Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis
Canan Kasikara, … , Muredach P. Reilly, Ira Tabas
Canan Kasikara, … , Muredach P. Reilly, Ira Tabas
Published February 25, 2021
Citation Information: J Clin Invest. 2021;131(8):e145275. https://doi.org/10.1172/JCI145275.
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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 4

PHACTR1 facilitates efferocytosis by increasing MLC phosphorylation.

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PHACTR1 facilitates efferocytosis by increasing MLC phosphorylation. (A)...
(A) Ratio of MFI of phospho-MLC (p-MLC) to total MLC (t-MLC) was quantified in Phactr1+/+ and Phactr1–/– BMDMs incubated in the absence or presence of ACs (AC–, AC+). (B) HMDMs were treated with scrambled RNA or PHACTR1 siRNA and assayed for p-MLC/t-MLC MFI ratio as in A. ***P < 0.001, ****P < 0.0001. (C) Ratio of p-MLC to t-MLC was quantified for MFI in AC+ rs9349379-GG and -AA HMDMs as in Figure 1. Images were quantified for p-MLC/t-MLC MFI ratio; n = 5 HMDMs per group; ***P < 0.001 by Student’s unpaired t test. (D) BMDMs treated with scrambled RNA or Mlc2 siRNA were then immunoblotted for MLC (18 kDa) and GAPDH (36 kDa) or assayed for efferocytosis. Results are shown as mean ± SEM; n = 3 experiments; *P < 0.05 by 2-tailed Student’s unpaired t test. (E) Phactr1+/+ and Phactr1–/– BMDMs were transfected with empty vector (Mock) or vector encoding WT MLC or S18/19D MLC. One set of cells was immunoblotted for MLC and GAPDH, and the other was assayed for efferocytosis. Results are shown as mean ± SEM, including individual data points; n = 3 experiments; *P < 0.05, **P < 0.01 by 2-way ANOVA with post hoc Tukey’s analysis.