Pro-inflammatory mediators released by activated monocytes promote aortic valve fibrocalcific activity

Figures

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  Fig. 1. Pam3 CM promotes collagen and calcium deposition in AVICs. A AVICs were cultured in the growth medium and treated with Pam3 (0.03 μg/ml), control CM or Pam3 CM for 14 days. Representative images of Picro-Sirius Red staining and spectrophotometric quantitation normalized by cell density show that increased collagen deposition occurred in AVICs exposed to Pam3 CM, not in cells exposed to control CM or a low concentration of Pam3. B AVICs were cultured in pro-osteogenic medium and treated with Pam3 (0.03 μg/ml), control CM or Pam3 CM for 10 days. Representative images of Alizarin red S staining and spectrophotometric quantitation show that Pam3 CM, not control CM or a low concentration of Pam3, increased calcium deposition in AVICs. *P < 0.05 vs. untreated control, Pam3 or control CM. All quantitative data are presented as mean ± SEM. Each experiment was performed with cells isolated from 5 different donors

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  Fig. 2. Pam3 CM up-regulates fibrogenic and osteogenic mediators in AVICs independent of TLR2. A–C AVICs were exposed to Pam3 (0.03 μg/ml), control CM or Pam3 CM for 48 h. Levels of collagen I, collagen IV, MMP-2, MMP-9, ALP and RUNX2 were determined by immunoblotting. Representative immunoblots and densitometric data show that Pam3 CM selectively up-regulated collagen I, MMP-2 and ALP in AVICs among the fibrogenic and osteogenic factors examined while control CM or a low concentration of Pam3 had no effect. D AVICs were pretreated with TLR2 inhibitor CU CPT 22 (10 µM) or DMSO for 1 h and then exposed to Pam3 CM treatment for 48 h. Inhibition of TLR2 in AVICs did not alter the effect of Pam3 CM on the upregulation of collagen I, MMP-2 and ALP. Data are presented as mean ± SEM. n = 5 cell isolates from distinct donor valves in each group. *P < 0.05 vs. untreated control

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  Fig. 3. RANTES contributes to the up-regulation of fibrogenic mediators, and TNF-α mediates Pam3 CM-induced up-regulation of both fibrogenic and osteogenic mediators. A ELISA assay found that Pam3 CM has greater levels of TNF-α and RANTES in comparison to control CM. Data are presented as mean ± SEM. n = 5, *P < 0.05 vs. control CM. B and C AVICs were treated for 48 h with Pam3 CM pre-incubated with neutralizing antibodies (10 μg/ml) against RANTES or TNF-α. Non-immune mouse IgG (10 μg/ml) was added to Pam3 CM as a control. Neutralization of RANTES reduced the potency of Pam3 CM on the expression of collagen I and MMP-2 while neutralization of TNF-α attenuated the effect of Pam3 CM on the levels of collagen I, MMP-2 and ALP. Data are presented as mean ± SEM. n = 5 cell isolates from distinct donor valves in each group. *P < 0.05 vs. untreated control. #P < 0.05 vs. Pam3 CM and Pam3 CM with IgG

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  Fig. 4. RANTES is involved in Pam3 CM-induced JNK phosphorylation, and TNF-α mediates the effect of Pam3 CM on JNK and NF-κB phosphorylation. A AVICs are treated with Pam3 CM for 0.5, 1, 2 or 4 h. Representative immunoblots and densitometric data show that Pam3 CM induces the phosphorylation of p38, ERK1/2, JNK and NF-κB. *P < 0.05 vs. 0 h. Data are presented as mean ± SEM. n = 5 cell isolates from distinct donor valves in each group. B AVICs were treated for 1 h with Pam3 CM pre-incubated with neutralizing antibodies (10 μg/ml) against TNF-α, RANTES or both. Neutralization of TNF-α attenuated the effect of Pam3 CM on JNK and NF-κB phosphorylation. Neutralization of RANTES attenuated the effect of Pam3 CM on JNK phosphorylation. *P < 0.05 vs. untreated control. #P < 0.05 vs. Pam3 CM or Pam3 CM treated with non-immune IgG. Data are presented as mean ± SEM. n = 5 cell isolates from distinct donor valves in each group

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  Fig. 5. JNK mediates fibrogenic mediator production, while NF-κB mediates the osteogenic mediator production. A and B AVICs were treated with JNK inhibitor SP600125 (10 μmol/L) or DMSO for 1 h followed by Pam3 CM treatment for 48 h. Inhibition of JNK abolished Pam3 CM-induced collagen I and MMP-2 up-regulation, but had no effect on ALP up-regulation. C and D AVICs were treated with NF-κB inhibitor Bay 11-7082 (10 μmol/L) or DMSO for 1 h followed by Pam3 CM treatment for 48 h. Inhibition of NF-κB suppressed ALP up-regulation by Pam3 CM, but had no effect on the up-regulation of collagen I and MMP-2 induced. All data are presented as mean ± SEM. n = 5 cell isolates from distinct donor valves in each group. *P < 0.05 vs. untreated control. #P < 0.05 vs. Pam3 CM with DMSO

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  Fig. 6. Schematic diagram depicting the mechanism underlying AVIC fibrocalcification induced by medium conditioned by activated monocytes. Pro-inflammatory mediators produced by monocytes activated by TLR-2 agonist induce AVIC fibrocalcification involving up-regulation of fibrogenic and osteogenic mediators. RANTES contributes to the fibrogenic response induced by Pam3 CM through activation of JNK, and TNF-α mediates the pro-fibrogenic and pro-osteogenic effects of Pam3 CM through activation of both JNK and NF-κB