two different outcomes of cigarette smoke condensate
Apoptosis and necrosis: two different outcomes of cigarette smoke condensate induced endothelial cell death
stores that carry cards against humanity, S Frotschnig2, A Steinacher Nigisch1, B Winter1, E Eichmair1, J Gebetsberger2,3, S Schwaiger4, C Ploner5, G Laufer1 and D Bernhard1Received 16 July 2012; Revised 28 August 2012; Accepted 4 September 2012
Edited by A Stephanou
Top of pageAbstractCigarette smoking is one of the most important and preventable risk factors for atherosclerosis. However, because of the complex composition of cigarette smoke, the detailed pathophysiological mechanisms are not fully understood. Based on controversial reports on the pro atherogenic activity of cigarette smoke condensate, also called tar fraction (CSC), we decided to analyse the effects of CSC on the viability of endothelial cells in vitro. The results of this study show that low concentrations of the hydrophobic tar fraction induces DNA damage resulting in a P53 dependent and BCL XL inhibitable death cascade. Western blot analyses showed that this cascade is caspase independent and immunofluorescence analysis have shown that the apoptotic death signalling is mediated by the release of apoptosis inducing factor. Higher CSC concentrations also induce apoptotic like signalling but the signalling cascade is then redirected to necrosis. Despite the fact that CSC induces a profound increase in cellular reactive oxygen species production, antioxidants exhibit only a minimal cell death protective effect. Our data indicates that not only hydrophilic constituents of cigarette smoke extract, but also CSC is harmful to endothelial cells. The mode and the outcome of CSC induced cell death signalling are highly concentration dependent: lower concentrations induce caspase independent apoptosis like cell death,
game cards against humanity, whereas incubation with higher concentrations interrupts apoptotic signalling and induces necrosis.
It is generally accepted that atherosclerosis is a multifactorial disease and many atherogenic processes lead to similar patho biological effects. However, of these effects, endothelial cell death (and therefore endothelial dysfunction) takes a central role in atherogenesis.7, 8 At the same time, the loss of endothelial cells has not only direct functional effects, but it is also crucial, which form of cell death is induced. Generally, cell death could be classified into two forms: apoptosis, also known as cell death and necrosis described as uncontrolled and accidental form of cell death.9, 10 Apoptosis is an energy dependent process characterised by cell shrinkage, nuclear condensation and fragmentation, as well as cellular fragmentation into the so called apoptotic bodies, which are removed by phagocytic cells. In contrast, anti apoptotic proteins of the Bcl 2 family (like BCl Xl) can inhibit mitochondrial membrane permeabilisation.17, 18 During mitochondrial membrane permeabilisation, two major groups of pro apoptotic proteins are released from the mitochondrial intermembrane space, determining the further signalling pathway.12, 19 Release of cytochrome c mediates the activation of a well known group of proteases, the caspases. Caspases are a group of cysteine proteases, which are divided into initiator and effector caspases. Among them, one of the crucial effector proteins is apoptosis inducing factor (AIF).12 In the course of caspase independent apoptotic signalling, AIF translocates into the nucleus where it induces chromatin condensation and the formation of large chromatin fragments, but the detailed mechanism remains unclear until now.22, 23
Over the last few years,
cards with humanity, two other types of programmed cell death were revealed: programmed necrosis and autophagy. The existence of a programmed necrosis is still controversially discussed as this form of cell death could only be demonstrated when apoptosis is inhibited.9, 11 Zong et al.24 demonstrated that DNA damage actively initiates a programmed form of necrosis, which is independent of well known apoptotic induction proteins like p53, Bax, Bak and caspases.
Also the classification of autophagy as an autonomous cell death mechanism is controversial.11 So far, autophagy is established as a survival mechanism of cells under nutrient deprivation by degradation and recycling of cellular components. Autophagic degradation of intracellular structures and organelles is induced by the formation of double membrane vesicles (autophagosomes) and the hydrolytic degradation of cellular structures after fusion with lysosomes (autolysosomes).25 One prominent marker to study autophagy is LC3 (microtubule associated protein light chain 3): conversion of LC3 I (cytoplasmatic form) to LC3 II (autophagosome specific form) is an indicator for the induction of autophagy.26
Based on different in vitro and in vivo studies, the hydrophilic fraction of cigarette smoke is well known to contain pro atherogenic compounds. Small hydrophilic components were shown to reach the circulation through the alveoli where they promote endothelial stress.27, 28 The resulting endothelial dysfunction as one of the first and critical steps in atherogenesis is caused by CSE induced endothelial cell contraction,27 the release of pro inflammatory cytokines,27 catalalysation of oxidative reactions,29 the increased expression of adhesion molecules30 and finally the induction of endothelial cell death (resulting in endothelial denudation).31, 32, 33
Conflicting and a sparse number of data exist on the atherosclerosis causing potential of the hydrophobic fraction. Penn et al.34 stated that intramuscularly injection of concentrated cigarette tar does not promote atherosclerotic plaque formation in cockerels. Indeed, studies investigating the effect of CSC in vivo are rare. However, various in vitro experiments revealed CSC induced cellular atherogenic alterations in endothelial cells as for instance: (a) genotoxic effects,35 (b) alteration in the cytokine expression,36 (c) upregulation of genes involved in matrix degradation,37 (d) CSC induced adherence of monocytes to the endothelial cell monolayer,38 (e) CSC induced surface expression of adhesion molecules on endothelial cells and transendothelial migration of monocytes39 and (f) decreased migration ability of endothelial cells in vitro.40 All the mentioned processes are well known to be involved in atherogenesis initiation and may contribute to vascular dysfunction and the atherogenic process in vivo. However, only few data are published addressing the mode and signalling pathway of CSC induced endothelial cell death in detail.
