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Assessment of the Role of Endothelial mTORC2 in FGF2-induced Angiogenesis in vivo


Georgiopoulou, Stavroula. Assessment of the Role of Endothelial mTORC2 in FGF2-induced Angiogenesis in vivo. 2015, University of Zurich, Faculty of Medicine.

Abstract

Angiogenesis is a process where new capillaries are formed from pre-existing ones. It involves endothelial cell activation, extracellular matrix degradation, endothelial cell proliferation and migration and fusion with neighboring sprouts. Stabilization and lumen formation of the new capillary are the last steps of sprouting angiogenesis. Sprouting angiogenesis is one the most prominent mechanism of tumor vascularization and is a growth factor-driven process with FGF2 and VEGF being the key regulators.
The mTOR signaling pathway is a central cellular regulator and consists of two distinct complexes with individual roles and functions: mTORC1 and mTORC2. mTORC1 is a regulator of cell proliferation, protein synthesis, and growth and conveys signals from growth factors, ATP and O2 levels. Until now mTORC2 is known to mediate growth factor signaling and regulates cell survival and cytoskeleton reorganization. In all cell types mTORC1 is sensitive to Rapamycin-based inhibition, while mTORC2 is not. However in certain cell type such as endothelial cells mTORC2 can be inhibited by prolonged Rapamycin treatment. In xenograft tumor models Rapamycin treatment reduced tumor growth and tumor vascularization. In the majority of these studies dual inhibition of both mTOR complexes results in greater inhibition of tumor growth and vascularization than mTORC1 inhibition alone, pointing to a specific role of mTORC2 in tumor vascularization. The distinct roles of mTORC1 and mTORC2 in angiogenesis have not been elucidated due to the absence of specific mTORC2 inhibitors.
In this thesis we examine the role of endothelial mTORC2 in physiologic and FGF2-induced angiogenesis. mTORC2 is a multi-protein complex composed by mTOR, Rictor and other accessory proteins. We employed an endothelial-specific and Tamoxifen-induced RictoriΔec mouse that ideally leads to the ceasing of mTORC2 signaling in the endothelium. We monitored FGF2 induced angiogenesis in vivo with intravital microscopy of the dorsal skinfold chamber and FGF2 Matrigel plug implantation. We observed that ceasing mTORC2 signaling had no impact in already developed vasculatures and in wound-healing angiogenesis. RictoriΔec mice showed limited FGF2-induced capillary diameter increase, in resident skin capillaries of the skin muscle vasculature and exhibited differential remodeling architecture in the skin muscle vasculature. Impaired neo-angiogenesis in FGF2-loaded Matrigel plugs was also seen in Matrigel plugs of the RictoriΔec mice. Thus, we demonstrate for the first time direct evidence that endothelial mTORC2 signaling is crucial for FGF2 induced angiogenesis.
This thesis also analyses the efficiency and stability of Rictor knockout induction in the endothelium and the optimization of multiple endothelial cell isolation techniques, which led in the optimization of future uses of Cre-Lox based deletion techniques in the laboratory.
In conclusion the present thesis demonstrates for the first time the role of endothelial mTORC2 in FGF2-induced angiogenesis, by regulating capillary diameter, vasculature architecture and neo-angiogenesis. A mechanistic insight is further needed to reveal the molecular and cellular aspects of this regulation

Abstract

Angiogenesis is a process where new capillaries are formed from pre-existing ones. It involves endothelial cell activation, extracellular matrix degradation, endothelial cell proliferation and migration and fusion with neighboring sprouts. Stabilization and lumen formation of the new capillary are the last steps of sprouting angiogenesis. Sprouting angiogenesis is one the most prominent mechanism of tumor vascularization and is a growth factor-driven process with FGF2 and VEGF being the key regulators.
The mTOR signaling pathway is a central cellular regulator and consists of two distinct complexes with individual roles and functions: mTORC1 and mTORC2. mTORC1 is a regulator of cell proliferation, protein synthesis, and growth and conveys signals from growth factors, ATP and O2 levels. Until now mTORC2 is known to mediate growth factor signaling and regulates cell survival and cytoskeleton reorganization. In all cell types mTORC1 is sensitive to Rapamycin-based inhibition, while mTORC2 is not. However in certain cell type such as endothelial cells mTORC2 can be inhibited by prolonged Rapamycin treatment. In xenograft tumor models Rapamycin treatment reduced tumor growth and tumor vascularization. In the majority of these studies dual inhibition of both mTOR complexes results in greater inhibition of tumor growth and vascularization than mTORC1 inhibition alone, pointing to a specific role of mTORC2 in tumor vascularization. The distinct roles of mTORC1 and mTORC2 in angiogenesis have not been elucidated due to the absence of specific mTORC2 inhibitors.
In this thesis we examine the role of endothelial mTORC2 in physiologic and FGF2-induced angiogenesis. mTORC2 is a multi-protein complex composed by mTOR, Rictor and other accessory proteins. We employed an endothelial-specific and Tamoxifen-induced RictoriΔec mouse that ideally leads to the ceasing of mTORC2 signaling in the endothelium. We monitored FGF2 induced angiogenesis in vivo with intravital microscopy of the dorsal skinfold chamber and FGF2 Matrigel plug implantation. We observed that ceasing mTORC2 signaling had no impact in already developed vasculatures and in wound-healing angiogenesis. RictoriΔec mice showed limited FGF2-induced capillary diameter increase, in resident skin capillaries of the skin muscle vasculature and exhibited differential remodeling architecture in the skin muscle vasculature. Impaired neo-angiogenesis in FGF2-loaded Matrigel plugs was also seen in Matrigel plugs of the RictoriΔec mice. Thus, we demonstrate for the first time direct evidence that endothelial mTORC2 signaling is crucial for FGF2 induced angiogenesis.
This thesis also analyses the efficiency and stability of Rictor knockout induction in the endothelium and the optimization of multiple endothelial cell isolation techniques, which led in the optimization of future uses of Cre-Lox based deletion techniques in the laboratory.
In conclusion the present thesis demonstrates for the first time the role of endothelial mTORC2 in FGF2-induced angiogenesis, by regulating capillary diameter, vasculature architecture and neo-angiogenesis. A mechanistic insight is further needed to reveal the molecular and cellular aspects of this regulation

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Additional indexing

Item Type:Dissertation
Referees:Gassmann Max, Grimm Christian, Christofori Gerhard, Battegay Edouard, Humar Rok
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic and Policlinic for Internal Medicine
05 Vetsuisse Faculty > Institute of Veterinary Physiology
07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2015
Deposited On:17 Nov 2015 14:02
Last Modified:05 Apr 2016 19:33

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