No abstract

Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. Objective Our goal was to investigate the role of smooth muscle Nox4 in atherosclerosis. Approach and results Atherosclerosis-prone conditions (disturbed blood flow and Western diet) increased Nox4 mRNA level in smooth muscle of arteries. To address whether upregulated smooth muscle Nox4 under atherosclerosis-prone conditions was directly involved in the development of atherosclerosis, mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), specifically in smooth muscle, were generated on a FVB/N ApoE deficient genetic background to counter the effect of increased smooth muscle Nox4. Nox4DN significantly decreased aortic stiffness and atherosclerotic lesions, with no effect on blood pressure. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly downregulated in Nox4DN smooth muscle cells (SMC), at both mRNA and protein levels. Downregulation of sEH by siRNA decreased SMC proliferation and migration, and suppressed inflammation and macrophage adhesion to SMC. Conclusions Downregulation of smooth muscle Nox4 inhibits atherosclerosis by suppressing sEH, which, at least in part, accounts for inhibition of SMC proliferation, migration and inflammation.

Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. To investigate the effect of celastrol on human multiple myeloma cell cycle arrest and apoptosis and explore its molecular mechanism of action. The activity of celastrol on LP-1 cell proliferation was detected by WST-8 assay. The celastrol-induced cell cycle arrest was analyzed by flow cytometry after propidium iodide staining. Nuclear translocation of the nuclear factor kappa B (NF-κB) was observed by fluorescence microscope. Celastrol inhibited cell proliferation of LP-1 myeloma cell in a dose-dependent manner with IC50 values of 0.8817 µM, which was mediated through G1 cell cycle arrest and p27 induction. Celastrol induced apoptosis in LP-1 and RPMI 8226 myeloma cells in a time and dose dependent manner, and it involved Caspase-3 activation and NF-κB pathway. Celastrol down-modulated antiapoptotic proteins including Bcl-2 and survivin expression. The expression of NF-κB and IKKa were decreased after celastrol treatment. Celastrol effectively blocked the nuclear translocation of the p65 subunit and induced human multiple myeloma cell cycle arrest and apoptosis by p27 upregulation and NF-kB modulation. It has been demonstrated that the effect of celastrol on NF-kB was HO-1-independent by using zinc protoporphyrin-9 (ZnPPIX), a selective heme oxygenase inhibitor. From the results, it could be inferred that celastrol may be used as a NF-kB inhibitor to inhibit myeloma cell proliferation.

Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. In this study, we investigated the effect of celastrol on lipopolysaccharide (LPS)-activated LP-1 human multiple myeloma cell-induced angiogenesis, and identified its molecular mechanism of action. Migration of human umbilical vein endothelial cells (HUVECs) was tested using a wound-healing assay. HUVEC invasion was assayed using a Transwell chamber. Cell surface expression of Toll-like receptor 4 (TLR4) was analyzed by flow cytometry. Angiogenic factor vascular endothelial growth factor (VEGF) level was quantified by LUMINEX and protein expression was analyzed by Western blot. Translocation of nuclear factor-kappa B (NF-κB) was observed by fluorescence microscopy. Celastrol inhibited LPS-stimulated LP-1 human multiple myeloma-induced HUVEC migration and invasion in a concentration-dependent manner. Wound diameters increased by 72.9, 165.4 and 246.2% at 0.025, 0.05 and 0.1 µM, respectively, compared to LPS alone. A 45-74% inhibition of LPS-dependent cell invasion was achieved in the presence of 0.025-0.1 µM celastrol. Celastrol significantly downregulated LPS-induced TLR4 expression and inhibited LPS-induced VEGF secretion in LP-1 cells. VEGF levels decreased by 64.8, 84.4 and 92.9% after coexposure to celastrol at 0.025, 0.05 and 0.1 µM, respectively, compared to LPS alone. Celastrol also inhibited the IκB kinase (IKK)/NF-κB pathway induced by LPS. Protein levels of NF-κB p65, IKKa and IκB-a decreased in a dose-dependent manner after coexposure to celastrol. Celastrol also blocked nuclear translocation of the p65 subunit. These results suggest that celastrol inhibits LPS-induced angiogenesis by suppressing TLR4-triggered NF-κB activation.

Induction of humoral anti-human high molecular weight melanoma-associated antigen (anti-HMW-MAA) immunity following active specific immunotherapy is associated with a statistically significant prolongation of survival in patients with melanoma. This association does not appear to be mediated by immunological mechanisms because anti-HMW-MAA antibodies are poor mediators of complement- and cell-mediated cytotoxicity of melanoma cells. Therefore, we have been investigating nonimmunological mechanisms by which anti-HMW-MAA antibodies (Abs) affect the biology of melanoma cells. We have demonstrated that anti-HMW-MAA mAbs interfere with the interaction of HMW-MAA with extracellular matrix (ECM) components, a process known to be crucial in the early phase of melanoma metastasis. Furthermore, anti-HMW-MAA mAbs appear to block the series of signal transduction events triggered by the interaction of HMW-MAA with ECM. They include the activation of the family of Rho GTPases, p130cas, and focal adhesion kinase (FAK). These findings parallel the inhibition of the rat homologue of HMW-MAA NG2 function by anti-NG2 antibodies. Taken together, all these results provide a mechanistic explanation not only for the therapeutic effect of anti-HMW-MAA antibodies in the treatment of melanoma, but also for the function of HMW-MAA in the biology of melanoma cells. This information is expected to serve as a useful background to design effective HMW-MAA-targeted immunotherapy in patients with melanoma.