Nicotine Induces Endothelial TNF-α Expression, Which Mediates Growth Retardation in Vitro

Abstract

Purpose. Atherosclerosis is understood as the common pathologic manifestation of arterial injury caused by a variety of etiologies. One well-established etiologic agent is nicotine. We hypothesized that cytokines of endothelial origin are involved with the pathologic changes found in atherosclerosis associated with smoking. We chose to assay for TNF-α due to its many biologic actions that are similar to those found in peripheral vascular disease.Methods. Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM-2) on plastic coverslips until 75% confluent. Free base nicotine (FBN) was diluted in EGM-2 to a concentration of 10−8 M and added to experimental cells. At 1, 3, and 24 h, coverslips were removed and fixed. Immunohistochemical staining was performed using anti-TNF-α. Digital image analysis (DIA) was performed to quantify expression of TNF-α. An intensity stain index measuring area and intensity of stain/total cellular area was determined for each time point (n = 5). Additional HUVEC were plated in 12-well plates in EGM-2 at 2 × 103 cells/cm2 on T−2 day. FBN was diluted in medium to 10−9 M and added to wells with and without 0.9 μg/ml anti-TNF-α on T0 day. Cell counts were performed in triplicate on days T2–5 utilizing hemocytometry. Data was analyzed using Student's t test and ANOVA, with a 95% confidence interval.Results. Dose response determinations showed that the minimal concentration required to show statistically significant cell retardation is 10 –9 M. Accordingly, this concentration was used for subsequent proliferation studies. DIA showed a threefold increase in TNF-α activity at 1 h and a twofold increase at 3 h. Activity returned to baseline by 24 h. Cell growth was significantly decreased in cells exposed to nicotine when compared to controls on days T2–T5 (P < 0.05). In cells exposed to anti-TNF-α and nicotine there was inhibition of the growth retardation seen in the cells containing nicotine alone. Differences between the control group and the anti-TNF-α group were not statistically significant.Conclusion. These data demonstrate the ability of endothelial cells to secrete TNF-α in response to nicotine at levels found in serum after smoking and also shows that endothelial cell growth retardation as a consequence of nicotine exposure may be TNF-α mediated.