Effects of air pollution on vascular thrombosis
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Tabor2011.doc (16.87Mb)
Date
05/07/2011Author
Tabor, Caroline Mary
Metadata
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.