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

Gregory Albaugh, Brian Kann, Louise Strande, Pratibha Vemulapalli, Charles Hewitt, James B. Alexander

Research output: Contribution to journalArticlepeer-review

31 Scopus citations


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 I 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.

Original languageEnglish
Pages (from-to)381-384
Number of pages4
JournalJournal of Surgical Research
Issue number2
StatePublished - 2001
Externally publishedYes


  • Atherogenesis
  • Cytokines
  • Endothelium
  • Nicotine


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