Abstract
Chronic hypoxia, in animals and man, results in remodelling of the pulmonary vasculature with consequent pulmonary hypertension. Pulmonary artery fibroblasts (PAF) have been observed to play an early and important role in hypoxic-induced pulmonary vascular remodelling. PAF proliferate excessively in an hypoxic environment. This is thought to be due to cell to cell signalling between the PAF and other vascular cells. These interactions initiate and progress the changes that occur in pulmonary vascular remodelling. Hypoxia induced proliferation of PAFs has been shown to be circulation specific and is dependent on phosphorylation of p38 mitogen-activated protein (MAP) kinase. Hypoxic activation of PAFs is a potential therapeutic target, however as p38 MAPK inhibitors display many off target effects. Therefore, the MAPK pathway needs to be better understood so other molecules can be targeted allowing indirect p38 inhibition.The method of cell to cell interactions that we are focused on is connexin signalling. Connexins are transmembrane proteins that can act as channels allowing the entry of small molecules into the cell. Connexin 43 signalling has been shown to be dysregulated in various models of pulmonary hypertension but no research has been done in PAFs. Cx43 has also been observed to drive cell proliferation in other cell types via the phosphorylation of p38 MAPK.
Based on this knowledge the main aims of this study were to investigate connexins in hypoxia-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) ii) assess whether Cx43 expression is dysregulated in the rat sugen/hypoxia model of pulmonary hypertension. The role of Cx43 in hypoxia-induced proliferation and migration in rPAFs was investigated using 18 connexin mimicking peptide Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic mediated proliferation and migration in rPAFs but not rPASMCs. Phosphorylation of p38 MAPK was increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen/hypoxic rat lung; this increased expression was not observed in sugen/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. The changes in connexin 43 expression correlated to an increase in cell proliferation and migration. PAFs from the sugen/hypoxic rats dosed with MAPK pathway inhibitor GS-444217 did not demonstrate changes in cell proliferation, migration or MAPK expression. In conclusion Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via effects mediated through the MAPK pathway.
| Date of Award | 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Yvonne Dempsie (Supervisor), David Welsh (Supervisor) & Patricia Martin (Supervisor) |