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Title: Mechanisms of vascular protection in chronic inflammatory disease
Author: Thornton, Catherine Clare
ISNI:       0000 0004 5365 9351
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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Premature cardiovascular disease (CVD) complicates chronic systemic inflammatory disease, and is initiated by endothelial dysfunction. Despite an ever-improving array of medications to treat these diseases, how to prevent CVD is not known. Studying the actions of endogenous mediators of endothelial cytoprotection and of disease-modifying drugs might establish how best to protect patients with systemic inflammatory diseases from atherosclerosis, if specific beneficial effects on vascular endothelium could be demonstrated. High flow laminar shear stress (LSS) and vascular endothelial growth factor (VEGF) are two well known endogenous drivers of endothelial cytoprotection. Both can induce expression of protective genes but it is not known how confluent endothelial cells (EC) respond to VEGF when conditioned with LSS. Methotrexate (MTX) is the most widely prescribed treatment for rheumatoid arthritis and clinical data suggests that it reduces CV mortality. Known mechanisms of action of MTX result in intracellular accumulation of activators of AMP-activated protein kinase (AMPK). AMPK regulates cytoprotective genes in EC and its activation is associated with diverse desirable effects. I hypothesised that EC conditioned with LSS would be primed to respond to VEGF, resulting in a synergistic induction of cytoprotective genes. Secondly I investigated whether MTX exerts beneficial protective effects on vascular endothelium via activation of AMPK, which enhance endothelial resistance to injury. Studies of human umbilical vein EC (HUVEC) exposed to LSS showed that responses to VEGF are not enhanced in these conditions. However, MTX phosphorylated AMPKαThr172 and induced expression of several cytoprotective genes, notably manganese superoxide dismutase (MnSOD). The addition of folic acid did not alter this; and it was also preserved when HUVEC were pre-treated with TNFα to mimic dysfunctional endothelium. siRNA depletion of AMPK attenuated MTX-mediated MnSOD induction. MTX treatment led to AMPK-dependent phosphorylation of the transcription factor CREBSer133. siRNA depletion of CREB also reduced MnSOD induction by MTX, and chromatin immunoprecipitation demonstrated binding of CREB to the MnSOD promoter in MTX-treated samples. Moreover, MTX protected HUVEC against apoptosis induced by glucose deprivation, demonstrating the functional importance of this pathway. Finally, treatment of the murine (BXSB x NZW)F1 SLE model of inflammatory vasculopathy with MTX improved the intramyocardial arterial vasculopathy and reduced end-organ damage, increased aortic MnSOD and phosphorylated AMPKαThr172, and reduced ICAM-1 expression. I have shown that MTX activates an AMPK-CREB pathway in vascular endothelium leading to enhanced expression of cytoprotective genes and protection against apoptosis in vitro and inflammatory vascular injury in vivo. This novel mechanism may explain its observed benefits in reducing CVD in chronic systemic inflammation.
Supervisor: Mason, Justin Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral