The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells.

AIM: To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs). METHODS: Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFß1, a critical profibrotic factor, in VSMCs. RESULTS: Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFß1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFß1. Spironolactone was also shown to significantly inhibit TGFß1 and Sp1 expression but not ERK1/2 phosphorylation. CONCLUSION: These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.

Simultaneous TLR2 inhibition and TLR9 activation synergistically suppress tumor metastasis in mice.

AIM: To develop a rational immunotherapy against tumor metastasis by combining a Toll-like-receptor 2 (TLR2)-neutralizing antibody with a TLR9 agonist CpG ODN, and then investigate the mechanism of action for this combinational regimen. METHODS: After mouse melanoma B16-F10 cell inoculation, female C57BL/6 mice were treated with either CpG ODN (0.5 mg/kg) or the anti-TLR2 antibody (200 µg/kg), or with a combination of the two agents. Pulmonary metastases were evaluated by counting metastatic nodes on the lung surface using anatomical microscopy. Flow cytometry was used to evaluate the cytotoxicity of the immune cells in tumor-draining lymph nodes, the cell population in the spleen, and the infiltration of immune cells within the lungs. Cytokine and enzyme expression in the lung tissue was evaluated using ELISA or immunostaining. RESULTS: Anti-metastatic effects were detected in mice treated with either CpG ODN or the anti-TLR2 antibody alone. However, treatment with CpG ODN plus the anti-TLR2 antibody synergistically suppressed the metastasis as compared with treatment with either single agent. The combinational treatment resulted in enhanced infiltration of natural killer cells and cytotoxic T cells, reduced recruitment of type 2 macrophages and Tregs, and decreased expression of immunosuppressive factors including TGF-ß1, cyclooxygenase-2 and indoleamine 2,3-dioxygenase, thus stimulated tumor cytotoxicity and suppressed metastasis. The anti-metastatic effect of the combinational regimen was further confirmed in spontaneous metastatic mouse model of Lewis lung carcinoma. CONCLUSION: Our studies suggest that combining a TLR9 agonist with an anti-TLR2 antibody, which eliminates immunosuppressive factors from the tumor environment, is critical for an effective anti-metastatic immunotherapy.