CD163+ macrophages restrain vascular calcification, promoting the development of high-risk plaque
Atsushi Sakamoto, … , Renu Virmani, Aloke V. Finn
Atsushi Sakamoto, … , Renu Virmani, Aloke V. Finn
Published January 31, 2023
Citation Information: JCI Insight. 2023;8(5):e154922. https://doi.org/10.1172/jci.insight.154922.
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Research Article Cell biology Vascular biology

CD163+ macrophages restrain vascular calcification, promoting the development of high-risk plaque

Abstract

Vascular calcification (VC) is concomitant with atherosclerosis, yet it remains uncertain why rupture-prone high-risk plaques do not typically show extensive calcification. Intraplaque hemorrhage (IPH) deposits erythrocyte-derived cholesterol, enlarging the necrotic core and promoting high-risk plaque development. Pro-atherogenic CD163+ alternative macrophages engulf hemoglobin:haptoglobin (HH) complexes at IPH sites. However, their role in VC has never been examined to our knowledge. Here we show, in human arteries, the distribution of CD163+ macrophages correlated inversely with VC. In vitro experiments using vascular smooth muscle cells (VSMCs) cultured with HH-exposed human macrophage — M(Hb) — supernatant reduced calcification, while arteries from ApoE–/– CD163–/– mice showed greater VC. M(Hb) supernatant–exposed VSMCs showed activated NF-κB, while blocking NF-κB attenuated the anticalcific effect of M(Hb) on VSMCs. CD163+ macrophages altered VC through NF-κB–induced transcription of hyaluronan synthase (HAS), an enzyme that catalyzes the formation of the extracellular matrix glycosaminoglycan, hyaluronan, within VSMCs. M(Hb) supernatants enhanced HAS production in VSMCs, while knocking down HAS attenuated its anticalcific effect. NF-κB blockade in ApoE–/– mice reduced hyaluronan and increased VC. In human arteries, hyaluronan and HAS were increased in areas of CD163+ macrophage presence. Our findings highlight an important mechanism by which CD163+ macrophages inhibit VC through NF-κB–induced HAS augmentation and thus promote the high-risk plaque development.

Authors

Atsushi Sakamoto, Rika Kawakami, Masayuki Mori, Liang Guo, Ka Hyun Paek, Jose Verdezoto Mosquera, Anne Cornelissen, Saikat Kumar B. Ghosh, Kenji Kawai, Takao Konishi, Raquel Fernandez, Daniela T. Fuller, Weili Xu, Aimee E. Vozenilek, Yu Sato, Hiroyuki Jinnouchi, Sho Torii, Adam W. Turner, Hirokuni Akahori, Salome Kuntz, Craig C. Weinkauf, Parker J. Lee, Robert Kutys, Kathryn Harris, Alfred Lawrence Killey, Christina M. Mayhew, Matthew Ellis, Leah M. Weinstein, Neel V. Gadhoke, Roma Dhingra, Jeremy Ullman, Armella Dikongue, Maria E. Romero, Frank D. Kolodgie, Clint L. Miller, Renu Virmani, Aloke V. Finn

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Figure 7

NF-κB signaling, HA, and RUNX2 expression in plaques of aged ApoE–/– versus ApoE–/– CD163–/– mice.

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NF-κB signaling, HA, and RUNX2 expression in plaques of aged ApoE–/– ver...
(A–D) Representative low- and high-power images of BCA sections with total p-65 immunofluorescence (red) and DAPI counterstaining obtained from aged ApoE–/– (A and B) and ApoE–/– CD163–/– (C and D) mice (1.5-year-old). Nuclei in the plaque showing t-p65 colocalization are indicated by yellow arrowheads. (E–G) Summary of plaque area (E), number of nuclei/plaque area (F), and % p65 nuclear colocalization (G) in the most stenotic BCA lesions were compared between ApoE–/– and ApoE–/– CD163–/– mice (n = 6 per group). (H) Schematic diagram of the methodology for plaque extraction from mouse aortas. Whole visible plaques from ascending (including aortic root) to descending (cut at the level of diaphragm) aorta were peeled out under dissecting microscope. Plaques in BCA, left common carotid, and subclavian arteries with the equivalent length with BCA were also included in the samples. Plaques were mechanically ground in equivalent volumes of PBS, and further ELISA and WB analyses were performed. (I) HA content in aortic plaque assessed by ELISA (n = 9 per group). (J–L) Representative WB images for mouse plaque samples including p-p65, t-p65, RUNX2, and β-actin (J). Summary of densitometry analysis is also shown in K (p-p65/t-p65) and L (RUNX2/β-actin) (n = 6 per group). (M–P) Representative low- and high-power images of BCA sections with RUNX2 staining (purple) obtained from aged ApoE–/– (M and N) and ApoE–/– CD163–/– (O and P) mice (1.5-year-old). (Q–S) Summary of % RUNX2+ cells in the plaque (Q), % RUNX2+ cells in the medial layer (R), and % RUNX2+ cells in the vessel (plaque+medial layer) (S) in the most stenotic BCA lesion were compared between ApoE–/– and ApoE–/– CD163–/– mice (n = 6 per group). *P < 0.05, **P < 0.01. Results are presented as the mean ± standard deviation (F, G, L, and Q–S) or median and interquartile range (E, I, and K). T test (F, G, L, and Q–S) or Mann-Whitney test (E, I, and K) was conducted for statistical analysis. Data normality was tested by Shapiro-Wilk test. Scale bars: 100 μm (A and C), 20 μm (B and D), and 100 μm (M–P).