CD163+ macrophages restrain vascular calcification, promoting the development of high-risk plaque
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
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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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 8

Effect of NF-κB inhibition by NBDpep on development of atheroma and VC in ApoE–/– mice with HFD feeding.

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Effect of NF-κB inhibition by NBDpep on development of atheroma and VC i...
(A) Schematic diagram of study design. HFD was started in ApoE–/– mice at the age of 8 weeks and continued to the end of the experiment. Treatment by continuous subcutaneous injection of CTLpep or NBDpep by Alzet osmotic pump (100 μg/kg/d) was started at the age of 14 weeks. Since the pump needed to be replaced every 6 weeks, pump replacing surgeries were performed, and Tx was continued for 18 weeks (by the age of 32 weeks). (B–E) Representative BCA sections with H&E and AR staining obtained from HFD feeding ApoE–/– mice with CTLpep (B; H&E, C; AR) or NBDpep Tx (D; H&E, E; AR). (F–J) Vessel size (EEL) (F), plaque area (G), % area stenosis (H), calcification area assessed by AR (I), and % area calcification (J) at the most stenotic BCA lesion were compared between CTLpep- and NBDpep-treated ApoE–/– mice (n = 9 per group). (K and L) Total aortic calcium contents assessed by colorimetric assay (n = 4 per group) (K) and HA content in aortic plaque assessed by ELISA (n = 5 per group) (L) with the comparison of CTLpep- and NBDpep-treated ApoE–/– mice. (M–O) Representative WB images of mouse plaque samples including p-p65 (Ser536), t-p65, RUNX2, and β-actin (M). Summary of densitometry analysis is shown in N (p-p65/t-p65) and O (RUNX2/β-actin) (n = 5 in each). Results are presented as the mean ± standard deviation (F, N, and O) or median and interquartile range (G–L). T test (F, N, and O) or Mann-Whitney test (G–L) was conducted for statistical analysis. Data normality was tested by Shapiro-Wilk test. Scale bars: 200 μm (B–E). Tx, treatment.