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 6

Augmented HA synthesis via NF-κB signaling by M(Hb) supernatants is responsible for its anticalcific effect on HASMCs.

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Augmented HA synthesis via NF-κB signaling by M(Hb) supernatants is resp...
(A–D) Representative WB images and summary of densitometry analysis for HASMCs after exposing to M(con)sup or M(Hb)sup without OS for 24 hours (whole-cell lysate); HAS1/β-actin (A), HAS2/β-actin (B), HAS3/β-actin (C), and CD44/β-actin (D) (n = 4 in each). (E) HASMCs were exposed to M(con)sup or M(Hb)sup with CTLpep or NBDpep (both 25 μM) without OS for 24 hours. Representative WB images and summary of densitometry analysis for HAS1/β-actin (whole-cell lysate, n = 4 per group). (F) Summary of HA concentration in M(con) and M(Hb) supernatant pre- and post-exposing (24 hours) to HASMCs (ELISA, n = 3 per group). (G) Summary of HA concentration in M(con) and M(Hb) supernatant post-exposing (24 hours) to HASMCs with CTLpep or NBDpep (both 100 μM) without OS for 24 hours (n = 3 per group). (H) Calcium content in HASMCs after 48 hours’ exposure in OS to HA supplementation (0, 50, and 200 μg/mL) (colorimetric assay, n = 3 per group, adjusted by protein amount). (I) Calcium content in HASMCs after 24 hours’ exposure to basal growth medium with OS and 4-MU, an HAS inhibitor, (0, 2.5, and 25 μM) (colorimetric assay, n = 4 per group, adjusted by protein amount). (J–L) HASMCs were transfected with Scr or siHAS1. The effect of siHAS1 was confirmed by WB (J). M(con) or M(Hb) supernatant with OS was exposed for 48 hours. Representative AR images (K) and summary of % AR-positive area (n = 4 per group) (L). *P < 0.05, **P < 0.01. Results are presented as the mean ± standard error (A–H and K). T test was applied to A–D. ANOVA followed by post hoc Tukey’s test was applied to E–I and L. Data normality was tested by Shapiro-Wilk test. All experiments were performed at least 3 times to confirm their reproducibility. HAS, hyaluronan synthase; siHAS1, HAS1 siRNA; 4-MU, 4-methylumbelliferone.