Illustrations
Figure 1
Increased IL-32 expression in human calcified aortic valve tissue. (A) Alizarin red staining of aortic valve tissue slides. Bright red staining shows calcification in calcified aortic valve cells. (10×) (B) IL-32 protein staining by immunohistochemistry in aortic valve tissue. (C) Relative expression level of IL-32 mRNA was significantly enhanced in calcified aortic valve tissue. The results are shown as mean relative abundance. SEM and quantified 4.79-fold change compared to control group. The data are expressed as mean ± SD. *P < 0.05 compared with control; n = 8.
Figure 1
Figure 2
Increased MAPK pathway activity in calcified aortic valve tissue. (A) Immunohistochemical (IHC) staining of ERK, JNK, p38 and their phosphorylated forms in aortic valve tissue (400×); the quantification results are presented graphically. *P < 0.05, ***P < 0.001; n = 12 (B) Western blotting of IL-32, ERK, JNK, p38 and their phosphorylated forms in aortic valve tissue. Quantification of protein levels was done by ImageJ, with the levels of each phosphor protein normalized to the relevant total protein and the level of IL-32 normalized to β-actin. *P < 0.05; **P < 0.01 and ***P < 0.001; n = 12 (C) Immunohistochemical staining of NF-κB p65 in aortic valve tissue (400×); the quantification results are presented graphically; n = 10 (D) Western blotting of NF-κB p65 in nuclear extracts of aortic valve tissue.
Figure 2
Figure 3
IL-32 is mainly induced via the MAPkinases/NF-κB Pathway. (A) Cell culture of human aortic valve interstitial cells in 10% FBS M199 medium, (100×, 400×) (B) IL-32 level determined by ELISA in primary cultured aortic valve interstitial cells; n = 8 (C) Effect of inhibitors of NF-κB, ERK, JNK, and p38 on IL-32 protein level (mean ± SEM); n = 4. Calcified human aortic valve interstitial cells were pretreated for 1 hour with various inhibitors as indicated (concentrations are given in 100 μM NF-κB inhibitor, 20 ng/mL ERK inhibitor, 1ng/mL JNK inhibitor, and 2.5 ng/mL p38 inhibitor). (*P < 0.05; **P < 0.01 and ***P < 0.001).
Figure 3
Figure 4
Signal transducer and phospho-activator of transcription 3 (p-STAT3)/IL-32 activity increased in human calcified aortic valve tissue. (A) Immunohistochemistry staining of p-STAT1 and p-STAT3 expression in aortic valve tissue (400×). (B) Immunoreactivity of p-STAT1 and p-STAT3 with over-expression of serum IL-32 in control (level 1: mean <10% nuclei or cytoplasma of human aortic valve interstitial cells stained, and level 2: mean ≧10% to <25% nuclei or cytoplasma of human aortic valve interstitial cells); n = 5 (C) Immunoreactivity of p-STAT1 and p-STAT3 with over-expression of serum IL-32 in calcified aortic valve tissue (level 1: mean <10% nuclei or cytoplasma of human aortic valve interstitial cells stained, and level 2: mean ≧10% to <25% nuclei or cytoplasma of human aortic valve interstitial cells); n = 5 (*P < 0.05).
Figure 4
Figure 5
IL-32 induced expression of downstream cytokines. IL-8, TNF-α, IL-6, and IL-1β levels determined by ELISA in primary cultured aortic valve interstitial cells treated with (IL-32) or without (Control) recombinant IL-32; n = 12 (*P < 0.05; **P < 0.005).
Figure 5
Figure 6
IL-32 plays an essential role in human calcified aortic valve via the regulation of the STAT3, MAPK, and NF-κB pathways.
Figure 6
Auteurs
1 Division of Cardiovascular Surgery, Taichung Veterans General Hospital, Taiwan
2 Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan
3 Division of Allergy, Immunology & Rheumatology, Changhua Christian Hospital, Changhua, Taiwan
4 Department of Nursing, College of Medicine & Nursing, Hungkuang University, Taichung, Taiwan
5 Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan
6 Department of Pediatrics, Lotung Poh-Ai Hospital, I-Lan, Taiwan
7 Division of Immunology and Rheumatology, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan
8 School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan
9 Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan
10 Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan
Interleukin-32 (IL-32) is an inflammatory cytokine produced mainly by T, natural killer, and epithelial cells. Previous studies on IL-32 have primarily investigated its proinflammatory properties. The IL-32 also has been described as an activator of the p38 mitogen-activated protein kinase (MAPK) and NF-κB, and induces several cytokines. In this study, we hypothesized that the inflammatory regulators NF-κB, MAP kinase, STAT1, and STAT3 are associated with the expression of the IL-32 protein in human calcified aortic valve cells. This study comprised aortic valve sclerotic patients and control group patients without calcified aortic valve. Increased IL-32 expression in calcified aortic valvular tissue was shown by immunohistochemical staining and western blotting. There was an increase in NF-κB p65 level, p-ERK, p-JNK, and p-p38 MAPK activation underlying IL-32 expression in the study. The level of p-STAT3 but not p-STAT1 was found to be increased in calcified aortic valve tissue. In cultured primary human aortic valve interstitial cells, inhibition of NF-κB or MAPK kinase pathways results in a decrease of IL-32 expression. Treatment of recombinant IL-32 induced the levels of TNF-α, IL-6, IL-1β, and IL-8. Our findings demonstrate that IL-32 may be an important pro-inflammatory molecule involved in calcific aortic valve disease.