| Objective: Dysfunction of vascular endothelial cells was one of the most important pathogeny on hypertension. Disequilibrium of diverse bioactive molecules synthesized and secreted by vascular endothelial cells would result in cardiovascular disorders. We recently showed that mitochondrial coupling factor 6 (CF6) was present as a pressor substance and a prostacyclin inhibitor in systemic circulation, it could make vascular contract and result in hypertension. Vascular endothelial cells could synthesize and secret coupling factor 6, and it would be the most important resource of circulating coupling factor 6. Recent results showed that the level of circulating CF6 was elevated in hypertension patients and CF6 would be a risk factor of hypertension. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-a (TNF-α) in the generation and release of CF6 in HUVEC and the effects oftroglitazone.Methods1. Primary cultured and identificated Endothelial Cells from Human Umbilical Vein.2. We used human umbilical vein endothelial cells as model, added TNF-α (100～250ng/ml) stimulator in the medium.The release and gene expression of CF6 in HUVEC were detected by radioimmunoassay and real-time RT-PCR, espectively. Flow cytometry analyzed the cell surface-associated CF6 level, and Immunostaining showed location of the protein p65, western blot showed the levels of the protein p65 in the nucleus and the protein IκBαin the cytoplasm.3. We used human umbilical vein endothelial cells as model, added TNF-α(100～250ng/ml) stimulator and troglitazone in the medium, detected the proteinsame as above.Results1. Teatment of HUVEC with TNF-a enhanced the release of CF6 in a dose - dependent manner. The RT-PCR results showed that the expression of coupling factor 6 mRNA was increased after 1.5-h treatment with TNF-a at 100 U/ml in HUVEC and increased after 1-h treatment with TNF-a at 250 U/ml in HUVEC. The RIA result showed that release of CF6 was enhanced after treatment with TNF-a of different concentration.(P＜0.05). Flow cytometry analysis revealed that the cell surfaceassociated CF6 was significantly increased at 24 h after treatment with TNF-a in a dose-dependent manner.2. Teatment of HUVEC with TNF-a enhanced the release of CF6 in a timedependent manner. The RIA result showed that release of CF6 was enhanced after treatment with TNF-a in the time course.(P＜0.05). But the level of CF6 in the cytoplasm did not change during the 24h.3. TNF-αcould activate NF-κB. Immunostaining showed that p65 translocated into nuclears after 6h treatment with TNF- a and the western blot telled us that the level of p65 in the nucleus was elevated. But the level of IκBαin the nucleus was decreased.4. troglitazone could inhibit the activation of IKK, block degradation of the Iκ Bαand inhibit the activation NF-κB. At last it resulted in expression blocking of CF6.Conclusions1. Teatment of HUVEC with TNF-a enhanced the release of CF6 in a dose and time-dependent manner.2. TNF-a enhanced the expression of CF6 in the HUVEC, and released to the medium and translocated onto the cellular membrane. The level of CF6 in the cells did not change.3. TNF-a enhanced the expression and release of CF6 in the HUVEC, and the high expression of CF6 would induce hypertension. So TNF-a could be a risk factor of hypertension.4. TNF-a induced increasing in the gene expression and release of CF6 is mediated by activation of NF- K B signaling pathway, so that interverntion of NF- K B signaling pathway can be the new target on the therapy of hypertension.5. troglitazone could inhibit the activation of IKK, block NF-κB signaling pathway and inhibited the expression of CF6. It may be the mechanism that TZDs cured hypertension...
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