Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6Chigh monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe−/− mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6Chigh monocytes and macrophages. SYK inhibition limited Ly6Chigh monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe−/− mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0535-8) contains supplementary material, which is available to authorized users.

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Background Guidelines recommend cholesterol lowering for primary and secondary prevention of cardiovascular disease. While lipid lowering has been reported to induce plaque regression, the underlying mechanisms have remained speculative. Purpose We hypothesize that lipid uptake triggers local macrophage proliferation in the plaque, and conversely, statin treatment inhibits local macrophage proliferation leading to plaque regression. Methods Mixed bone marrow chimeras were generated in LDLR−/− mice reconstituted with wild type and scavenger receptor deficient or cholesterol exporter deficient bone marrow cells to study cell autonomous effects on macrophage proliferation. APOE*3-Leiden.huCETP mice with established atherosclerosis were randomized to three groups: Continued cholesterol diet, cholesterol diet supplemented with 0.01% atorvastatin, and cholesterol free diet for 4 weeks to study mechanisms of plaque regression. Results Proliferation of scavenger receptor A and CD36 deficient macrophages with impaired lipid uptake was reduced by 30–50% in the plaque, while ABCA1/ABCG1 exporter deficiency resulted in cholesterol overloading and apoptosis. Oral atorvastatin treatment decreased total plasma cholesterol levels by 50% to the same extend as cholesterol free diet feeding in APOE*3-Leiden.huCETP. Cholesterol lowering resulted in a 50% reduction in local macrophage proliferation and plaque regression with reduced macrophage and lipid contents and increased collagen. GFP bone marrow reconstitution of APOE*3-Leiden.huCETP mice in which the aortas were shielded from irradiation showed infiltrating monocytes to contribute only 11% to the plaque macrophage pool during plaque progression, thereby underscoring the relevance of targeting macrophage proliferation for plaque regression. Finally, rates of macrophage proliferation in human carotid artery plaques correlated with serum LDL-cholesterol levels, in line with our experimental studies. Conclusion Foam cell formation in atherosclerotic plaques triggers their proliferation. Targeting macrophage proliferation leads to plaque regression.

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