The multi-functional cytokine Leukaemia Inhibitory Factor (LIP) has been ascribed many roles in both disease and homeostatic physiological conditions. Its ability to induce cellular differentiation and proliferation and regulate the response to physical injury in a variety of different tissues is now well accepted. Previous studies performed in the Centre of Research in Vascular Biology demonstrated the novel role of LIP as an anti-atherogenic/-restenotic agent. Systemic administration of LIP in a model of vascular disease inhibited the formation of a vascular smooth muscle cell (VSMC)-rich occlusive neo-intima, prevented leucocyte infiltration into the vessel wall and maintained vascular reactivity in response to contractile agonists.
The aim of the present study was to elucidate the molecular mechanisms by which LIP exerts its anti-atherogenic/-restenotic action on cells of the vasculature. To this end the activation of signal transduction pathways, the induction of down-stream gene expression and the subsequent biological end-points in response to LIP treatment were investigated.
Chapter 3 demonstrates the RNA and protein expression of LIP receptor components (LIFR-α and gp130) within cells of the vasculature, and the induction of LIF expression in response to pro-atherogenic stimuli. It further demonstrates that the expression of LIP receptor components are up-regulated with the progression of vascular disease following endothelial denudation and in VSMC following modulation to the 'synthetic' (diseased, developmental, repair) phenotype.
In Chapter 4, LIF-mediated signal transduction in vascular cells is shown to be tightly controlled with maximal activation of STAT3, ERKl/2 and Akt after 10 minutes, but down-regulated by 1 hour; activation of p38 is moderate, but sustained for at least 24 hours. In Chapter 5, the capacity of pro-atherogenic molecules such as IL-1β for 'cross-talk' inhibition of LIF-mediated STAT3 activation in VSMC is found to require the activation of p38 and involve the down-regulation of LIFR-α protein expression independent of the action of phosphatases and SOCS molecules. Investigation of the molecular mechanisms by which LIF-mediated signalling pathways are regulated in VSMC (Chapter 6) demonstrates that STAT3 and p38 signalling are regulated by de novo protein synthesis and that all pathways, with the exception of STAT3, are regulated by both tyrosine and serine/threonine phosphatases. The down-regulation of LIFR-α protein expression in the continued presence of LIF also contributes to the attenuation of LIF-mediated signalling in VSMC. Analysis of gene expression downstream of LIP signalling in vascular cells demonstrates the contribution of individual signal transduction pathways to the induction of anti-atherogenic and repression of pro-atherogenic genes. The results suggest that the therapeutic actions of LIF may be facilitated by inducible nitric oxide synthase and the consequent inhibition of VSMC proliferation by nitric oxide. In addition, the induction of TIM-1 and the down-regulation of PAI-1 expression may contribute to a reduction in vascular thrombosis and VSMC migration.
Further investigations demonstrated LIF is capable of exerting multiple biological effects in the vasculature. In isolated cells LIF is demonstrated to inhibit VSMC proliferation induced by serum (Chapter 9). This inhibition is dependent upon the timing of LIF stimulation, with LIF having no effect if administered at either the same time or following serum stimulation. LIF is also capable of inhibiting VSMC migration in an in vitro scratch wound assay, an action dependent upon the inhibition of PI3-K/Akt activity (Chapter 9). Other cellular actions of LIP include the ability to inhibit the extent of apoptosis within the vessel wall and promote the survival of VSMC in response to hydrogen peroxide treatment and following serum deprivation. LIP also inhibits the apoptosis of endothelial cells (BC) following serum deprivation. The anti-apoptotic effects of both VSMC and EC require the activation of ERK1I2, and the phosphorylation of the pro-death factor BAD (Chapter 10). In EC the promotion of survival by LIP correlates with the maintenance of the cytoskeleton and requires the activation of ERKl/2, p38 and PI3-K/Akt (Chapter 11). Finally, LIP administration via osmotic mini-pump in a rabbit model of atherosclerosis was shown to inhibit progression of a pre-existing lesion, influence macrophage accumulation and the deposition of extracellular matrix proteins associated with vascular disease (Chapter 12).
In summary, the action of LIP as an anti-atherogenic/-restenotic agent is governed by the activation of multiple signalling pathways that exert both positive and negative actions on various cellular components that contribute to the establishment and progression of vascular disease. In contrast to many current intervention strategies, which exert their effect on only a single facet of vascular disease, LIP exerts multiple actions on the vasculature and thus exhibits great potential as a therapeutic agent.