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 4

Effect of M(Hb) supernatant on HASMC calcification in vitro.

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Effect of M(Hb) supernatant on HASMC calcification in vitro. (A–C) HASMC...
(A–C) HASMCs were exposed for 2 days to control basic culture media with OS (containing CaCl2, β-glycerophosphate, l-ascorbic acid, insulin, and dexamethasone) with or without HH, or with M(con)sup or M(Hb)sup. Representative macro- (inset) and microscopic AR findings (4× original magnification) in each condition (A). Summary of % AR-positive area in each condition (B) (n = 3 per group). Amount of calcium examined by colorimetric assay adjusted by protein amount (C) (n = 4 per group). (D and E) HASMCs were cultured for 24 hours in M(con) or M(Hb) with or without OS. Representative immunoblotting image of RUNX2 and β-actin (D). Summary of densitometry analysis (E) (n = 3 per group). (F–K) HASMCs were cultured for 6 hours in M(con) or M(Hb) with OS. Extracted RNA samples were analyzed by Affymetrix Clariom S microarray (n = 4 per group). We demonstrated 548 upregulated and 249 downregulated DEGs in M(Hb)+OS versus M(con)+OS condition (threshold: FC ≥ 2.0 or ≤ –2.0 with P ≤ 0.05). Heatmap of 797 DEGs visualized by row z score scaling (F). Volcano plots detailing the magnitude of expression difference (G). GO enrichment analysis of unbiased DEGs was conducted by DAVID v6.8 bioinformatics resources. Top 25 GO BP terms with highest statistical significance in up- and downregulated DEGs (H and I). Selected genes related to inflammation and NF-κB signaling from upregulated genes (J) and these related to calcification/osteogenic differentiation-related genes from downregulated DEGs (K) were visualized by heatmap with row z score scaling of log2 FC. *P < 0.05, **P < 0.01. Results are presented as the mean ± standard error and ANOVA followed by post hoc Tukey’s test was applied (B, C, and E). Data normality was tested by Shapiro-Wilk test. The experiments of A–E were performed at least 3 times to confirm their reproducibility. AR, Alizarin Red; BP, biological process; CTL, control; DEGs, differentially expressed genes; FC, fold change; GO, gene ontology; HH, hemoglobin-haptoglobin complex; M(con)sup, control macrophage supernatant; M(Hb)sup, HH-differentiated macrophage supernatant; Neg-reg, negative regulation; OS, osteogenic components supplementation; Pos-reg, positive regulation; TC, transcription; TF, transcription factor.