Hyaluronan synthase 3 promotes plaque inflammation and atheroprogression

Abstract

Objective: Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied.

Approach and results: It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 β (IL-1β) in an NFkB dependent manner and blocking antibodies to IL-1β abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation.

Conclusions: Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1β, our results suggest that therapeutic anti-IL-1β antibodies, recently tested in human clinical trials (CANTOS), may exert their beneficial effects on inflammation in post-myocardial infarction patients in part via effects on HAS3. TOC categorybasic study TOC subcategoryarteriosclerosis.

Keywords: Atherosclerosis; Hyaluronan; Inflammation; Interleukin 1 beta; Macrophage; T-cells; Vascular Biology.

Fig. 1

Detection of HAS isoenzymes in human atherosclerotic lesions and association of Has3 expression with early macrophage accumulation in murine lesions. A Representative images of HAS1,-2 and -3 detection via immunohistochemical stainings and Ct values of 18S, HAS1, HAS2, and HAS3v1 in human atherectomy specimen as determined by qPCR; mean ± SEM; n = 6–12. B Left, mRNA expression of Has1, Has2, and Has3 in aortas of Apoe^−/− mice at different ages; n = 2–3; means ± SEM. Right, quantification of the area fraction of HA staining and Mac2 in aortic root sections at different ages of Apoe^−/− mice; mean ± SEM; n = 3–7. C,D Depiction of HA (C) and Mac2 (D) stainings of aortic roots of 6-, 10-, 14-, and 19-week-old Apoe^−/− mice.

Fig. 2

Activated macrophages induce HAS3 expression in human vascular SMCs via IL-1β and NF-κB signaling. A Human vascular SMCs (VSMC) were stimulated with IL-1β and subsequently RNA was extracted and analyzed via qPCR. Has isoenzyme mRNA expression is expressed as fold of unstimulated controls. Left, Has mRNA expression after 3, 6, 12, and 24 h of stimulation with IL-1β (10 ng/ml). Right, IL-1β dose-dependent Has isoenzyme expression. RNA was isolated after 3 h of IL-1β stimulation; means ± SEM, n = 3–10; mRNA expression is expressed as fold of unstimulated controls; *p < 0.05 vs. control. B In a transwell insert LPS-activated U937 macrophages were co-cultured with VSMCs in the presence and absence of a control mIgG (10 μg/ml), a neutralizing IL-1β antibody (10 μg/ml), and the NF-κB inhibitor BAY11-7082 (10 μM), respectively. After 24 h of co-culture, Has3 mRNA expression was analyzed in VSMCs; means ± SEM; n = 3; *p < 0.05. C VSMCs were incubated for 3 h with IL-1β, Bay11-7082 (Bay), or Bay11-7082 and IL-1β. Thereupon, Has3 expression was assessed using qPCR; n = 3; *p < 0.05. Data are shown as mean ± SEM.

Fig. 3

Has3^−/−/Apoe^−/− mice have reduced aortic plaque burden and reduced lesion size A Aortic plaque score was determined by Oil-Red-O staining of aortas from Apoe^−/− and Has3^−/−/Apoe^−/− mice after 15 weeks of feeding WD (23 weeks of age). Representative images and quantification of en face and Oil-Red-O stained aortas are depicted; n = 7; mean ± SEM; *p < 0.05. B Representative images of an H&E stained brachiocephalic artery section from Apoe^−/− and Has3^−/−/Apoe^−/− mice, respectively. Scale bar: 100 μm. C Quantification of the area within the external elastic lamina (EEL), the internal elastic lamina (IEL), and the lesion area. Data are shown as mean ± SEM; n = 6/5; *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

