Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.805432
Title: An investigation into the mechanism by which cholesterol induces interleukin-1 secretion from the vascular endothelium
Author: Almansouri, Majid
ISNI:       0000 0004 8510 7172
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2020
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Abstract:
Background: Atherosclerosis is a chronic vascular inflammatory disease characterised by disturbed blood flow due to an atheromatous plaque buildup within arterial layers. These plaques contain various forms of modified cholesterol, such as oxidised low-density lipoprotein (OxLDL). OxLDL enters vascular cells, contributing towards foam cell formation and initiating over exuberant repair processes, leading to arterial occlusion and life threatening myocardial infarction or stroke. Endothelial cells, by producing proinflammatory cytokines, such as interleukin-1 beta (IL-1ß), are also strongly involved in the pathogenesis of atherosclerosis. However, little is known of the link between OxLDL and endothelial cell actions. Hypothesis: I hypothesised that OxLDL enters the arterial endothelium and induces IL-1ß secretion, potentially via a caspase-1/NLRP3 mechanism. Methods: Human coronary artery endothelial cells (HCAEC), isolated from different donors, were cultured and stimulated with pro-inflammatory cytokines TNFa and IL1a (10 ng/ml each, for 48 hours), followed by incubation with native human OxLDL cholesterol at multiple concentrations (10-200 ug/ml) for 6 hours. Inhibitors of Caspase-1 (YVAD), NLRP3 (MCC950) and P2X7 receptor (A438079 HCl) were also used. Cell lysates and culture supernatants were collected and analysed for IL-1ß using ELISA and Western Blot. Cell viability and caspase-1 activity were also measured. Results: Brightfield microscopy imaging showed OxLDL enters HCAECs, and forms particles. HoweverBackground: Atherosclerosis is a chronic vascular inflammatory disease characterised by disturbed blood flow due to an atheromatous plaque buildup within arterial layers. These plaques contain various forms of modified cholesterol, such as oxidised low-density lipoprotein (OxLDL). OxLDL enters vascular cells, contributing towards foam cell formation and initiating over exuberant repair processes, leading to arterial occlusion and life threatening myocardial infarction or stroke. Endothelial cells, by producing proinflammatory cytokines, such as interleukin-1 beta (IL-1ß), are also strongly involved in the pathogenesis of atherosclerosis. However, little is known of the link between OxLDL and endothelial cell actions. Hypothesis: I hypothesised that OxLDL enters the arterial endothelium and induces IL-1ß secretion, potentially via a caspase-1/NLRP3 mechanism. Methods: Human coronary artery endothelial cells (HCAEC), isolated from different donors, were cultured and stimulated with pro-inflammatory cytokines TNFa and IL1a (10 ng/ml each, for 48 hours), followed by incubation with native human OxLDL cholesterol at multiple concentrations (10-200 ug/ml) for 6 hours. Inhibitors of Caspase-1 (YVAD), NLRP3 (MCC950) and P2X7 receptor (A438079 HCl) were also used. Cell lysates and culture supernatants were collected and analysed for IL-1ß using ELISA and Western Blot. Cell viability and caspase-1 activity were also measured. Results: Brightfield microscopy imaging showed OxLDL enters HCAECs, and forms particles. However, uptake of OxLDL was not seen following inhibition of the P2X7 receptor. OxLDL induced the release of IL-1ß from stimulated HCAECs with maximum release at concentration of 50 ug/ml compared to control. This release was not caused by toxicity of the OxLDL: cell viability was confirmed by lactate dehydrogenase cell viability assay. Inhibition of either NLRP3 or Caspase-1 significantly reduced the release of extracellular IL-1ß (4-fold, p < 0.0001; 14- fold, p<0.0001, respectively). Release of IL-1ß was associated with expression of active GSDMD, suggesting the release via a pyroptosis mechanism. Conclusion: HCAEC uptake of OxLDL occurs via the P2X7 receptor. OxLDL elicits IL-1ß release in activated HCAECs by a Caspase1/NLRP3 mechanism, without causing toxicity. The released IL-1ß probably exits the cells via a GSDMD-induced membrane pore. These data show that the study of modified lipids linked to the release of inflammatory cytokines such as IL-1 from endothelial cells could lead to the discovery and development of a useful strategy for the prevention/reduction of atherosclerosis.
Supervisor: Francis, Sheila ; Chamberlain, Janet Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.805432  DOI: Not available
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