The Effects And Mechanisms Of Nicotine On Apoptosis In Huvecs And Adhesion Of Monocytes To Huvecs

THE EFFECTS AND MECHANISMS OF NICOTINE ON APOPTOSIS IN HUVECS AND ADHESION OF MONOCYTES TO HUVECSEndothelial injury and the adhesion of monocytes to endothelial cells are considered to be key initiating events in the pathogenesis of cardiovascular disease. Smoking causes endothelial cell (EC) injury and enhances the adhesion between monocytes and endothelial cells; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury and adhesion are understood. It has been recently reported that nicotine, the addictive component of tobacco, is an important modulator at the level of programmed cell death and may be involved in smoking-induced endothelial cells injury and adhesion of monocutes to endothelial cells. This paper investigated the effects and the mechanisms of action of nicotine on apoptosis in HUVECs and the adhesion of monocytes to HUVECs mediated by macrophages (Ana-1).Part I: The effect of nicotine on apoptosis in human umbilicalvein endothelial cells.Recently, evidence has been accumulated that disturbance of balance between endothelial cell proliferation and apoptosis contributes to the endothelial cells injury and the development of atherosclerosis. Nicotine appears to play a role in the modulation of, but controversy exits in terms of how nicotine affects this process. Whereas some studies have demonstrated that nicotine induces apoptosis, other studies have demonstrated that nicotine inhibits apoptosis. We firstly investigated the effects and the mechanisms of action of nicotine on apoptosis. The human umbilicalvein endothelial cells (HUVECs) were grown in RPMI-1640. HUVECs, 20 X 105/ml HUVECs, were incubated at 37? in a humid chamber under 5%CO2 atmosphere, with the cells in culture medium serving as the negative control, and with DEX plus TNF-a serving as a positive control. In order to assess the impact of nicotine on apoptosis, cells were incubated with nicotine at different concentrations (0.1/iM, 0.5/iM, 1/iM and 2/iM) that correlates with the tissue levels of smokers,concurrently with tumor necrosis factor-alpha (TNF-a) and dexamethasone to induce apoptosis. The results showed that nicotine significantly inhibited apoptosis in HUVECs, as verified by flow cytometry, DNA agarose gel electrophoresis and TUNEL assay. Apoptosis is a process of paramount importance in maintaining homeostasis. The key mechanism that controls the outcome of atherogenesis is in maintaining the critical balance between cell survival and cell death during vascular repair. Inhibition of nicotine on apoptosis in HUVECs suggests that nicotine could lead to disruption of the critical balance between cell death and proliferation and may have an impact on endothelial injury during cardiovascular pathology and atherogenesis.Part II: The mechanisms of nicotine on apoptosis in human umbilicalvein endothelial cells.Nicotine can inhibit apoptosis induced by TNF- a and dexamethasone as described above. However, the intracellular mechanisms involved in nicotine suppression of apoptosis are unclear. Bcl2 is the first identified survival gene involved in the control of apoptosis. Because phosphorylation of Bcl2 can positively regulate the anti-apoptotic function of Bcl2, it is possible that nicotine-inhibited apoptosis may be associated with Bcl2 phosphorylation. To test this possibility, HUVECs expressing high levels of endogenous Bcl2 were metabolically labeled with 32P-orthophosphoric acid and treated with nicotine. Our findings indicated that nicotine can potently stimulate endogenous Bcl2 phosphorylation in HUVECs. However, the protein kinases involved is not clear. Because PKCa and MAPK ERKl/2 can directly phosphorylate Bcl2 in vitro and in vivo, we tested whether nicotine might activate either of these kinases to phosphorylate Bcl2 in HUVECs. Time course experiments indicated that nicotine induced activation of PKCa and ERKl/2. By contrast, nicotine has no effect on either JNK or p38. These findings suggested that nicotine-induced Bcl2 phosphorylation may occur thro...