Immunoglobulin E (lgE) activates immunity by binding to mast cells and basophils. It is well-known that IgE and its receptor, FcɛR1, play a key role in the development of airway inflammation and remodeling in allergic asthma. Recent studies show that IgE also plays an important role in abdominal aortic aneurysm (AAA) pathogenesis. However, the mechanism by which IgE promotes AAA remains unclear. Here we report that in our mouse model, asthma-induced high level of IgE aggravated AAA, but IgE lost this effect on AAA in FcɛR1-/- mice. Our in vitro study revealed that IgE induced smooth muscle cell senescence via upregulating lincRNA-p21 against p21 without altering expression of p53. By this mechanism, IgE accelerated AAA in ApoE-/- mice, which was blocked by knockdown of lincRNA-p21 in both vitro and vivo. This study suggests that IgE actives the lincRNAp21-p21 pathway to induce SMC senescence, which contributes to the formation of AAA, and lincRNA-p21 is a potential therapeutic target for AAA aggravated by asthma.
Figure 1. Effect of IgE receptor, FcɛR1, on asthma-accelerated AAA
(A) Schematic diagram for the mouse model of asthma-accelerated AAA. (B) Representative pictures from Giemsa staining for bronchial-alveolar lavage fluid (BALF) cell, and Masson staining and H&E staining for lung tissue. Eosinophils Scale bars: 100μm. Collagen Scale bars: 50μm. Inflammation cells Scale bars: 50μm; (C) Anti-OVA IgE levels in serum; (D) Total number of cells in BALF; (E) Percentage of eosinophils in BALF; (F) Percentage of lymphocytes in BALF. (G) Images of mouse aorta of ApoE-/-mice treated with or without OVA, and ApoE-/-FcɛR1-/- mice treated with OVA. (H) The average diameters of the AAA. (I) The incidence rate of AAA in ApoE-/-, OVA-treated ApoE-/-, and OVA-treated ApoE-/-FcɛR1-/- mice. (J) Representative staining for elastin at AAA lesions where the most severe elastin degradation occurred. Scale bars: 100μm. (K) Scores from the elastin degradation classification for AAA lesions of the mice with the indicated genotype. Data information: In (C-F, H, I, K), data are presented as mean ± SEM (n=15 per group). *P < 0.05; **P<0.01
Figure 2. Inflammation and remodeling in AAA lesion
(A-G) AAA lesion macrophage content (A); CD4+ T-cell content (B); major histocompatibility complex (MHC) class-II-positive area (C); mast cell content (D); and chemokine TNF-α-positive area (E); IL-6-positive area (F); and MCP-1-positive area (G) from ApoE-/-mice treated with or without OVA, and from OVA-treated ApoE-/-FcɛR1-/- mice. (H-J) The expression of plasma inflammatory factors, TNF-α (H), IL-6 (I), and IFN-γ (J) in serum from PBS ApoE-/-, OVA ApoE-/- and OVA ApoE-/-FcɛR1-/- mice; (K) Statistic analysis of TUNEL staining for apoptotic cells in the AAA tissue from ApoE-/-mice treated with or without OVA, and from OVA-treated ApoE-/-FcɛR1-/- mice. (L) Cell content of apoptotic vascular smooth muscle cell in AAA lesion from PBS ApoE-/-, OVA ApoE-/- and OVA ApoE-/-FcɛR1-/- mice. (M) Representative picture of β-galactosidase staining for senescent cells in AAA lesions. Scale bars: 25μm. (N) Counts for senescent cells/mm2 in the AAA lesions. Data information: Data are presented as mean ± SEM (n=15 per group). *P < 0.05; **P<0.01.
Figure 3. IgE effect on senescence of vascular SMCs in vitro and in vivo
(A) RT-PCR detection of FceR1α expression at different doses of IgE stimulation. (B) RT-PCR detection of FceR1α expression at different times of IgE stimulation. (C) Representative images from β-galactosidase staining of human vascular smooth under the indicated conditions. Scale bars: 100 μm. (D) The statistics of the results from C. (E) RT-PCR detection of p21 and p53 mRNA expression in IgE-stimulated HSMCs. (F) Western blotting analysis of p21 and p53 expression in human vascular smooth muscle cells stimulated by IgE. (G) Statistical analysis on the relative intensities of p21 and p53 bands in the Western blotting of human vascular smooth muscle cells stimulated by IgE. (H) The expression of p21 and p53 mRNA in human vascular smooth muscle cells stimulated by IgE. (I) RT-PCR detection of p21 and p53 mRNA expression in human vascular smooth muscle cells stimulated by IgE (J) Representative pictures of p21 immunohistochemical staining in AAA lesions. Scale bars: 25μm. (K) The statistical analysis of p21 immunohistochemical staining in AAA lesions. Data information: Data are presented as mean ± SEM (animal tissues; n=15 per group). *P < 0.05; **P<0.01
Figure 4. The effect of lincRNA-p21 on senescence of human vascular SMCs
(A) The expression of lincRNA-p21 in human vascular smooth muscle cells stimulated by different doses of IgE. (B) The expression of lincRNA-p21 in human vascular smooth muscle cells stimulated by 10 μg/ml IgE at different time. (C) Detection of lincRNA-p21 expression in HSMC after transfectioned with lincRNA-p21 siRNA. (D) Representative images of HSMC β-galactosidase staining of control, IgE, si-lincRNAp21 and si-lincRNA-p21+IgE treatment groups. Scale bar: 100μm. (E) The senescent cell counts of HSMC after control, IgE, si-lincRNA-p21 or si-lincRNA-p21+IgE treatment. (F) PCR detection of p21 and p53 expression in HSMCs treated with IgE and si-lincRNA-p21. (G) Statistical analysis of p21 and p53 in HSMC treated with IgE and si-lincRNA-p21. (H) Western blot analysis of p21 and p53 in HSMC after IgE and si-lincRNA-p21 treatments. (I) Statistical analysis on the relative intensities of p21 and p53 protein expression in HSMC treated with IgE and si-lincRNA-p21. Data information: Data are presented as mean ± SEM. *P < 0.05; **P<0.01.
Figure 5. The effect of lincRNA-p21 on formation of AAA
(A) Schematic diagram of animal model procedures. (B) Expression of lincRNA-p21 in AAA lesion of mice in control (n=8) and si-mlincRNA-p21 (n=5) groups. (C) Representative images of aortas from mice in control and si-mlincRNA-p21 groups of mice. (D) The average diameters of abdominal aortic aneurysm of two groups of mice. (E) The incidence rate of AAA in two groups of mice. (F) Representative pictures of β-galactosidase staining in AAA lesion. Scale bars: 25μm. (G) Senescent cells number per mm2 in AAA lesions. (H) Representative pictures of immunohistochemical staining for p21 in AAA lesions. Scale bars: 25μm. (I) The statistical analysis of p21 positive areas in immunohistochemical staining in AAA lesions. (J) RT-PCR detection of p21 and p53 mRNA expression in AAA lesions. (K) Schematic diagram of this article: IgE aggravates AAA mainly by upregulating lincRNA-p21 contributing to HSMC senescence. Data information: Data are presented as mean ± SEM. *P < 0.05; **P<0.01.
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