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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 2, 2003 as doi:10.1096/fj.03-0312fje. |
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* Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria; and
Institute of Analytical Chemistry and Radiochemistry, and
Institute for Zoology and Limnology, Leopold-Franzens-University of Innsbruck, Innsbruck, Austria
2Correspondence: Institute for Pathophysiology, University of Innsbruck, Fritz-Pregl-Str, 3/4, OG., A-6020 Innsbruck, Austria. E-mail: David Bernhard{at}uibk.ac.at
SPECIFIC AIMS
By the year 2020 smoking will be the largest single health problem worldwide. The proatherogenic activity of tobacco smoking is well defined from the medical point of view, but the mechanisms by which smoking contributes to atherosclerosis are largely unknown. Here we set out to examine the effect of cigarette smoking on the vascular endothelium.
PRINCIPAL FINDINGS
1. Human vascular endothelial cells exposed to tobacco smoke undergo cell death by necrosis
First we analyzed whether cigarette smoke exerts toxic effects on vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated with aqueous cigarette smoke extracts (TS) for 24 h in order to determine various cell death-associated phenotypes (Fig. 1
). Upon TS treatment, HUVECs show 1) a necrotic phenotype with the annexin method (Fig. 1a
), 2) reduced cell viability (Fig. 1b
), 3) no functional involvement of caspases in TS-mediated cell death (Fig. 1c
), 4) no caspase-3 activation (Fig. 1d
), and 5) no PARP cleavage (Fig. 1e
). These data demonstrate that TS causes a necrotic cell death of human vascular endothelial cells. Further, TS treatment kills HUVECs in a dose- and time-dependent fashion. As little as 8.4% TS comprising only 8.5% the nicotine from the blood of average smokers causes HUVEC cell death starting 12 h after the addition of TS.
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2. Vascular endothelial morphology changes dramatically upon treatment with cigarette smoke extracts
In response to TS treatment, HUVECs show cytoskeletal disruption and the breakdown of cell–cell junctions. Time dependence studies reveal that dramatic changes in cell morphology begin only a few minutes after the addition of TS. Scanning electron microscopic analyses support these findings by revealing that TS-treated HUVECs lose their typical "flat" morphology and intact junctional complexes by contracting to round "detachment-like" cells with cytoplasmic protrusions, indicating open junctions. The effect of TS on intact endothelial tissue is shown in Fig. 2
. Although endothelial cells (stained for CD31, green fluorescence) of whole tissue were more resistant to TS morphology effects, 33.2% TS led to the detachment of endothelial cells and consequently to leakiness of the endothelium.
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3. The damage causing cigarette smoke constituents are not stable and wash out rescues endothelial cells
To determine chemical characteristics of the bioactive tobacco smoke constituents, we performed experiments allowing us to draw conclusions on the stability of the "key player" compounds. The data gathered thereby clearly show that the extracts have lost their activity (to induce necrosis and to modulate cell morphology) after 6 h of incubation at 37°C. Time dependence analysis of the "point of no return" (the time of HUVEC incubation with TS necessary to cause cell death and irreversible morphological changes) showed that cells fully recover even after 6 h of incubation with TS.
4. Cigarette smoke effects can be blocked by pharmacological inhibitors
Finally, we screened for and found pharmacological inhibitors for TS effects. Vitamin C, curcumin, resveratrol, N-acetyl cysteine, and the statin atorvastatin (in a HMG-CoA reductase inhibition-independent fashion) protected HUVECs against TS-mediated morphology effects. However, only N-acetyl cysteine and atorvastatin were able to rescue HUVECs from TS-mediated necrosis.
CONCLUSIONS
A recent study from our lab revealed that smoking is the most important risk factor for the development of early atherosclerotic lesions in clinically healthy young males, highlighting the important contribution of smoking to the initiation of atherosclerosis. The present project was designed to reveal potential mechanisms involved in (accelerated) atherosclerosis initiation by cigarette smoking. We have shown that tobacco smoke induces necrosis of vascular endothelial cells (Fig. 1)
. Necrosis is defined as a passive form of cell death that includes the release of proinflammatory cytosolic constituents to the extracellular environment. Since inflammation is an important process contributing to atherogenesis, necrosis mediated by tobacco smoke might contribute to and promote atherogenesis. Another hallmark of atherosclerosis is thickening of the media, caused by proliferation of vascular smooth muscle cells (SMC). Leakiness of the endothelium, which we observed in response to TS treatment of cultured and tissue endothelium (Fig. 2)
, allows plasma-derived growth factors to directly access the SMC layer and stimulate SMC proliferation. The fact that morphological changes could be observed within minutes after the addition of TS makes it feasible that TS directly (chemically) affects the cell surface. Therefore, the participation of complex signal transduction cascades in this process can be excluded. Our results showing the biorelevant chemical characteristics of the noxious tobacco smoke constituents will be extended to identify hazardous raw materials. This approach may facilitate the reduction of tobacco product toxicity. Finally, we show that N-acetyl cysteine and the statin atorvastatin are capable of blocking TS effects on HUVECs. Inhibition of TS effects by atorvastin is of particular interest, because statins are the current state-of-the-art pharmaceuticals for the prevention and treatment of atherosclerosis. Our data may provide a biological explanation for the finding that smokers who take statins have a similar risk for cardiovascular events as untreated nonsmokers. However, the authors would like to stress that prevention is always better than treatment.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0312fje; doi; 10.1096/fj.03-0312fje 
This article has been cited by other articles:
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  D. Bernhard, A. Rossmann, B. Henderson, M. Kind, A. Seubert, and G. Wick Increased Serum Cadmium and Strontium Levels in Young Smokers: Effects on Arterial Endothelial Cell Gene Transcription Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 833 - 838. [Abstract] [Full Text] [PDF]
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 D. Bernhard, A. Csordas, B. Henderson, A. Rossmann, M. Kind, and G. Wick Cigarette smoke metal-catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules FASEB J, July 1, 2005; 19(9): 1096 - 1107. [Abstract] [Full Text] [PDF]
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