Phosphodiesterase 4 expression and proliferation rates in a cellular model of pulmonary hypertension

Millen, Jennifer Elena (2004) Phosphodiesterase 4 expression and proliferation rates in a cellular model of pulmonary hypertension. PhD thesis, University of Glasgow.

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Pulmonary arterial hypertension (PAH) is characterised by increased vascular resistance which leads to pulmonary artery remodelling and increased smooth muscle cell proliferation (Fishman 2004; Humbert et al., 2004). Chronic hypoxia treated rats (to induce PAH) display alterations in cyclic nucleotide signalling pathways. Both cAMP and cGMP levels are reduced (MacLean et al., 1997), with a corresponding increase in PDE3A/B and PDE5A2 in pulmonary arteries of the chronic hypoxic rat (Murray et al., 2002). The objective of this study was to investigate the expression of PDE4 isoforms in the cellular model of PAH as the PDE4 family represents the major cAMP-hydrolysing activity within these cells (Palmer et al., 1998). It was shown that in human pulmonary artery smooth muscle cells (hPASMC), the PDE4A10, PDE4A11, PDE4B2 and PDE4D5 isoforms all show significant increases in expression after 14 days exposure of chronic hypoxia (10% O2). The increase in PDE4 expression did not correspond to an increase in PDE4 activity levels however. Levels of cAMP were tripled and PKA activity was doubled after 7 days in 10% O2. The hypoxia induced increase in cAMP was determined to be ERK dependant as use of the MEK inhibitor, U0126, could reduce hypoxic cAMP levels to that of normoxic cells. The autocrine loop shown previously in aortic smooth muscle cells (Baillie et al., 2001), was demonstrated to be in effect in hPASMC. In this loop, active ERK led to the production of PGE2 through PLA2. This increase in PGE2 stimulated adenylyl cyclase and increased the generation of cAMP. Using the COX-2 inhibitor, indomethacin, completely ablated the rise in cAMP levels in hypoxia. Exogenous PGE2 mimicked the hypoxia induced rise in cAMP in normoxic cells. Inhibition of ERK also led to a decrease in PDE4 activity through the reduced level of cAMP generated and thus decreased the amount of PKA able to activate PDE4. This effect was not witnessed in hypoxia, suggesting desensitisation to cAMP stimulation had occurred. Indeed, it was observed that hypoxic cells produced less cAMP in response to cAMP agents such as rolipram or PGE2 than normoxic cells. Vascular smooth muscle cells have been reported to proliferate in response to hypoxia. Indeed, it was demonstrated in this study that hPASMC do increase their proliferation in response to hypoxia in both serum starved cells and cells cultured in serum. The PKA RII subunit was demonstrated to be essential for normal proliferation of these cells. Both PKA RI and PKA RII can significantly reduce proliferation in both normoxic and hypoxic cells. Also, the recently discovered cAMP substrate Epac (de Rooij et al., 1998), elicits anti-proliferative effects on serum induced proliferation in hPASMC.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Pharmacology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Houslay, Prof. M. and MacLean, Prof. M.
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-71433
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 16 Jul 2021 15:37

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