Thesis

The role of mitochondria in regulating smooth muscle cell proliferation and migration in pulmonary hypertension

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Awarding institution
  • University of Strathclyde
Date of award
  • 2020
Thesis identifier
  • T15651
Person Identifier (Local)
  • 201390432
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • Remodeling of the pulmonary arteries with proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is the hallmark of pulmonary hypertension, a proliferative disease with poor prognosis, which is exacerbated by hypoxic conditions. Hyperproliferation of PASMC underlies the failure of current therapy directed at vasodilation. Therefore, more direct anti-proliferative treatment, which targets the pulmonary vasculature is urgently sought. Signalling mechanisms that cause these cells to proliferate during hypoxia remain unclear. Recent studies highlight the role of mitochondrial alteration in PASMC of pulmonary hypertension, which is characterized by the glycolysis shift in metabolism and the increase in mitochondrial fragmentation that is also observed in cancer. The work presented in this thesis describes the influence of the mitochondria (particularly mitochondrial fission) on cell proliferation and migration of PASMCs during a short period of hypoxia and identifying the mitochondrial-dependent signalling in response to platelet-derived growth factor (PDGF) and hypoxia. Aims were first to determine the appropriate model of hypoxia-induced cell proliferation among different degrees of hypoxia. The second aim was to investigate the role of the mitochondria in regulating cell proliferation and migration using the established hypoxic cell model (3% O2). PASMCs were isolated from Sprague-Dawley rats. Cells were maintained under hypoxia for 24 h and compared with normoxic cultures. Cell proliferation was measured by [3H]-thymidine incorporation assay. The effect of mitochondrial inhibition was studied using mitochondrial dynamin-related protein-1 (DRP1) inhibitor Mdivi-1 (10 μM). The signalling proteins and genes were measured by Western blot and real-time qPCR techniques, respectively. Mitochondrial functions were assessed by measuring ATP level, reactive oxygen species (ROS; superoxide and H2O2) production, and cellular apoptosis. Mitochondrial morphology was examined using an epi-fluorescence microscope. During hypoxia, data show PDGF significantly enhances cell proliferation, HIF1α expression, and promotes mitochondrial dysfunction by decreasing mitochondrial fission, ATP, ROS (H2O2 release) and cellular apoptosis, which all exhibit the proliferative/apoptotic-resistant phenotype of PASMCs compared to PDGF stimulated cells in normoxia. In comparison to hypoxic background cells (cells quiesced with 0.1% FCS and maintained in 3% O2), PDGF caused a reduction in DRP1 expression and mitochondrial fragmentation, an increase in mTORC1 expression and a decrease in hypoxic genes (HIF1α and FIH-1). Interestingly, despite the reduced DRP1 expression and the presence of elongated mitochondria that were observed in the PDGF stimulated cells during hypoxia, inhibiting mitochondrial fission in these cells with Mdivi-1 slows cell proliferation and migration. It causes further reduction in HIF1α and DRP1 expression, promotes hyper-fused mitochondria, upregulates FIH-1 gene expression, inhibits mTORC1 expression and recovers the mitochondrial function possibly via increasing mitochondrial COX-II and ATPase 6 gene expression. Taken together, these data suggest PDGF under hypoxic conditions increases mTORC1 which could control mitochondrial fission and hypoxic genes (HIF1α and FIH-1) and significantly stimulates cell proliferation. However, DRP1 still seems essential in regulating cell proliferation. Therefore, the effect of mTORC1 knockdown in PDGF stimulated cells during hypoxia was studied to confirm the role of mTORC1 in mediating cell proliferation. Knockdown of the mTORC1 gene caused a significant decrease in cell proliferation associated with an increase in DRP1, HIF1α and FIH-1 gene expression. Finally, the inhibitory mechanism of Mdivi-1 following mTORC1 knockdown was studied. Mdivi-1 caused further reduction in cell proliferation, an increase in HIF1α gene and a decrease in FIH-1 expression in cells treated with siRNA against mTORC1. The results also showed Mdivi-1 enhanced HIF1α and FIH-1 expression and decreased mTORC1 expression in hypoxic background cells. Taken together, Mdivi-1 inhibits PDGF induced cell proliferation during hypoxia by increasing FIH-1, which is responsible for HIF1α degradation, via its direct inhibitory effect on mTORC1.
Advisor / supervisor
  • Coats, Paul
  • Plevin, Robin
Resource Type
Note
  • This thesis was previously held under moratorium from 08/07/2020 to 08/07/2022
DOI
Date Created
  • 2020
Former identifier
  • 9912896293402996

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