Marshall, Kirsty M
Investigation of the intracellular pathways required for 5HT-induced mitogenesis and their role in pulmonary hypertension.
PhD thesis, University of Glasgow.
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Pulmonary arterial hypertension (PAH) is a rare and progressive disease characterised by increased pulmonary vascular resistance and elevated pulmonary artery pressure, leading to right ventricular failure and eventually death. The monoamine 5-hydroxytryptamine (5HT) has been implicated in the processes of pulmonary vasoconstriction and pulmonary artery remodelling that contribute to the development of the PAH. However, the signalling mechanisms utilised by 5HT that contribute to pulmonary vascular remodelling are still unclear and appear to be cell-type specific, with much of the work having been carried out in pulmonary artery smooth muscle cells (PASMCs). Fibroblasts also contribute significantly to the pulmonary vascular remodelling that occurs during PAH, however little is known of the role 5HT plays in this cell type. Using Chinese Hamster Lung Fibroblast (CCL-39) cells as a model system to investigate the mitogenic effects of 5HT, this study has characterised potential 5HT-mediated signalling pathways in fibroblasts that may contribute to pulmonary vascular remodelling.
5HT was found to induce a rapid and transient activation of extracellular regulated mitogen-activated protein kinase (ERK), a process central to the mitogenic effects of 5HT in CCL-39 cells. Furthermore, the 5HT transporter (5HTT), 5HT1B and 5HT2A receptors were all required for optimal ERK-dependent proliferation. Pharmacological inhibition of the Rho/ROCK (Rho-associated kinase) pathway significantly inhibited 5HT-stimulated ERK activation, cyclin D1 accumulation and proliferation. Inhibition of ROCK had no effect on the translocation of active ERK to the nucleus, but did however selectively inhibit 5HT-induced activation of a cytoplasmic pool of ERK. Additionally, ROCK inhibition had no effect on the ability of 5HT to activate mitogen-activated protein kinase kinase (MEK), suggesting ROCK is required for maintaining functional interactions between MEK and ERK. Sensitivity to ROCK inhibition is restricted to 5HT1B receptor activation of the ERK pathway. Moreover, the role of ROCK in maintaining cytoskeletal integrity is important in mediating 5HT-induced ERK activation, as disruption of the actin cytoskeleton markedly and specifically reduces 5HT- stimulated ERK activation.
Using a model of PAH, arising from overexpression of 5HTT (5HTT+), the effects of ROCK inhibition in vivo were investigated. ROCK 1 and ROCK 2 transcripts were upregulated in response to chronic hypoxia, with the upregulation of ROCK 1 potentiated in 5HTT+ mice. Administration of the ROCK inhibitor Y27632 had significantly greater effects in 5HTT+ mice compared to WT, highlighting the functional importance of the increase in ROCK 1 transcript. Hypoxia-induced pulmonary vascular remodelling and elevated right ventricular pressure were attenuated more significantly by ROCK inhibition in 5HTT+ mice than in WT. Furthermore, ROCK inhibition only reduced hypoxia-derived right ventricular hypertrophy significantly in 5HTT+ animals and not WT.
In conclusion, this study highlights a role for ROCK in the pulmonary vascular changes that occur during PAH and proposes a new mechanism by which cross-talk between ROCK and 5HT signalling systems occurs.
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