Investigation of the regulatory role of heme oxygenase-1 and its products during VEGF-induced angiogenesis, using in vitro and in vivo models
Author(s)
Bauer, Andrea
Type
Thesis
Abstract
Angiogenesis is fundamental to many physiological processes, and associated with various pathologies, including atherosclerosis and malignant disease.
Increasing evidence suggests a role for the cytoprotective enzyme heme oxygenase-1 (HO-1) and its products in angiogenesis. However, the mechanisms through which HO-1 exerts its effects remain elusive. This study aims to identify signalling pathways and novel HO-1 downstream targets regulating angiogenesis.
I show that inhibition of HO-1 with synthetic antagonist (ZnPP) or specific siRNA alters the angiogenic process at various levels. HO-1 inhibition significantly reduced vascular endothelial growth factor A (VEGF)-mediated human endothelial cell (EC) proliferation and inhibited capillary-like formation on 2D-Matrigel. Further, I demonstrate that VEGF-induced EC cell cycle progression is inhibited by HO-1 siRNA; an observation associated with decreased expression of cell cycle regulators cyclin A1 and cyclin E1. In contrast, HO-1-deficient cells were still protected from apoptosis by VEGF, most likely through induction of anti-apoptotic genes Bcl-2 and A1. Interestingly, HO-1 depletion negatively affected directional migration of EC towards a VEGF gradient; a phenotype reversed by HO-1 over-expression using an adenoviral vector. Moreover, migrating HO-1-deficient cells showed decreased cyclin A1 protein accompanied by decreased cyclin-dependent kinase 2 activity. Importantly, a combined proteomics and microarray approach has identified downstream targets of HO-1 and their potential roles in HO-1-driven angiogenesis have been investigated. For instance, HO-1 depletion results in impaired assembly of the intermediate filament vimentin. HO-1-deficient cells show reduced activity of the calcium-dependent protease calpain in response to VEGF; this observation was accompanied by a decrease in vimentin cleavage. The differences in vimentin cleavage and filament assembly may in turn account for the impaired angiogenic phenotype of HO-1-deficient cells.
Identification of HO-1 downstream target genes may reveal potential therapeutic approaches for enhancing angiogenesis at sites of ischaemia or wound healing, or alternatively inhibiting angiogenesis associated with atherosclerosis or tumourogenesis.
Increasing evidence suggests a role for the cytoprotective enzyme heme oxygenase-1 (HO-1) and its products in angiogenesis. However, the mechanisms through which HO-1 exerts its effects remain elusive. This study aims to identify signalling pathways and novel HO-1 downstream targets regulating angiogenesis.
I show that inhibition of HO-1 with synthetic antagonist (ZnPP) or specific siRNA alters the angiogenic process at various levels. HO-1 inhibition significantly reduced vascular endothelial growth factor A (VEGF)-mediated human endothelial cell (EC) proliferation and inhibited capillary-like formation on 2D-Matrigel. Further, I demonstrate that VEGF-induced EC cell cycle progression is inhibited by HO-1 siRNA; an observation associated with decreased expression of cell cycle regulators cyclin A1 and cyclin E1. In contrast, HO-1-deficient cells were still protected from apoptosis by VEGF, most likely through induction of anti-apoptotic genes Bcl-2 and A1. Interestingly, HO-1 depletion negatively affected directional migration of EC towards a VEGF gradient; a phenotype reversed by HO-1 over-expression using an adenoviral vector. Moreover, migrating HO-1-deficient cells showed decreased cyclin A1 protein accompanied by decreased cyclin-dependent kinase 2 activity. Importantly, a combined proteomics and microarray approach has identified downstream targets of HO-1 and their potential roles in HO-1-driven angiogenesis have been investigated. For instance, HO-1 depletion results in impaired assembly of the intermediate filament vimentin. HO-1-deficient cells show reduced activity of the calcium-dependent protease calpain in response to VEGF; this observation was accompanied by a decrease in vimentin cleavage. The differences in vimentin cleavage and filament assembly may in turn account for the impaired angiogenic phenotype of HO-1-deficient cells.
Identification of HO-1 downstream target genes may reveal potential therapeutic approaches for enhancing angiogenesis at sites of ischaemia or wound healing, or alternatively inhibiting angiogenesis associated with atherosclerosis or tumourogenesis.
Date Issued
2012-11
Date Awarded
2013-05
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Randi, Anna
Wait, Robin
Mason, Justin
Sponsor
National Heart and Lung Institute Foundation
Publisher Department
National Heart and Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)