Gene transfer of prolyl hydroxylase domain 2 inhibits hypoxia‐inducible angiogenesis in a model of choroidal neovascularization

Abstract

Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF).In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Hypoxia is a stress situation triggering a multitude of responses that ensure survival of organisms to oxygen deprivation. Adaptation to hypoxia occurs by transcriptional upregulation of multiple genes involved in responses such as angiogenesis (e.g. vascular endothelial growth factor; VEGF), formation of red blood cells (e.g. erythropoietin), anaerobic metabolism (e.g. glycolytic enzymes and glucose transporters), and multiple others 1,2 . Gene induction in hypoxia is mediated by hypoxia-inducible factors (HIF), a family of heterodimeric transcription factors composed of an α -and a β -subunit capable of recognizing hypoxia-response elements (HRE) in the regulatory regions of hypoxia-inducible genes [3][4][5] . In contrast to the constitutive HIF-β , oxygen levels regulate HIF-α activity and protein stability. At normoxia, an asparagine residue within the C-terminal transactivation domain of HIF-α is hydroxylated by the factor inhibiting HIF-1 (FIH-1), impairing the recruitment of the coactivator CBP (cAMP response element binging protein) 6,7 . An additional modification by hydroxylation regulates HIF-α protein stability, in this instance by a family of prolyl hydroxylase domain proteins (PHD), that hydroxylate two distinct proline residues within HIF-α [8][9][10][11][12] . Hydroxylated prolines are the recognition signature for the E3 ubiquitin-ligase von Hippel-Lindau protein (VHL), leading to proteasome-mediated degradation of HIF-α [13][14][15][16][17][18][19] . HIF dioxygenases (PHDs and FIH-1) require molecular oxygen to hydroxylate HIF-α , and are considered the cellular oxygen sensors. Upon oxygen deprivation, the dioxygenases are rendered inactive allowing formation of the transcriptional active HIF. In certain tissues, as the cornea in the eye, avascularity is maintained under hypoxic conditions, illustrating a supplementary regulatory mechanism of HIF-α proteins. In the hypoxic cornea, the tissue-specific inhibitory PAS protein (IPAS; inhibitory Period-Arnt-Sim domain) binds HIF-α subunits and creates a DNA-abortive complex incapable of activating transcription 20,21 .