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Investigating the endothelial PI3 kinase signalling pathway in vascular repair Open Access


Other title
Endothelial cells
Type of item
Degree grantor
University of Alberta
Author or creator
Farhan, Maikel AA
Supervisor and department
Murray, Allan (Department of Medicine)
Examining committee member and department
Murray, Allan (Department of Medicine)
Touret, Nicolas (Department of Cell Biology)
McMurtry, Sean (Department of Medicine)
West, Lori (Department of Pediatrics)
Anderson, Deborah (University of Saskatchewan)
Department of Medicine
Experimental medicine
Date accepted
Graduation date
2016-06:Fall 2016
Doctor of Philosophy
Degree level
Thrombotic microangiopathy (TMA) is a broad term for a range of diseases that usually manifest with rapid failure of the affected organ. Although different in etiology, these diseases share a common pattern of injury originating in the vascular endothelium. In turn, the injured vasculature elicits a physiological response in a trial to repair the damage. Accumulating evidence support the significance of vascular repair but the key regulators, and the underlying mechanism are still elusive. We have shown before that key aspects of the genetic programming involved in angiogenesis are required for vascular repair. In this thesis: First, we characterized the PI3K/AKT pathway, the main angiogenic pathway, outputs in the endothelial cell (EC). We approached the pathway at three different levels; upstream (mTORC2), midstream (Akt) and downstream (mTORC1). Our results indicate sustained inactivation of mTORC1 activity, up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and vascular endothelial growth factor (VEGF). mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt. We concluded mTORC2 may have a superior role to Akt and mTORC1 in angiogenesis and vascular repair. Second, we identified the role of Facio-genital dysplasia-5 (FGD5) in regulation of the PI3K/AKT pathway. FGD5 is selectively expressed in EC and was reported to regulate angiogenesis. FGD5 deficiency reduced the number of angiogenic sprouts and their filopodia. These defects were accompanied by down regulation of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting, as well as VEGF-stimulated, EC, FGD5 formed a complex with VEGFR2 and was enriched at the leading edge of the cells and among endosomes. Further, FGD5 loss decreased endosomal VEGFR2 coupling to PI3K and diverted VEGFR2 to lysosomal degradation. This indicates FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3K/mTORC2-dependent cytoskeletal remodeling. Third, we investigated the role of FGD5 in regulation of G protein-coupled receptors (GPCRs) signaling. GPCRs operate in conjunction with VEGFR2 to activate PI3K pathway. We showed dual stimulation of GPCRs and VEGFR2 had synergic effect on angiogenesis. FGD5-loss abolished the GPCRs angiogenic effect and signaling to PI3K. Cdc42 inhibition, a RHO GEF required for PI3K activity, recapitulated the same signaling defects of FGD5 deficiency indicating that FGD5 may control PI3K activity through Cdc42. Subcellular localization of PI3K and its downstream Akt showed no change in PI3K localization to the early endosomes in case of FGD5 deficiency. However, failure of recruitment of active Akt to the PI3K positive endosomes suggests a defect in PI3K activity after FGD5 loss. This study investigated a novel role of FGD5 in regulating GPCRs signaling to PI3K, and suggests FGD5 as a convergence node regulating multiple angiogenic pathways that can spark hope for novel anti-angiogenic therapy. Finally, we studied vascular injury in animal model of chronic allograft vasculopathy (CAV). We showed that deficiency of Apelin, a peptide involved in angiogenesis that signals through PI3K in the endothelium, accelerated the vascular lesions in CAV and markedly affected the function of the transplanted grafts. This indicated that apelin may protect against the vascular injury produced in CAV. In summary, this work identifies potential targets that can regulate angiogenesis and vascular repair; and expands our understanding of the underlying mechanisms involved in angiogenesis. Further, it suggests novel candidates for antiangiogenic therapy to regulate pathological angiogenesis.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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