Effect of adenosine A2A receptor gene transfer on nuclear factor-kappaB-regulated inflammatory responses in vascular endothelial cells

Strong, Elaine Wenda (2006) Effect of adenosine A2A receptor gene transfer on nuclear factor-kappaB-regulated inflammatory responses in vascular endothelial cells. PhD thesis, University of Glasgow.

Abstract

Inappropriate or prolonged inflammation contributes to the pathogenesis of many diseases including atherosclerosis, rheumatoid arthritis, sepsis, heart disease and cancer. It is widely accepted that the A2A adenosine receptor (A2aAR) is a critical non-redundant suppressor of inflammatory responses in vivo. Therefore understanding the mechanisms behind the suppression of inflammation would be beneficial in providing future drug targets for chronic inflammatory diseases. While the receptor is known to elevate intracellular cAMP levels, the molecular mechanisms underlying its inhibitory effects on defined pro-inflammatory signalling pathways remains obscure. We have demonstrated that adenovirus-mediated human A2aAR gene transfer into approximately 75% of virally-infected human umbilical vein endothelial cells (HUVECs) results in an expression level of 0.3-0.4 pmol/mg membrane protein as determined by 125I-ZM241385 antagonist radioligand binding studies. A2AAR-expression results in a constitutive suppression of TNFalpha-mediated induction of NFkB target genes such as cyclooxygenase-2 (COX-2) and the adhesion molecule E-selectin. To assess the functional significance of this phenomenon, the effects of A2aAR gene transfer in HUVECs on the accumulation of three important gene products known to be controlled at least in part by NFkB, namely COX-2, E-selectin and vascular cell adhesion molecule-1 (VCAM-1) was determined. Characterisation of TNFalpha-mediated COX-2 induction revealed that while sustained induction (24hr) was solely dependent on the activation of p38 MAP kinase, earlier time-points at which induction was first detectable (8hr) were also sensitive to inhibition of the NFkB pathway. At this time-point, A2aAR gene transfer alone was sufficient to reduce TNFalpha-mediated COX-2 induction compared with controls, whilst no effect of the A2aAR was detectable at 24hr. Surprisingly, addition of the A2AAR-selective agonist CGS21680 to A2aAR-expressing HUVECs actually reversed the effect observed with receptor expression alone. Time-course experiments in control cells using elevated concentrations of the adenylyl cyclase activator forskolin demonstrated that cAMP elevation was sufficient to promote the transient induction of COX-2 even in the absence of TNFalpha. However, in the presence of TNF?, exposure to different concentrations of forskolin exerted a biphasic effect, suppressing COX-2 induction at low concentrations while enhancing it at higher concentrations. These data suggest that the effects of A2aAR expression with or without agonist on TNFalpha-mediated COX-2 induction can be accounted for completely by changes in cAMP. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Immunology, Physiology
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Palmer, Dr. Tim
• Date of Award: 2006
• Depositing User: Enlighten Team
• Unique ID: glathesis:2006-71121
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 21 May 2021 08:24
• URI: https://theses.gla.ac.uk/id/eprint/71121