Top of pageResultsCSC inhibits the proliferation and induces cell death in human umbilical vein endothelial cellsTo examine the potential toxic effects of CSC on endothelial cells, we performed in vitro viability assays and tested for the induction of cell death by CSC. Based on the fact that the XTT assay cannot differentiate between the inhibition of proliferation and the induction of cell death, we also directly tested for the induction of cell death by annexin V iodide staining by FACS analysis. Figure 1b shows that CSC leads to an increase in the number of apoptotic and to a much lesser extent to an increase in necrotic cells. Another indicator of cell toxicity is the release of lactate dehydrogenase (LDH) through porous membranes. The contradictory observations between annexin V iodide staining and LDH assay was further explained by analysing the amount of DNA in treated cells. Mean values of a representative experiment performed in quadruplicates are shown. (b) FACS analysis of CSC induced cell death. Mean values of a representative experiment performed in triplicates (annexin V staining) or quadruplicates (XTT assay and LDH assay) are shown. (d) Shows images of scanning electron microscopic analysis of control and CSC treated endothelial cells (arrow: necrotic cell; star: apoptotic cell). Representative images are shown. The DNA content was analysed by FACS analysis. It is already known that DNA damage leads to the stabilisation of the tumour suppressor P53, and its activation, leading to pro apoptotic signalling in affected cells. The involvement of P53 in CSC induced cell death cascade was studied by western blot analyses. The massive P53 protein degradation observed may be caused by necrosis associated proteolytic processes. In conclusion, lower CSC concentrations activate the typical P53 dependent apoptotic death signalling pathway resulting in mitochondrial membrane depolarisation. (a) Shows data from a DNA damage analysis by the comet assay. Percent comet positive cells compared with the control are shown. Representative blots are shown. Data in (c) shows JC 1 based FACS analysis of intracellular mitochondrial membrane potential after CSC treatment for 2, 6, 12 and 24 Mean values of a representative experiment performed in triplicates (JC 1 staining) or quadruplicates (Comet assay) are shown. (d) Shows western blot analysis of caspase 3 in CSC treated endothelial cells. Representative blots are shown. Mean values are shown.
As P53 activated death signalling is known to be mediated through caspase activation,
which cards against humanity expansion is best, we performed western blot analysis of caspase 3, an important effector caspase. Endothelial cells were incubated with or without ursolic acid (UA) to obtain cytosolic extracts with active caspase 3. These extracts were then incubated with or without CSC and caspase activity was analysed.
Involvement of P53, BCL XL, AIF and autophagy in CSC induced cell death: differences between lower and high CSC concentrationsTo further analyse the involvement of P53 and the anti apoptotic mitochondrial protein BCL XL in CSC induced cell death, we infected endothelial cells with retroviruses containing a P53 shRNA vector and as P53 activated death signalling is known to be mediated through pro apoptotic BCL 2 family members we generated human umbilical vein endothelial cells (HUVECs) overexpressing the anti apoptotic protein BCL XL. Figure 3a shows that the knock down of P53 significantly inhibited CSC induced cell death at low and high concentrations. (a) Shows the result of annexin V iodide staining and FACS analysis of CSC treated P53 knock down and BCL XL overexpressing cells. (b) Shows also immunofluorescence pictures of endothelial cells (controls and CSC incubated cells) incubated with an isotype control (negative control; cells stained with FITC rabbit anti human IgG isotype). (c) FACS based analysis of lysosomal integrity. (d) Shows western blot analysis of LC 3 (autophagy marker). Mean values are shown. MFI fluorescence intensity
Full figure and legend (406K)
To analyse the caspase independent cell death signal downstream of the mitochondria, we performed immunofluorescence based analysis of CSC incubated endothelial cells with anti AIF antibody. In addition, we also tested for autophagic signalling in CSC treated endothelial cells as the lysosomes are central organelles in autophagic signalling. Therefore, we assessed the ratio of LC3 II per LC3 I. To further analyse the involvement of autophagy in CSC induced cell death, we incubated endothelial cells with 3 methyladenine (3 MA) and analysed CSC induced cell death. FACS based analyses showed that inhibition of autophagosome formation by 3 MA was not able to inhibit CSC induced cell death.
Cigarette smoke induced oxidative stress is partially preventable by antioxidantsTo test for the potential occurrence of oxidative stress, we performed DHR 123 based analyses of oxidative stress. To do so, endothelial cells were incubated with different CSC concentrations as well as 1 H2O2 for 24 (since H2O2 is a potent inducer of oxidative stress this incubation serves as positive control). In order to test for a potential ROS preventive effect of antioxidants (vitamin E, vitamin C and N acetyl cysteine (NAC)), we pre incubated endothelial cells with antioxidants and performed DHR 123 based analysis. Figure 4a shows that none of the above antioxidants was capable of preventing CSC induced ROS formation in endothelial cells. In addition, FACS based analyses of cell death revealed that neither vitamin C nor NAC were able to inhibit CSC induced reduction in endothelial cell viability (Figure 4b).
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