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 journalArticle

28 Citations (Scopus)

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 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 (US)
Pages (from-to)381-384
Number of pages4
JournalJournal of Surgical Research
Volume99
Issue number2
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Nicotine
Growth
Human Umbilical Vein Endothelial Cells
Atherosclerosis
Coloring Agents
Endothelial Cells
Smoking
Aptitude
Peripheral Vascular Diseases
In Vitro Techniques
Plastics
Analysis of Variance
Cell Count
Confidence Intervals
Staining and Labeling
Cytokines
Students
Control Groups
Wounds and Injuries
Serum

Keywords

  • Atherogenesis
  • Cytokines
  • Endothelium
  • Nicotine

ASJC Scopus subject areas

  • Surgery

Cite this

Albaugh, G., Kann, B., Strande, L., Vemulapalli, P., Hewitt, C., & Alexander, J. B. (2001). Nicotine induces endothelial TNF-α expression, which mediates growth retardation in vitro. Journal of Surgical Research, 99(2), 381-384. https://doi.org/10.1006/jsre.2001.6215

Nicotine induces endothelial TNF-α expression, which mediates growth retardation in vitro. / Albaugh, Gregory; Kann, Brian; Strande, Louise; Vemulapalli, Pratibha; Hewitt, Charles; Alexander, James B.

In: Journal of Surgical Research, Vol. 99, No. 2, 2001, p. 381-384.

Research output: Contribution to journalArticle

Albaugh, G, Kann, B, Strande, L, Vemulapalli, P, Hewitt, C & Alexander, JB 2001, 'Nicotine induces endothelial TNF-α expression, which mediates growth retardation in vitro', Journal of Surgical Research, vol. 99, no. 2, pp. 381-384. https://doi.org/10.1006/jsre.2001.6215
Albaugh, Gregory ; Kann, Brian ; Strande, Louise ; Vemulapalli, Pratibha ; Hewitt, Charles ; Alexander, James B. / Nicotine induces endothelial TNF-α expression, which mediates growth retardation in vitro. In: Journal of Surgical Research. 2001 ; Vol. 99, No. 2. pp. 381-384.
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title = "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 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.",
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AU - Strande, Louise

AU - Vemulapalli, Pratibha

AU - Hewitt, Charles

AU - Alexander, James B.

PY - 2001

Y1 - 2001

N2 - 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.

AB - 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.

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