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Cardiovascular disease

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Atherosclerotic cardiovascular disease (CVD) is the major cause of mortality in the Western world. The major defining characteristic of this disease is the formation of atherosclerotic lesions or plaques which can occlude blood vessels and disrupt blood flow.

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 [5]. 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].

Schematic cross-section of the vessel wall

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.

Endothelial gene and protein expression

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 [7].

           Immunocytochemistry analysis of protein expression in HUVECs exposed to TPM. *, statistical differences based on P<0.05, unpaired Student's t-test

Endothelial migration

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

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).

Smoke extracts inhibit in vitro angiogenesis

Picture on the upper left is the untreated control and picture on the upper right is cigarette smoke TPM treated(48 µg/ml).

Endothelial gene expression and adhesion responses to disturbed flow

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 [17]. 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 [21].  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.

 Monocyte function

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 [3]. 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 [22].

Adhesion of monocytic THP-1 cells to HUVECs taken form Cockcroft et al (2009). [21]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 Opens new window.

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 [21].


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  12. Bishop, E., Fearon, I.M., Gaca, M. (2008). The effects of tobacco smoke on vascular endothelial cells-a potential model. Poster presented at Vasculata, Seattle, August 2008. Link to poster PDF. Opens new window
  13. Bishop, E., Fearon, I.M., Gaca, M. (2008). A potential model to examine the effects of tobacco smoke on vascular endothelial cells. Poster presented at Multiple Risk factors in Cardiovascular Disease, Venice, October 2008. Link to poster PDF. Opens new window
  14. Snajdar, R.M., Busuttil, S.J., Averbook, A., Graham, D.J. (2001). Inhibition of endothelial cell migration by cigarette smoke condensate. The Journal of Surgical Research. 96: 10 16.
  15. Fearon, I.M., Bishop, E., Gaca, M.D. (2009) Examination of the role of oxidative stress in cigarette smoke extract-induced impairment of endothelial migration in vitro. Poster, 2009. Link to poster PDF. Opens new window
  16. Acheampong, D., Bishop, E., Fearon, I.M. (2009). Effects of cigarette smoke extracts on endothelial migration are independent of oxidative stress. Oral Presentation at The Physiological Society Main Meeting, Dublin, July 2009. Link to PDF presentation. Opens new window
  17. Stone, P.H., Coskun, A.U., Kinlay, S., Clark, M.E., Sonka, M., Wahle, A., Ilegbusi, O.J., Yeghiazarians, Y., Popma, J.J., Orav, J., Kuntz, R.E., Feldman, C.L. (2003). Effect of endothelial shear stress on the progression of coronary artery disease, vascular remodeling, and in in-stent restenosis in humans: in vivo 6-month follow up study. Circulation. 108: 438‑444.
  18. Caro, C.G., FitzGerald, J.M., Schroter, R.C. (1969). Arterial wall shear and distribution of early atheroma in man. Nature. 223: 1159‑1161.
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  20. Prado, C.M., Ramos, S.G., Elias, J., Rossi, M.A. (2008). Turbulent blood flow plays an essential localizing role in the development of atherosclerotic lesions in experimentally induced hypercholesterolaemia in rats. International Journal of Experimental Pathology. 89: 72‑80.
  21. Cockcroft, N., Oke, O., Cunningham, F., Bishop, E., Fearon, I., Zantl, R., Gaca, M. (2009). An in vitro perfusion system to examine the responses of endothelial cells to simulated flow and inflammatory stimulation. Alternatives to animal testing (ATLA). 37: 657-669. Link to manuscript abstract and citation. Opens new window
  22. Gough, P.J., Gomez, I.G., Wille, P.T., Raines, E.W. (2006). Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. The Journal of Clinical Investigation. 116: 59-69.
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