bay-11-7082 and Atherosclerosis

Hyperuricemia is an important risk factor for atherosclerosis, yet the potential mechanisms are not well understood. Migration and adhesion of leukocytes to endothelial cells play key roles in initiation and development of atherosclerosis. We investigated monocyte-endothelial cell interactions and potential signaling pathways under uric acid (UA)-stimulated conditions.. Primary human umbilical vein endothelial cells (HUVECs) were cultured and exposed to different concentrations of UA for various periods. Experimental hyperuricemia rat models were established. Expression of chemoattractant protein-1 (MCP-1), interleukin 8 (IL-8), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were evaluated. Monocyte-endothelial cell interactions were elucidated by chemotaxis and adhesion assays, and nuclear factor-kappa B (NF-κB) pathway was studied using fluorescent microscopy and electrophoretic mobility shift assay. Results showed that high concentration of UA stimulated generation of chemokines and adhesion molecules in ex vivo and in vivo experiments. Migration and adhesion of human monocytic leukemia cell line THP-1 cells to HUVECs were promoted and activated NF-κB was significantly increased. UA-induced responses were ameliorated by organic anion transporter inhibitor probenecid and NF-κB inhibitor BAY11-7082. It was also observed that human endothelial cells expressed urate transporter-1, which was not regulated by UA.. High concentration of UA exerts unfavorable effects directly on vascular endothelium via the NF-κB signaling pathway, the process of which requires intracellular uptake of UA.

Topics: Animals; Atherosclerosis; Cell Adhesion; Cell Adhesion Molecules; Cell Survival; Chemokine CCL2; Chemokines; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Hyperuricemia; Intercellular Adhesion Molecule-1; Interleukin-8; Male; Monocytes; NF-kappa B; Nitriles; Organic Anion Transporters; Organic Cation Transport Proteins; Rats; Rats, Wistar; Signal Transduction; Sulfones; Uric Acid; Vascular Cell Adhesion Molecule-1

Tumor necrosis factor-α (TNF-α) is an established pro-atherosclerotic factor, but the mechanism is not completely understood. We explored whether TNF-α could promote atherosclerosis by increasing the transcytosis of lipoproteins (e.g., LDL) across endothelial cells and how NF-κB and PPAR-γ were involved in this process. TNF-α significantly increased the transcytosis of LDL across human umbilical vein endothelial cells (HUVECs) and stimulated an increase of subendothelial retention of LDL in vascular walls. These effects of TNF-α were substantially blocked not only by transcytosis inhibitors, but also by NF-κB inhibitors and PPAR-γ inhibitors. In ApoE(-/-) mice, both NF-κB and PPAR-γ inhibitors alleviated the early atherosclerotic changes promoted by TNF-α. NF-κB and PPAR-γ inhibitors down-regulated the transcriptional activities of NF-κB and PPAR-γ induced by TNF-α. Furthermore, cross-binding activity assay revealed that NF-κB and PPAR-γ could form an active transcription factor complex containing both the NF-κB P65 subunit and PPAR-γ. The increased expressions of LDL transcytosis-related proteins (LDL receptor and caveolin-1, -2) stimulated by TNF-α were also blocked by both NF-κB inhibitors and PPAR-γ inhibitors. TNF-α promotes atherosclerosis by increasing the LDL transcytosis across endothelial cells and thereby facilitating LDL retention in vascular walls. In this process, NF-κB and PPAR-γ are activated coordinately to up-regulate the expression of transcytosis-related proteins. These observations suggest that inhibitors of either NF-κB or PPAR-γ can be used to target atherosclerosis.

Topics: Anilides; Animals; Atherosclerosis; Benzamides; Caveolin 1; Caveolin 2; Cinchona Alkaloids; Filipin; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins, LDL; Mice; Mice, Knockout; NF-kappa B; Nitriles; PPAR gamma; Proline; Pyridines; Receptors, LDL; RNA, Small Interfering; Signal Transduction; Sulfones; Thiocarbamates; Transcytosis; Tumor Necrosis Factor-alpha

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE(-/-) mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (-34%; p<0.005) and uptake (-38%, p<0.05). rHSP27 reduced SR-A mRNA (-39%, p=0.02), total protein (-56%, p=0.01) and cell surface (-53%, p<0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p<0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE(-/-) and ApoE(-/-) SR-A(-/-) mice fed with a high fat diet were treated for 3weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE(-/-) mice by 39% and 36% (p<0.05), respectively, but not in ApoE(-/-)SR-A(-/-) mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.

Topics: Animals; Aorta; Apolipoproteins E; Atherosclerosis; Cell Line; CHO Cells; Cricetulus; Diet, High-Fat; Disease Models, Animal; Female; Foam Cells; Gene Expression Regulation; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Mice; Mice, Knockout; Molecular Chaperones; Neoplasm Proteins; NF-kappa B; Nitriles; Scavenger Receptors, Class A; Signal Transduction; Sulfones

Previous studies have shown that elevated homocysteine (Hcy) levels promote the development of atherosclerotic lesions in atherosclerosis-prone animal models. There is evidence that oxidant stress contributes to Hcy's deleterious effects on the vasculature. The accumulation and adhesion of monocytes to the vascular endothelium is a critical event in the development of atherosclerosis. We investigated the effects of Hcy on the interaction between human endothelial cells (EC) (EC line EA.hy 926 and primary human umbilical vein EC [HUVEC]) and the monocytic cell line THP-1, and the impact of vascular oxidant stress and redox-sensitive signaling pathways on these events.. L-Hcy, but not D-Hcy, increases the production of reactive oxygen species inside EC, enhances nuclear factor(NF)-kappaB activation, and stimulates intercellular adhesion molecule-1 (ICAM-1) RNA transcription and cell surface expression. This leads to a time- and dose-dependent increase in monocyte adhesion to ECs. Pretreatment of ECs with superoxide scavengers (MnTBAP and Tiron) or with an inhibitor of NF-kappaB activation abolished Hcy-induced monocyte adhesion, ICAM-1 expression, and nuclear translocation of NF-kappaB.. These findings suggest that reactive oxygen species produced under hyperhomocysteinemic conditions may induce a proinflammatory situation in the vessel wall that initiates and promotes atherosclerotic lesion development.

Topics: Atherosclerosis; Cell Adhesion; Cell Line, Tumor; Dose-Response Relationship, Drug; Endothelium, Vascular; Homocysteine; Humans; Intercellular Adhesion Molecule-1; Isomerism; Leukemia, Monocytic, Acute; Molecular Conformation; Monocytes; NF-kappa B; Nitriles; Oxidation-Reduction; Reactive Oxygen Species; Signal Transduction; Sulfones; Superoxides; Umbilical Veins; Vasculitis