Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis
Canan Kasikara, Maaike Schilperoort, Brennan Gerlach, Chenyi Xue, Xiaobo Wang, Ze Zheng, George Kuriakose, Bernhard Dorweiler, Hanrui Zhang, Gabrielle Fredman, Danish Saleheen, Muredach P. Reilly, Ira Tabas
Canan Kasikara, Maaike Schilperoort, Brennan Gerlach, Chenyi Xue, Xiaobo Wang, Ze Zheng, George Kuriakose, Bernhard Dorweiler, Hanrui Zhang, Gabrielle Fredman, Danish Saleheen, Muredach P. Reilly, Ira Tabas
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Research Article Cardiology Cell biology

Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis

Abstract

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte–derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet–fed Ldlr–/– mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps — all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Authors

Canan Kasikara, Maaike Schilperoort, Brennan Gerlach, Chenyi Xue, Xiaobo Wang, Ze Zheng, George Kuriakose, Bernhard Dorweiler, Hanrui Zhang, Gabrielle Fredman, Danish Saleheen, Muredach P. Reilly, Ira Tabas

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Figure 7

WD-fed Ldlr–/– mice lacking hematopoietic PHACTR1 show defective macrophage efferocytosis and increased plaque necrosis in atherosclerotic lesions.

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WD-fed Ldlr–/– mice lacking hematopoietic PHACTR1 show defective macroph...
(A–C and G) Bone marrow from Phactr1+/+ or Phactr1–/– mice was transplanted into irradiated Ldlr–/– mice. After 4 weeks, the mice were fed a Western-type diet (WD) for 8 weeks, and then aortic root lesional cross sections were analyzed. (A) Ratio of Mac2+ macrophage–associated to free TUNEL+ cells. (B) Ratio of p-MLC to t-MLC MFI in macrophages associated with TUNEL+ cells. (C) Images of aortic root sections stained with H&E (dashed lines indicate necrotic area), with quantification of necrotic and lesion areas. (D–F and H) Bone marrow from Phactr1+/+ or Phactr1+/– mice was transplanted into irradiated Ldlr–/– mice. After 4 weeks, the mice were fed the WD for 12 weeks. (D–F) Lesional endpoints were quantified as above. (G and H) Aortic root cross sections were stained with Picrosirius red. For each section, cap thickness was measured at the lesional midpoint and both shoulder regions and then averaged and quantified as the ratio of collagen cap thickness to lesion area, relative to the Phactr1+/+ group. (I) Bone marrow from Phactr1–/– or Phactr1+/+ mice was transplanted into irradiated Phactr1–/– Ldlr–/– mice. After 4 weeks, the mice were fed the WD diet for 8 weeks, and lesional endpoints were quantified as above. In all panels, results are shown as individual data points with lines indicating mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001 by 2-tailed Student’s unpaired t test. Scale bars: 100 μm.