Abstract
Pathological activation of the hypoxia-inducible-factor (HIF) pathway leading to expression of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF), is the fundamental cause of neovascularization in ocular ischemic diseases and cancers. We have shown that pure honokiol inhibits the HIF pathway and hypoxia-mediated expression of pro-angiogenic genes in a number of cancer and retinal pigment epithelial (RPE) cell lines. The crude extracts, containing honokiol, from Magnolia plants have been used for thousands of years in the traditional oriental medicine for a number of health benefits. We have recently demonstrated that daily intraperitoneal injection of honokiol starting at postnatal day (P) 12 in an oxygen induced retinopathy mouse model significantly reduced retinal neovascularization at P17. Here, we evaluate the mechanism of HIF inhibition by honokiol in RPE cells. Using chromatin immunoprecipitation experiments, we demonstrate that honokiol inhibits binding of HIF to hypoxia-response elements present on VEGF promoter. We further show using a number of in vitro angiogenesis assays that, in addition to anti-HIF effect, honokiol manifests potent anti-angiogenic effect on human retinal micro vascular endothelial cells. Our results suggest that honokiol possesses potent anti-HIF and anti-angiogenic properties. These properties of honokiol make it an ideal therapeutic agent for the treatment of ocular neovascular diseases and solid tumors.
Highlights
Pathological neovascularization (NV), where abnormal vasculature originate from existing blood vessels, is observed in most solid tumors and in retinal ischemic diseases, such as diabetic retinopathy (DR), retinopathy of prematurity (ROP), retinal vein occlusion (RVO), etc. [1,2,3]
We further show using a number of in vitro angiogenesis assays that, in addition to anti-hypoxia inducible factor (HIF) effect, honokiol manifests potent anti-angiogenic effect on human retinal micro vascular endothelial cells
In the reporter plasmid (5HRE-ODD-luc), 5 copies of the hypoxia response element (HRE) (5HRE) and an oxygendependent degradation (ODD) domain of HIF-1a regulate the expression of luciferase
Summary
Pathological neovascularization (NV), where abnormal vasculature originate from existing blood vessels, is observed in most solid tumors and in retinal ischemic diseases, such as diabetic retinopathy (DR), retinopathy of prematurity (ROP), retinal vein occlusion (RVO), etc. [1,2,3]. Despite different primary cause and clinical manifestations, these diseases share a common molecular feature, i.e. hypoxia mediated activation of the hypoxia inducible factor (HIF) pathway. Central to this pathway is the heterodimeric HIF transcription factor. If any HIF-a escapes cytoplasmic degradation and translocates to the nucleus, asparagine hydroxylation prevents its binding to co-activator (p300/CBP), thereby hindering HIF mediated transcription [11]. Under hypoxic conditions HIF hydroxylases become inactive, ceasing HIF-a hydroxylation. This allows translocation of HIF-a in to the nucleus and its dimerization with HIF-b, forming the active HIF complex. Activated HIF binds to hypoxia response element (HRE) present in the promoters of hypoxia response genes including many critical proteins involved in angiogenesis [12]
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