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. 2007 May;27(5):1087-94.
doi: 10.1161/ATVBAHA.0000261548.49790.63.

Expression of tumor necrosis factor receptor-1 in arterial wall cells promotes atherosclerosis

Lisheng Zhang  1 , Karsten Peppel, Perumal Sivashanmugam, Eric S Orman, Leigh Brian, Sabrina T Exum, Neil J Freedman
Affiliations

Expression of tumor necrosis factor receptor-1 in arterial wall cells promotes atherosclerosis

Lisheng Zhang et al. Arterioscler Thromb Vasc Biol. 2007 May.

Abstract

Objective: Mechanisms by which tumor necrosis factor-alpha (TNF) contributes to atherosclerosis remain largely obscure. We therefore sought to determine the role of the arterial wall TNF receptor-1 (TNFR1) in atherogenesis.

Methods and results: Carotid artery-to-carotid artery interposition grafting was performed with tnfr1-/- and congenic (C57Bl/6) wild-type (WT) mice as graft donors, and congenic chow-fed apolipoprotein E-deficient mice as recipients. Advanced atherosclerotic graft lesions developed within 8 weeks, and had 2-fold greater area in WT than in tnfr1-/- grafts. While the prevalence of specific atheroma cells was equivalent in WT and tnfr1-/- grafts, the overall abundance of cells was substantially greater in WT grafts. WT grafts demonstrated greater MCP-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression at both early and late time points, and proliferating cell nuclear antigen expression at early time points. Aortic atherosclerosis was also reduced in 14-month-old apoe(-/-)/tnfr1(-/-) mice, as compared with cognate apoe-/- mice. In coculture with activated macrophages, smooth muscle cells expressing the TNFR1 demonstrated enhanced migration and reduced scavenger receptor activity.

Conclusions: TNFR1 signaling, just in arterial wall cells, contributes to the pathogenesis of atherosclerosis by enhancing arterial wall chemokine and adhesion molecule expression, as well as by augmenting medial smooth muscle cell proliferation and migration.

