Cardiovascular disease is the principal cause of mortality in developed societies. Studies have identified a correlation between radiotherapy for cancer and atherosclerosis in later life1. Endothelial cell dysfunction is a defining feature of atherosclerosis initiation. This study aims to investigate the effect of radiation on endothelial cell intracellular signalling networks, including the Nuclear Factor-kappaB (NFκB) and c-Jun N-terminal kinase (JNK) pathways. Human umbilical vein endothelial cells (HUVECs) were exposed to 10 Gy X-rays using an X-RAD 225 biological irradiator. Western blotting was used to detect NFκB and JNK pathway activation in whole cell lysates using anti-phospho-p65, anti-IκBα, anti-tp100/p52 and anti-phospho-JNK antibodies and in nuclear extracts using anti-NFκB-p65 antibodies. An in vitro kinase assay utilising 32P enabled detection of JNK-mediated phosphorylation of c-Jun. X-irradiation induced p65 phosphorylation (fold increase at 2hrs; 2.5±0.42, p<0.05, n=3) and IκBα degradation (fold at 8hrs; 0.29±0.05, n=3) in HUVECs, markers of canonical NFκB pathway activation. Surprisingly, nuclear p65 levels were not increased suggesting absence of p65 nuclear translocation. Interestingly, tp100/p52 levels were unaltered after irradiation implying that the non-canonical pathway is not activated. X-irradiation also triggered JNK phosphorylation (fold increase at 4hrs; 2.73±0.66, n=3) and JNK kinase activity, observed as increased phosphorylated c-Jun expression 4 hours after irradiation. Thus stress-associated signalling pathways (canonical NFκB and JNK pathways) are activated in irradiated endothelial cells. Further investigations will confirm the cellular events that link radiotherapy and cardiovascular disease. Project supported by a British Heart Founation PhD studentship (no.Fs/13/25/30155).

Previous research from our laboratory has demonstrated a novel phenomenon whereby GPCRs play a role in inhibiting cytokine-mediated c-Jun N-terminal kinase (JNK) signalling. So far this novel phenomenon seems to have been vastly overlooked, with little research in the area. Therefore, in this study we explored this further; by assessing the potential of P2YRs to mediate inhibition of cytokine-mediated JNK signalling and related functional outcomes in human endothelial cells. We utilised primary endothelial cells, and employed the use of endogenous activators of P2YRs and well characterised pharmacological inhibitors, to assess signalling parameters mediated by P2YRs, Interleukin-1β (IL-1β), TNFα and JNK. Activation of P2YRs with adenosine tri-phosphate (ATP) resulted in a time- and concentration-dependent inhibition of IL-1β-mediated phosphorylation of JNK and associated kinase activity. The effect was specific for cytokine-mediated JNK signalling, as ATP was without effect on JNK induced by other non-specific activators (e.g. sorbitol, anisomycin), nor effective against other MAPK pathways such as p38 and the canonical NFκB cascade. Pharmacological studies demonstrated a role for the P2Y receptor in mediating this effect, but not the P2Y nor the adenosine receptors (A1, A2A, A2B & A3). The novel Gα inhibitor YM254890 and a protein kinase A (PKA) inhibitor H89 both partially reversed ATP-mediated inhibition of IL-1β-stimulated JNK indicating involvement of both Gα and Gα mediated pathways. ATP also partially reversed IL-1β-mediated induction of cyclo‑oxygenase-2 (COX-2) and E-selectin. Collectively, these studies indicate the potential for activation of purinergic receptors to protect the endothelium from inflammatory driven JNK activation and may be a new target for inflammatory disease therapy.

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