CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis - PubMed (original) (raw)

CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis

Yvonne Baumer et al. Atherosclerosis. 2017 Jan.

Abstract

Background and aims: Vascular smooth muscle cells (VSMC) migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. CD98 is a transmembrane protein made of two subunits, CD98 heavy chain (CD98hc) and one of six light chains, and is known to be involved in cell proliferation and survival. Because the influence of CD98hc on atherosclerosis development is unknown, our aim was to determine if CD98hc expressed on VSMC plays a role in shaping the morphology of atherosclerotic plaques by regulating VSMC function.

Methods: In addition to determining the role of CD98hc in VSMC proliferation and apoptosis, we utilized mice with SMC-specific deletion of CD98hc (CD98hcfl/flSM22αCre+) to determine the effects of CD98hc deficiency on VSMC function in atherosclerotic plaque.

Results: After culturing for 5 days in vitro, CD98hc-/- VSMC displayed dramatically reduced cell counts, reduced proliferation, as well as reduced migration compared to control VSMC. Analysis of aortic VSCM after 8 weeks of HFD showed a reduction in CD98hc-/- VSMC proliferation as well as increased apoptosis compared to controls. A long-term atherosclerosis study using SMC-CD98hc-/-/ldlr-/- mice was performed. Although total plaque area was unchanged, CD98hc-/- mice showed reduced presence of VSMC within the plaque (2.1 ± 0.4% vs. 4.3 ± 0.4% SM22α-positive area per plaque area, p < 0.05), decreased collagen content, as well as increased necrotic core area (25.8 ± 1.9% vs. 10.9 ± 1.6%, p < 0.05) compared to control ldlr-/- mice.

Conclusions: We conclude that CD98hc is required for VSMC proliferation, and that its deficiency leads to significantly reduced presence of VSMC in the neointima. Thus, CD98hc expression in VSMC contributes to the formation of plaques that are morphologically more stable, and thereby protects against atherothrombosis.

Keywords: Apoptosis; Atherosclerosis; CD98; Cell proliferation; Vascular smooth muscle cell.

Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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Conflict of interest statement

The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

Figures

Fig. 1

Fig. 1. VSMC and CD98 expression in various stages of atherosclerotic plaque

(A) TEM analysis of _ldlr_−/− mouse aorta sections showing elastin layers (E), medial VSMC (mVSMC) and intimal VSMC (iVSMC) at areas of breaks (*) in the elastin. (B) Immunofluorescence staining for SM22α in aortic root sections showing the distribution of mVSMC and iVSMC covering the necrotic core (NC) and their quantification in plaque over time. (C) Quantification of SM22α staining in aortic root sections (shown in E, n=4/time point). (D) RT-qPCR data showing expression pattern of CD98 in aortas of _ldlr_−/− mice on HFD from 0–16 weeks (n=4/time point). (E) Aortic root sections showing expression of CD98 (red), SM22α (red, SMC), and MOMA-2 (green, macrophages) at different stages of atherosclerosis. CD98 is expressed robustly in macrophages (*) after 4 weeks and in VSMC after 8 weeks of HFD. iVSMC and mVSMC are shown by white arrows and arrow heads respectively.

Fig. 2

Fig. 2. CD98hc controls VSMC proliferation and migration

(A) After 5 days of culture, the number of proliferating cells is significantly reduced in _CD98hc_−/− VSMC. (B) Proliferation of VSMC (SMA positive), measured ± PDGF-BB using EdU assay, shows significantly impaired proliferation of _CD98hc_−/− VSMC compared to control cells, especially with added PDGF-BB. (red=EdU, blue=DAPI). (C) Defective proliferation of _CD98hc_−/− VSMC is shown by ECIS measurement of impedance across an electrode as cells adhere and proliferate (D) and after wounding and subsequent recovery (E). (F and G) _CD98hc_−/− and control mice were put on HFD for 8 weeks and injected with EdU (100 μg/mouse) 3 days before sacrifice. The aorta of each mouse was digested analyzed for EdU+ (proliferating) (F) and cleaved-Casp3+ (apoptotic). (G) VSMC using flow cytometry. *p<0.05.

Fig. 3

Fig. 3. VSMC-CD98hc deficiency alters atherosclerotic plaque morphology

After 16 weeks of HFD measurement of atherosclerosis in whole aorta (A) and aortic root sections (B), using Oil Red-O staining revealed no significant differences in plaque content between _CD98hc_−/− and control mice. Quantification of necrotic core area (C) revealed significantly increased necrotic core in _CD98hc_−/− animals compared to controls. (D) Immunofluorescence staining for SM22α (red) to visualize VSMC and MOMA-2 (green) to visualize macrophages in aortic root sections. Nuclei are stained with DAPI and shown in blue. SM22α and MOMA-2 positive areas were analyzed with ImageJ as a percent of total plaque area and are quantified in E and F respectively. *p<0.05, n=15.

Fig. 4

Fig. 4. Characterization of plaque composition and VSMC proliferation

(A) Aortic root sections from _CD98hc_−/− and control mice were stained with Sirius red for quantification of collagen content. (B) Sirius red was also analyzed by circular polarized light to detect collagen fibers of varying size with thick fibers shown in red and orange, while thinner fibers are shown in yellow and green. (C) Masson Trichrome staining reveals keratin and muscle in red, collagen in blue, cytoplasm in pink, and black nuclei. (D) Aortic root serial sections were stained with αSMA to detect smooth muscle cells, and Ki67 to detect proliferating cells. Ki67+ proliferating VSMC were quantified for _CD98hc_−/− and control mice (E). αSMA+ area was also quantified and is expressed as a percentage of total plaque area (F). *p<0.05, ***p<0.001, n=8.

Fig. 5

Fig. 5. VSMC content and ultrastructural details of atherosclerotic plaque from control and _CD98hc_−/− mice

TEM images display thoracic aorta sections stained with Richardson stain showing the neointima and elastin layers interspersed within VSMC layers (top row) after 8 weeks (A) or 16 weeks (B) of HFD in control or _CD98hc_−/− mouse aortas. The bottom row for both time points and genotypes depicts TEM photos of junctions between the last elastin layer and neointima. (C and D) TEM analysis of breaks in the elastin layers of the thoracic aorta (yellow arrowheads) was performed by counting the number of breaks in at least 3 images taken for each mouse aorta. *p<0.05, n=3.

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