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Figures

Figure 1
Figure 1
Atherosclerosis progresses rapidly in congenic carotid grafts implanted in apoe−/− mice. A, The 8-mm carotid interposition graft (1), thickened by atherosclerosis, was harvested 8 weeks postoperatively, along with the aortic arch and contralateral carotid (2). Cross-sections of carotid grafts are shown (original magnification ×220) from specimens harvested 0 (B), 4 (C), 6 (D), and 8 (E) weeks postoperatively, and stained with a modified connective tissue stain. Scale bar=200 μm. F, Carotid grafts harvested 4 or 8 weeks postoperatively were frozen, and serial sections were immunostained for either macrophages (Mac3) or SMC α-actin, and counterstained with Hoechst 33342 (DNA). The luminal surfaces are oriented upward in each panel. Images of single specimens are representative of 3 grafts of each postoperative age. Scale bar=50 μm (original magnification ×440). G, The 8-week-old carotid grafts were stained for DNA and apoE, the staining pattern for which corresponds to SMC actin and SMC myosin heavy chain (supplemental Figure IIA and data not shown, n=3). Scale bar=100 μm.
Figure 2
Figure 2
Arterial wall expression of TNFR1 contributes to atherosclerosis. Carotid arteries from WT or tnfr1−/− (tnfr1 KO) mice were grafted as in Figure 1 into apoe−/− mice, and harvested at the indicated times. A, Cross-sections from the middle of each graft were stained with a modified connective tissue stain. Specimens shown represent ≥3 obtained at each time point. B, Computerized morphometry was performed on 2 sections from the middle of each 8-week-old carotid graft specimen prepared as in (A); means±SE of 6 specimens/group are displayed. Average external diameter (ext diameter) was calculated from the perimeter of the external elastic lamina. Scale bar=100 μm (original magnification ×220). Compared with WT: *P<0.05.
Figure 3
Figure 3
Arterial wall expression of TNFR1 does not affect the prevalence of atherosclerotic lesion cells derived from the arterial wall or circulating progenitors. Carotid grafting was performed as in Figure 2, except that either donor or recipient mice were transgenic for ubiquitous GFP expression (/GFP). A, Frozen sections of grafts were stained with Hoechst 33342 and imaged sequentially for endogenous GFP fluorescence (green) and DNA fluorescence. Images are representative of ≥4 grafts of each type (original magnification ×220); scale bar=100 μm. B, Neointimal green fluorescence in non-GFP specimens (nonspecific) was subtracted from that in GFP specimens (total fluorescence) to obtain specific GFP fluorescence. This value was divided by the cognate value for neointimal DNA fluorescence to obtain GFP/DNA (arbitrary units). These neointimal GFP/DNA values were averaged among grafts of each indicated type, and the mean±SE of ≥4 independent graft specimens is presented.
Figure 4
Figure 4
TNFR1 expression by arterial wall cells promotes chemokine and adhesion molecule expression. Carotid grafts harvested postoperatively at 2 weeks (A, with only minimal neointimal inflammatory cells) or 8 weeks (B) were stained with a connective tissue stain (A, top, ×220), or with antibodies against the indicated proteins and Hoechst 33342 (original magnification ×440, proliferating cell nuclear antigen) or ×1100; scale bars=50 μm). Immunofluorescence was quantitated as in Methods, and normalized to that obtained in WT grafts to obtain % of control; fluorescence in serial sections stained with nonimmune IgG (not shown) was subtracted from each specimen. Plotted are the means±SE from ≥3 specimens of each genotype. Compared with control: *P<0.02; #P<0.05.
Figure 5
Figure 5
Equivalent apoE expression by arterial wall cells in WT and tnfr1−/− carotid arteries accelerates carotid graft atherosclerosis in apoe−/− mice. A, Carotid grafting with harvest at 8 weeks was performed in apoe−/− mice as in Figure 2, except that graft donors were apoe−/−. Depicted are sections from mid-graft (left) and the anastomosis (right), representative of 5 grafts harvested (original magnification ×220). B, ApoE immunofluorescence (with Hoechst 33342 counterstain) was performed on frozen sections from normal carotids of 20-week-old female mice of the indicated genotype. Results are representative of 3 carotid arteries of each genotype (original magnification ×1100). C, Carotid grafts with the indicated donor and recipient mice were harvested 8 weeks postoperatively, sectioned at mid-graft, and stained as in Figure 2. Native carotid arteries were prepared equivalently. Images are representative of results from ≥3 specimens of each type (original magnification ×1100). Scale bars=50 μm.
Figure 6
Figure 6
Macrophage-induced SMC migration and scavenger receptor activity: effects of SMC TNFR1 expression. A, TNFR1 expression in SMCs (WT[+/+] and tnfr1−/−) and macrophages is demonstrated by SDS-PAGE/immunoblots (IB), with 30 μg of membrane protein from each cell type. Parallel blots were probed serially with TNFR1-specific or nonimmune IgG (IgG), and then anti-actin IgG. Shown are results from a single experiment, representative of ≥4 performed. B, Migration of aortic SMCs from congenic WT and tnfr1−/− (KO) mice was assessed in response to murine TNF, human PDGF-BB (each 10 ng/mL), or cholesterol-loaded murine macrophages as described in Methods. Within each experiment (n=4), the number of SMCs migrated was normalized to the number of WT SMCs that migrated in response to PDGF (17±5 fold/basal), to obtain % of control. Basal migration values for WT and tnfr1−/− SMCs were (OD562) 0.06±0.03 and 0.06±0.04, respectively. Compared with WT: *P<0.05 (repeated measures ANOVA). For a list of cytokines secreted by these macrophages, please see http://atvb.ahajournals.org. C, WT and tnfr1−/− SMCs cultured in the absence (none)or presence of activated murine macrophages were incubated with fluorescently labeled acetylated low-density lipoprotein, and subjected to flow cytometry. Specific uptake of fluorescently labeled acetylated low-density lipoprotein is plotted (total – nonspecific) as the means±SE of 4 experiments performed in duplicate. Relative to WT SMCs: *P<0.05.
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