ImageStreamX^™ imaging flow cytometry platform. Data depict actively dividing macrophages. (h) IF of aortic root sections demonstrating Ki-67 staining of Mac3⁺ intimal macrophages. (i) CD45.1⁺ and CD45.2⁺ Apoe^−/− HCD mice were joined in parabiosis and then implanted with BrdU containing osmotic pumps. Data show chimerism in newly made (BrdU⁺) aortic tissue macrophages. One representative experiment of two is shown. Monocyte chimerism in peripheral blood is also shown. (j) Relative contribution of in situ proliferation and monocyte recruitment to macrophage accumulation in early (2–3 month old) and established (4–5 months old) atherosclerosis.

Figure 4. Lesion microenvironment dictates macrophage proliferation

(a) WT (CD45.1⁺ C57BL6/J) and CD45.2⁺ Apoe^−/− HCD mice were joined in parabiosis for 5 weeks. Data show DAPI staining in CD45.1⁺ and CD45.2⁺ aortic tissue macrophages in each parabiont. One representative experiment of 3 is shown. (b) Enumeration of data in a. (mean ± SEM, n = 8–9). * P < 0.05. (c) Cell cycle analysis of intimal versus adventitial aortic macrophages. (d) WT/Msr1^−/−mixed chimeras were generated by reconstituting lethally irradiated Ldlr^−/− mice with CD45.1⁺ wild type (Msr1^+/+) and CD45.2⁺ Msr1–deficient (Msr1^−/−) bone marrow cells. (e) Data show number of BrdU⁺ Msr1^+/+ (WT) and BrdU⁺ Msr1^−/−Ly6C^high monocytes in blood 2 h following BrdU pulse labeling. (mean ± SEM, n = 5). (f) Percentage of WT and Msr1^−/− aortic macrophages that are BrdU⁺. (mean ± SEM, n = 5).* P < 0.05. (g) Number of BrdU⁺ Msr1^+/+and BrdU⁺ Msr1^−/− macrophages in aortic tissue of WT/Msr1^−/− mixed chimeras. (mean ± SEM, n = 5).* P < 0.05. (h) Cartoon depicting macrophage expansion in established atherosclerosis.