This leads to acute manifestations such as myocardial infarction and stroke in which tissue oxygen and nutrient supply are severely compromised. A complication of these plaques is their vulnerability to rupture, giving rise to a thrombus with the ability to occlude vessels away from the initial plaque site [1,2,3]. Although atherosclerosis was initially considered a simple disease involving arterial lipid accumulation, it is now known to involve a cascade of inflammatory processes [1,2,3]. The initiating step in the development of an atherosclerotic lesion is damage to the endothelium [4,5], a monolayer of endothelial cells lining blood vessels which is a master regulator of vascular function. In a healthy individual and prior to the onset of CVD, the endothelium plays a homeostatic role in maintaining vascular tone and blood flow . In the early stages of CVD progression, endothelial damage and dysfunction triggers a chronic inflammatory process in the vessel wall. This ultimately involves numerous other cell types including vascular smooth muscle cells, monocytes (which become tissue macrophages and subsequently foam cells) and platelets [6,7,8,9].
Cigarette smoking is a well-described cardiovascular disease risk factor, and this is at least in part due to the pro-atherogenic effects of smoke and smoke toxicants within the cardiovascular system [10,11]. The primary focus of the cardiovascular disease group is to develop in vitro models of disease processes in order to test reduced toxicant prototypes and to contribute to the assessment of whether such products are likely to present reduced risks. These models will also be used to drive forward our knowledge of the effects of individual and collective smoke toxicants on cardiovascular function, with the aim of identifying pathogenic toxicants for reduction and/or removal by new technologies. A further focus of the group’s work is to identify and characterise clinical biomarkers of smoke-induced cardiovascular disease which align to the in vitro models workstreams and which will be used for the assessment of reduced harm potential in smokers in clinical studies.
The effects of smoking on the cardiovascular system are complex and involve the interplay of a series of up- and down-regulations of the expression of a host of both pro- and anti-atherogenic genes and proteins which ultimately leads to altered disease susceptibility. Prominently, altered endothelial gene and protein expression may enhance the susceptibility to both initiation and progression of atherosclerosis. These genes and proteins fall into different functional categories, for example those involving monocyte adhesion, inflammation, responses to oxidative stress or extracellular matrix regulation. We have developed an in vitro model of the endothelium, using human umbilical vein endothelial cells (HUVECs), in which we examine changes in the expression of genes and proteins involved in atherosclerosis following exposure to both tar and vapour phase cigarette smoke extracts. This work is carried out using multiple and integrated technologies including the PCR SuperArray platform to monitor gene expression changes and the MesoScale Discovery (MSD®) electrochemiluminescence detection platform to monitor the expression of proteins such as the adhesion molecules ICAM, VCAM and E and P selectins .
Endothelial damage and dysfunction is a critical initiating step in atherosclerosis. While endothelial injury may initiate atherosclerosis, endothelial repair by the processes of migration and proliferation re establish endothelial integrity and are atheroprotective. Smoking has long been recognised for its ability to cause gross structural damage to the endothelium and endothelial dysfunction. Various data support the hypothesis that an impaired migratory capacity of endothelial cells in smokers supports cardiovascular disease initiation and development [e.g.14], and this has led to our development of an in vitro model of endothelial migration. In this assay, confluent HUVECs are ‘scratched’ using a pipette and the migration of endothelial cells into the damaged region is monitored by imaging the scratch wound at periodic intervals using the IncuCyte apparatus. Data from this assay show a concentration dependent inhibition of endothelial migration by both particulate and vapour phase cigarette smoke extracts.
Angiogenesis is defined as the growth of new blood vessels from the pre-existing vasculature and is a critical component of both physiological and pathological processes which ensure tissue oxygen and nutrient demands are met. Angiogenesis is clinically beneficial, for example in recovery after the reperfusion of ischemic tissue or cardiac repair and also following systemic wounding and inflammation. Angiogenesis is further critical to the restoration of the blood supply to the brain following ischemic stroke. The processes underlying angiogenesis are complex and involve endothelial proliferation, migration, differentiation and structural re-arrangement (tube formation).
Many studies have examined the detrimental effects of smoking on endothelial angiogenic responses. We are currently utilising a 2-dimensional Matrigel assay to examine the effects of cigarette smoke extracts on endothelial angiogenesis. Our data have demonstrated inhibition of angiogenesis in this model by cigarette smoke extracts (below).
Picture on the upper left is the untreated control and picture on the upper right is cigarette smoke TPM treated(48 µg/ml).
Branches and curvatures of the vascular tree generate disturbed blood flow within vessels. At these sites, endothelial cells are physically exposed to disturbed blood flow, leading to stress and injury . Precise mapping of atherosclerotic lesions in damaged arteries has led to a hypothesised association between CVD and haemodynamics. In support of this, clinical and computational studies have demonstrated that certain arterial segments are prone to the development of atherosclerotic lesions or accumulation of indicators of lesion formation, while the adjacent areas remain unaffected [18,19,20]. Using a microfluidics platform, we can simulate haemodynamic stresses and expose endothelial cells to laminar or oscillatory shear stress in combination with cigarette smoke . This model allows us to investigate morphological changes and assess expression of disease mediators such as pro-inflammatory markers and adhesion of monocytes to endothelial cells.
Circulating monocytes adhere to the damaged endothelium and migrate through the endothelial monolayer into the subendothelial space and differentiate to become tissue macrophages. Macrophages participate in atheroma formation and stability by accumulating lipids, thus becoming foam cells . By producing free radicals and cytokines, these cells also participate strongly in the inflammation associated with vascular atherosclerosis. Furthermore, a body of evidence suggests that macrophages secrete matrix metalloproteinases leading to the breakdown of the advanced plaque and predisposing it to rupture .
Using the monocytic cell line THP-1 and also by developing external collaborations we are currently examining changes in gene expression, protein secretion and free radical production in order to advance our understanding of the role of monocytes/macrophages in smoking-induced cardiovascular disease. This work is being carried out using a suite of integrated techniques which are described in more detail on the inflammation and oxidative stress page .
Picture to the right shows adhesion of monocytic THP-1 cells to HUVECs. HUVECs were seeded on microslides and conditioned with oscillatory flow (6 ml/min at a frequency of 1 Hz for 20 hours) prior to adding THP-1 cells to the perfusion system and monitoring .