The interaction between cigarette smoke, oxidative stress, inflammation and cardiovascular disease has been widely reported [14
] and there is increasing evidence of a role for osteopontin as an inflammatory mediator in atherosclerosis [6
]. Here, we report that exposure of endothelial cells to cigarette smoke PM in vitro
induced the expression of osteopontin, a previously undescribed effect which may have important implications for our understanding of the association of smoking with cardiovascular disease. While a similar effect has previously been reported in human bronchial epithelial cells [17
], this is the first instance where osteopontin production has been reported in response to cigarette smoke exposure in endothelial cells. Thus, the endothelium is a potential source of smoke-induced inflammatory osteopontin protein and this may play a role in cardiovascular disease. The mechanism of this endothelial response likely involves oxidative stress since osteopontin induction was diminished by treating cells with the antioxidant, ascorbate. Furthermore, our studies highlight a potential role for MMP cleavage in regulating the levels of cleaved forms of osteopontin, which have different functional attributes [11
The analysis of gene expression was important to verify that cigarette smoke-induced changes in protein expression were due to transcriptional regulation and not transient changes in local protein concentration. This finding of altered osteopontin gene expression is in accordance with the increased osteopontin mRNA expression seen in atherosclerotic plaques in vivo
, a measurement which closely associates with the severity of atherosclerosis and plaque calcification [13
]. This suggests that the in vitro
model used in this study to examine smoke-induced alteration of gene expression may be an appropriate one in which to further examine the inflammatory and atherogenic effects of cigarette smoke extracts.
Subsequent to the gene expression analysis we demonstrated that osteopontin protein levels were elevated following cigarette smoke extract exposure and that this effect was reduced by antioxidant pre-treatment. Cigarette smoke is both a source and a cause of the production of cellular reactive oxygen species. The pro-inflammatory effects of cigarette smoke have been well described [17
] and these may occur through activation of oxidative stress-regulated transcription factors such as NFκB or AP-1, which regulate the expression levels of inflammatory mediators. Given our current data, we propose that endothelial-derived osteopontin may be a mediator of smoke- and oxidative stress-induced inflammation with a role in the development and/or progression of cardiovascular disease.
The functional effects of osteopontin are determined not only by its expression levels but also by its cleavage by various enzymes into functionally diverse fragments [12
]. The matrix metalloproteinase family of enzymes regulate the accumulation of extracellular matrix and contribute to the growth of the atherosclerotic plaque and therefore its stability through excessive degradation of plaque cellular matrix. In particular, MMP-3 has been implicated as a strong regulator of vascular re-modelling and polymorphisms in the MMP-3 gene are linked to an increased risk of cardiac events [22
]. As well as being co-localised at sites of tissue injury [12
], osteopontin has been described as a substrate for MMP-3 and this may modify its integrin binding properties as well as its ability to initiate downstream signalling events [11
]. In this study, we demonstrated that PM enhanced the expression of both the full-length and the MMP-3-cleaved osteopontin fragments. Interestingly, PM-induced elevation of MMP-3 itself was ascorbate-insensitive. Oxidative stress therefore may be differentially involved in the production of functionally-dissimilar osteopontin fragments and functional changes in osteopontin effects can be mediated by cigarette smoke acting on distinct and non-oxidative stress-dependent parts of the pathway.
Oxidative stress has been proposed to play a prominent role in numerous smoking-related diseases including atherosclerotic cardiovascular disease [14
] and it has also been shown that the oxidative stress induced by smoking is rapidly reversible on cessation [15
]. Given our data obtained using an in vitro
cardiovascular disease model we hypothesised that, in a short-term study in subjects who refrained from smoking, osteopontin levels would be reduced. Our data presented here support this hypothesis by demonstrating a decrease in serum osteopontin levels in healthy subjects who quit smoking for 5
days, compared to a control group who continued to smoke 20 cigarettes per day. While further studies are needed to examine the longer-term effects of smoking cessation on both biomarkers of oxidative stress/inflammation and on the levels of osteopontin in serum, this study indicates that osteopontin can be a potential short-term biomarker of inflammation/oxidative stress.