Nicotine induces endothelial TNF-alpha expression, which mediates growth retardation in vitro

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-alpha due to its many biologic actions that are similar to those found in peripheral vascular disease.

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-alpha. Digital image analysis (DIA) was performed to quantify expression of TNF-alpha. 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 x 10(3) cells/cm(2) on T(-2) day. FBN was diluted in medium to 10(-9) M and added to wells with and without 0.9 microg/ml anti-TNF-alpha on T(0) day. Cell counts were performed in triplicate on days T(2-5) utilizing hemocytometry. Data was analyzed using Student's t test and ANOVA, with a 95% confidence interval.

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-alpha 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 T(2)-T(5) (P < 0.05). In cells exposed to anti-TNF-alpha 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-alpha group were not statistically significant.

These data demonstrate the ability of endothelial cells to secrete TNF-alpha 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-alpha mediated.