Investigation of the regulatory role of heme oxygenase-1 and its products during VEGF-induced angiogenesis, using in vitro and in vivo models

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.