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. 2013 May;33(5):886-93.
doi: 10.1161/ATVBAHA.112.300941. Epub 2013 Feb 21.

Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention

Amarylis Wanschel  1 , Tara Seibert, Bernd Hewing, Bhama Ramkhelawon, Tathagat D Ray, Janine M van Gils, Katey J Rayner, Jonathan E Feig, Edward R O'Brien, Edward A Fisher, Kathryn J Moore
Affiliations

Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention

Amarylis Wanschel et al. Arterioscler Thromb Vasc Biol. 2013 May.

Abstract

Objective: The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques.

Approach and results: Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe(-/-) mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque.

Conclusions: Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.

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Figures

Figure 1
Figure 1. Sema3E and its Receptor PlexinD1 are Expressed by Macrophages in Advanced Atherosclerotic Plaques
Immunofluorescent staining of CD68 (green), DNA (DAPI, blue) and (a) Sema3E (red) or (b), , PlexinD1 (red) in aortic root atherosclerotic plaques of Apoe−/− mice fed a Western diet for 12 weeks. Areas of co-localization are shown in yellow in the merged image (arrows). Scale bar, 50 µm. Images are representative of n ≥ 3 mice.
Figure 2
Figure 2. Sema3E Expression by Lesional Macrophages is Downregulated during Atherosclerosis Regression
(a) Immunofluorescent staining of CD68 (green) and Sema3E (red) in aortic arch plaques transplanted into a progressive (Apoe−/−- recipient) or regressive (C57BL6 recipient) environment. Areas of co-localization (yellow) are shown in the merged image. Images are representative of n ≥ 3 mice. (b) qPCR analysis of mRNA from laser captured CD68+ cells in aortic arch plaques from mice in (a) (n=4 mice/group). (c) qPCR analysis of Sema3e mRNA in BMDMs polarized towards M1, M2 or unpolarized (M0). (d) qPCR analysis of Sema3e mRNA from laser captured CD68+ cells in aortic root plaques from Ldlr−/- mice fed a western diet for 14 weeks (baseline) and then switched to a chow diet for 4 weeks (regression conditions) (n=5 mice/group). Data in b-d are the mean of triplicate samples ± SEM. Statistical analyses were performed by Student’s t-test (b,d) or ANOVA (c). *P0003C;0.05
Figure 3
Figure 3. Sema3E Expression is Up-regulated by Physiological Drivers of Plaque Inflammation
qPCR analysis of Sema3e mRNA in BMDMs treated with (a) 50 µg/ml of AcLDL or (b) oxLDL for the indicated times, or (c) CoCl2 for 24 h. (d-e) qPCR analysis of Sema3e mRNA expression in peritoneal macrophages treated with (d) oxLDL (50 µg/ml) or (e) CoCl2 (200 µM) with or without HIF-1α inhibitor (100 µmol/L). (f) Western blot of full-length and cleaved Sema 3E in supernatants from BMDMs stimulated with 50 µg/ml oxLDL, 10 µg/ml HDL, or both for 24 h. (g) Western blot of PlexinD1 in cell lysates of BMDMs stimulated with 50 µg/ml oxLDL, 10 µg/ml HDL, or both for 24 h. Data in a-c are the mean of triplicate samples ± SEM. Statistical analysis was performed by ANOVA; (a-c) * P<0.05 compared to untreated, (d-e) * P<0.05 compared to OxLDL or CoCl2 alone.
Figure 4
Figure 4. Sema3E Inhibits the Migration of Macrophages
(a) Transwell migration of peritoneal macrophages to CCL19 (500 ng/ml) with or without recombinant Sema3E at the concentrations indicated. (b) Real-time migration (xCelligence) of peritoneal macrophages to CCL19 with or without 250 ng/ml Sema3E. (c) Transwell migration of peritoneal macrophages pre-treated with 250 ng/ml Sema3E for 45 minutes, washed and then exposed to 500 ng/ml CCL19. (b) Transwell migration of peritoneal macrophages to 100 ng/ml CCL2 with or without recombinant Sema3E at the concentrations indicated. Data are the mean ± s.d. of triplicate samples in a single experiment and are representative of an experimental n=3. Statistical analysis was performed by ANOVA followed by Tukey test; (a,c,d) * P<0.01.
Figure 5
Figure 5. Sema3E Affects the Organization of the Actin Cytoskeleton
Peritoneal macrophages stained with Phalloidin to detect polymerized actin after treatment with (a) 500 ng/ml CCL19 or (b) 100 ng/ml CCL2 in the presence or absence of recombinant Sema3E (250 ng/mL). Arrows indicate membrane ruffles, scale bar 10 µm. Mean cell surface area of macrophages is graphed at right. Statistical analysis was performed by ANOVA; * P<0.05 compared to CCL19 (a) or CCL2 (b) alone.
Figure 6
Figure 6. Sema3E impairs Rho GTPAse signaling
Immunoblot of activated (a) Rac1 and (b) CDC42 in macrophages treated with 100 ng/ml CCL2 with and without 250 ng/ml Sema3E. Densitometry quantification of blots is shown graphically. Data shown is from one experiment, and is representative of an experimental n=3.
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