In early atherosclerotic lesions of Has3/Apoe-double deficient mice monocyte/macrophage accumulation is reduced After 4 weeks of WD feeding (12 weeks of age), aortic immune cell accumulation was analyzed in Apoe^−/− and Has3^−/−/Apoe^−/− mice by flow cytometry. Immune cells were detected by surface CD45 expression and then further discriminated into (A) CD45⁺SSC^low lymphocytes (B) and CD11b⁺ myeloid cells. CD11b⁺ cells were subdivided into CD11b⁺Ly6C⁺ monocytes (D), CD11b⁺F4/80⁺ macrophages (E), and CD11b⁺Ly6G⁺ neutrophils (F). (G) Representative plots of the flow cytometric analyses are depicted. (H) Ratio of F4/80⁺CD86⁺ M1 macrophages and F4/80⁺CD206⁺ M2 macrophages in atherosclerotic lesions was calculated. Data are represented as mean ± SEM; n = 3–7; *p < 0.05.

Fig. 5

Reduced circulating Th1 cells in Has3^−/−/Apoe^−/− mice Immune cells in the blood were investigated after 4 weeks of WD by flow cytometry. (A) The number of circulating CD11b⁺CD115⁺ monocytes and the three monocyte subsets that differentially express Ly6C are depicted. (B) CD19⁺ B cells and CD3⁺ T cells; (C) different CD4⁺ T cell subsets, such as T-bet⁺IFNγ⁺ Th1 cells, Gata3⁺IL4⁺ and Gata3⁺IL5⁺ Th2 cells, RORγT⁺IL17A⁺ Th17 cells, and FoxP3⁺ T_reg cells. Data are shown as mean ± SEM; n = 5–7; *p < 0.05. (D) Representative plots for the determination of Th1 cells by flow cytometric analysis. (E) Plasma concentration of IL-12(p70) was analyzed after 7 weeks of WD feeding in Has3^−/−/Apoe^−/− and control mice. Data are shown as mean ± SEM; n = 7,9; *p < 0.05.

Fig. 6

Has3-deficiency affects the cytokine expression in aortas. After 4 weeks of WD feeding, RNA was extracted from aortas of Has3^−/−/Apoe^−/−and Apoe^−/− mice and analyzed using qPCR. (A) Cd3e, (B) Tbx21, (C) Il12b, (D) Ccl5, (E) Csfr1; n = 3–5; data are shown as mean ± SEM; *p < 0.05.

Fig. 7

HAS3 in human and murine T cells. A, mRNA expression of the three hyaluronan synthases, Has1, Has2, and Has3 by activated T cells normalized to 18S expression levels. Data are for pooled CD4⁺ T cells isolated from 4 mice, activated using anti-CD3/28 beads, and measured in triplicate. B, HAS3 mRNA expression by activated, well characterized T_H1 and T_H2 clones. The clones were previously described in Bollyky et al., Cellular and Molecular Immunology, 2010. Panel A includes 12 measurements for each gene; 3 mRNA assessments from each of 4 animals. The error bars shown represent the standard error of the mean. Panel B includes a total of 12 measurements; 3 mRNA assessments from a total of 4 human T-cell clones; 2 clones for each condition. The error bars shown represent standard deviation; *p < 0.05. C, CD3⁺ T-cells were isolated from spleens of Has3^−/−/Apoe^−/− deficient mice and Apoe^−/− mice (8–12 weeks old) and stimulated with IL-1β (10 ng/ml) for 3 h. Subsequently Has3 mRNA was analyzed by qPCR; means ± SEM; n = 6; *p < 0.05.

Fig. 8

Has3-deficiency does not affect CD3-/CD28-induced stimulation of isolated T cells. In 12 week old mice (4 weeks on WD), T lymphocytes were isolated from the spleen and axillary lymph nodes of Apoe^−/− and Has3^−/−/Apoe^−/− mice and stimulated for 24 h with anti-CD3/CD28 beads. Activation of CD4⁺ and CD8a⁺ T cells was assessed by flow cytometric measurements of the surface antigens. As controls T cells were cultured without anti-CD3/CD28 beads for 24 h. Data are shown as mean ± SEM; n = 4–3.