High mobility group box 1 induces calcification of aortic valve interstitial cells via toll-like receptor 4

Figures

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  Figure 1.. Expression of HMGB1 and TLR4 in the calcification region of CAVD arterial valves. Human aortic valves with calcific and normal aortic valve leaflets were collected from explanted hearts of patients and were assessed by histological and IHC analyses. (A) Sections were stained with HE or with specific antibodies against HMGB1 and TLR4. The black boxes represent the area presented in the IHC staining images. (B) The expression levels of HMGB1 and TLR4 were detected by IHC and were quantified using Image Pro-Plus version 6.0. Data are presented as integrated optical density/area. Three different sections and five different fields of view were analyzed for each section (n=3). **P<0.01 vs. Con. HMGB1, high mobility group box 1; TLR4, toll-like receptor 4; CAVD, calcific aortic valve disease; IHC, immunohistochemistry; HE, hematoxylin and eosin; Con, control.

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  Figure 2.. HMGB1 induced calcification of AVICs. Primary AVICs were incubated with 0.5, 1 and 2 µg/ml HMGB1 for 3 weeks. Quantification of (A) ALP activity and (B) alizarin red S staining in AVICs. (C) Western blotting was used to determine the protein expression levels of Runx2, Msx2, BMP2 and OPN. (D) Quantification of band densities by densitometry in 3 independent experiments relative to β-actin (n=3). *P<0.05 and **P<0.01 vs. Con. HMGB1, high mobility group box 1; AVICs, aortic valve interstitial cells; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; Msx2, msh homeobox 2; BMP2, bone morphogenetic protein 2; OPN, osteopontin; Con, control.

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  Figure 3.. TLR4 mediates HMGB1-induced calcification in AVICs. Primary AVICs were derived from TLR4−/− and C57BL/6 mice. They were subsequently pretreated with a TLR4-specific antibody, and a mouse IgG was used as a control. (A) TLR4 expression in TLR4−/− and C57BL/6-derived AVICs was determined using western blotting (n=4 per genotype). Quantification of (B) ALP activity and (C) alizarin red S staining. (D) Western blotting was used to determine the protein expression levels. (E) Quantification of western blotting by densitometry in 3 independent experiments relative to β-actin (n=3). **P<0.01 vs. C57BL/6 group; ##P<0.01 vs. the mouse IgG treatment group. TLR4, toll-like receptor 4; HMGB1, high mobility group box 1; AVICs, aortic valve interstitial cells; ALP, alkaline phosphatase; Runx 2, runt-related transcription factor 2; Msx2, msh homeobox 2; BMP2, bone morphogenetic protein 2; OPN, osteopontin.

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  Figure 4.. HMGB1 activates p38 and NF-κB in AVICs via TLR4 signaling. Primary AVICs were incubated with 0.5, 1 and 2 µg/ml HMGB1 for 60 min. The group without HMGB1 treatment was used as a control. Primary AVICs derived from TLR4−/− mice or C57BL/6 mice, and C57BL/6-derived AVICs treated with a specific anti-TLR4 antibody (or mouse IgG as the control), were treated with 2 µg/ml HMGB1 for 60 min to determine the activation of p38 or NF-κB. The phosphorylation of p38 and NF-κB was detected by (A and C) western blotting and (B and D) quantified by densitometry in 3 independent experiments. *P<0.05 and **P<0.01 vs. 0 µg/ml HMGB1 control. The results are expressed as relative units (p38 or NF-κB phosphorylated protein/total protein). **P<0.01 vs. the C57BL/6 group; ##P<0.01 vs. the IgG group. HMGB1, high mobility group box 1; AVICs, aortic valve interstitial cells; p-p38, phosphorylated-p38; t-p38, total-p38; p-NF-κB, phosphorylated-nuclear factor κB; t-NF-κB, total-NF-κB; TLR4, toll-like receptor 4.