Hypoxia-mediated human pulmonary arterial fibroblast proliferation is dependent on p38 mitogen-activated protein kinase activity.

Mortimer, Heather Jane (2010) Hypoxia-mediated human pulmonary arterial fibroblast proliferation is dependent on p38 mitogen-activated protein kinase activity. MD thesis, University of Glasgow.

Abstract

Abstract

Background:

Pulmonary hypertension (PH) is a rare condition that can occur as a primary disease process, Idiopathic Pulmonary Hypertension (IPH) or secondary to other disorders. In Familial IPH mutations have been identified in the bone morphogenetic protein receptor II gene (BMPRII) (chromosome 2q32-31) a member of the Transforming Growth Factor  (TGF) (Lane et al, 2000). Despite the mutation being present in all cells, vascular wall remodelling is only seen in the pulmonary circulation with marked thickening of the intima and neointimal formation, muscularisation of small-generation resistance vessels and thickening of the adventitial layer together with increased ECM deposition. Similar appearances are noted in the pulmonary circulation’s response to hypoxia. for this projectProlonged exposure of the pulmonary circulation to hypoxia results in vasoconstriction and subsequent vascular wall remodelling.

The hypothesis of this work is that the pulmonary circulation’s response to hypoxia may be partially explained by the existence of differences exist in cell signalling pathways in between adventitial fibroblasts from pulmonary and systemic arteries in HPAF. Studies from the Scottish Pulmonary Vascular (SPVU) Laboratory have shown that pulmonary arterial fibroblasts (PAFB) in bovine and rat models of acute hypoxic exposure preferentially proliferate to hypoxia, whereas systemic arterial fibroblasts (SAFB) do not , that the stress mitogen activated protein kinase p38 MAPK is consistently activated in PAFB exposed to acute hypoxia, and is constitutively upregulated in PAFB cultured from rats exposed to chronic hypoxia (Welsh et al, 1998; Welsh et al; 2001). This response to hypoxic exposure has been shown to be dependent on p38 MAPK activity, as use of SB203580 can block the hypoxia-mediated proliferative response to acute hypoxia (Scott et al, 1998; Welsh et al, 2001).

Aims and methods:
We wished to establish whether the pro-proliferative response of PAFB to acute hypoxic exposure previously noted in bovine and rat models could also be demonstrated in a human model. We wished to establish a role for both classic MAPK and stress MAPKs in hypoxia-mediated PAFB proliferation. We also wished to examine the role of hypoxia inducible factor 1 (HIF1) in human arterial fibroblast responses to acute hypoxia. There is a body of literature that documents cross talk between p38 MAPK and the Bone Morphogenetic Protein (BMPR) signalling pathways. We wished to establish whether Smad proteins (involved in the downstream signalling cascade from BMPR) might play a role in human pulmonary and systemic arterial fibroblast proliferation to acute hypoxia.

Following approval from the local Ethics Committee, PAFB were harvested from patients undergoing lobectomy for the treatment of lung cancer. Left internal mammary arteries (SAFB) were harvested from patients undergoing coronary artery bypass grafting. Cells from systemic and pulmonary arterial fibroblasts were grown in conditions of normoxia or acute hypoxia (PO2 35 mmHg ~ 5% O2). Cellular proliferation was assessed using [3H]Thymidine uptake as a surrogate. p38, p44/p42 - ERK1/2 and JNK MAPKs and Smad protein activity was assessed using Western Blotting Techniques with the use of appropriate primary and secondary antibodies and Chemiluminescence to detect the presence of protein. p38 MAPK isoform activity was assessed using Catch and Release® immunophoresis techniques.

Findings and conclusions:
We demonstrated that acute hypoxic exposure results in human PAFB proliferation, associated with increased p44/p42 – ERK 1/2 MAPK activity, but dependent on p38 MAPK  activity. We also found that the p38 MAPK  isoform was expressed in human PAFB following hypoxic exposure but this did not appear to be involved in the hypoxia-mediated proliferative response. p38 MAPK  activity appeared to occur in a bi-phasic pattern with peaks of activity at t = 6 and 16 hours, the second peak was found to be responsible for the hypoxia-mediated proliferation seen in these cells in agreement with previous work from the SPVU laboratory (Scott et al, 1998; Welsh et al., 2001). The second peak in p38 MAPK  activity was synchronous with peak HIF1 activity (between t = 8 –16 hours). We demonstrated that HIF1 activity can be abrogated by pre-incubation of human PAFB with SB203580 suggesting a mechanistic link between p38 MAPK  activation and HIF1 in a human model of acute hypoxic exposure.

We have also demonstrated that that BMPR2-associated Smad 1, 5 and 8 activation is increased in hypoxic human SAFB, suggestive of the activation of an anti-proliferative pathway in these cells that is not associated with p38 MAPK activity. To our knowledge this is the first demonstration of an active response in SAFB to acute hypoxic exposure that involves the active upregulation of an anti-proliferative pathway in these cells. In addition we have demonstrated that in hypoxic pulmonary arterial fibroblasts phospho Smad 1, 5 and 8 expression is reduced (suggestive of the down-regulation of an anti-proliferative pathway) and can be further abrogated by pre-incubation with SB203580. This suggests that in SAFB Smad 1, 5 and 8 activation occurs independent of p38 MAPK activation while in PAFB, p38 MAPK activity augments Smad 1, 5 and 8 activation.

• Item Type: Thesis (MD)
• Qualification Level: Doctoral
• Keywords: Hypoxia, fibroblasts, cell culture, p38 MAPK, proliferation, human
• Subjects: R Medicine > R Medicine (General); R Medicine > RB Pathology; R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Peacock, Professor Andrew J. and Welsh, Dr. David
• Date of Award: 2010
• Depositing User: Dr Heather J Mortimer
• Unique ID: glathesis:2010-1409
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 08 Jan 2010
• Last Modified: 10 Dec 2012 13:39
• URI: https://theses.gla.ac.uk/id/eprint/1409