Effects of air pollution on vascular thrombosis

Abstract

Increases in air pollution, especially the particulate component, are associated with increased cardiovascular mortality, possibly through increases in thrombogenic mechanisms. The research presented in this thesis addresses the hypothesis that diesel exhaust particulates (DEP) increase thrombogenicity by impairing the release of tissue plasminogen activator (t-PA) from vascular endothelial cells, thus inhibiting the endogenous fibrinolytic mechanisms that promote thrombus breakdown. The initial aims of this work were to develop an in vivo model of thrombosis, to determine whether exposure to DEP did alter clotting. Initial attempts to develop the Folts’ model (which stimulates thrombus formation via arterial stenosis and mechanical injury), first in male C57/Bl6 mice and later in male Wistar rats, were unsuccessful. An alternative approach, using ferric chloride (FeCl3) to induce chemical injury to the rat carotid artery was found to produce reliable and reproducible thrombotic occlusion: this model was used for all subsequent experiments. The effects of DEP on thrombus formation were assessed in vivo by applying the FeCl3 model. DEP were administered via intratracheal instillation or tail vein injection 2, 6 or 24 hours prior to induction of thrombosis. The effects of DEP were compared with vehicle and suitable controls: carbon black (a clean carbon nanoparticle); quartz (a large non-carbon particle that causes well-characterised pulmonary inflammation). The time to thrombotic occlusion was significantly reduced 6h after intra-pulmonary instillation of DEP (0.5ml of a 1mg/ml suspension). In contrast, instillation of carbon black or quartz had no significant effect on thrombosis, despite causing greater pulmonary (increased neutrophils and levels of interleukin-6, tumour necrosis factor-α and C-reactive protein in bronchoalveolar lavage fluid) and systemic (C-reactive protein in plasma) inflammation than DEP. Direct administration of DEP (0.5mg/kg) to the blood stream resulted in an acute (2 hours after injection) increase in time to thrombotic occlusion in the absence of pulmonary inflammation. A similar (but less pronounced) effect was observed following administration of carbon black (0.5mg/kg). These data suggest that the DEP-mediated increase in thrombosis is independent of pulmonary and systemic inflammation. The mechanisms involved were addressed by measuring platelet-monocyte interactions (flow cytometry) and markers of the endogenous fibrinolytic system (ELISA). Exposure (either instillation of injection) to DEP significantly increased platelet-monocyte aggregation. Carbon black and quartz produced no such effect (but did increase platelet-platelet aggregation). t-PA antigen and activity were reduced, whilst PAI-1 and fibrinogen were increased, following either instillation or injection of DEP. The final aim was to develop a suitable dispersant for use in cell culture to determine whether DEP alter the expression (real-time polymerase chain reaction; rtPCR) and generation (enzyme-linked immunosorbent assay; ELISA) of t-PA and plasminogen activator inhibitor (PAI-1). Cell culture medium containing bovine serum albumin (0.5mg/ml; BSA) provided the best combination for DEP dispersal and maintenance of small particle size (<200nM), without detrimental effects on human umbilical endothelial cells (HUVECs). Exposure (6 and 24 hours) of HUVECs to DEP resulted in reduced basal and thrombin stimulated t-PA and PAI-1 expression. This was mirrored by reduced detection of t-PA and PAI-1 in culture medium. In conclusion, these investigations confirm that exposure to DEP is capable of increasing the rate of thrombus formation and that this is, in part, mediated by an alteration in the endogenous fibrinolytic system. These changes did not appear to be secondary to pulmonary or systemic inflammation. Whilst cell culture experiments suggested DEP could directly alter endogenous fibrinolytic activity in endothelial cells, there was no evidence from these experiments of DEP translocation into the systemic circulation. Thus, this work suggests that DEP is capable of increasing thrombus formation in vivo via several mechanisms. Similar changes may account for the increased thrombus formation in humans exposed to diesel exhaust in air pollution.