Curcumina en la inhibición de la angiogénesis inducida por epitelio pigmentario de la retina, estudio comparativo con Bevacizumab

Abstract

Choroidal neovascularization is the third leading cause of blindness in the western world. Its etiology remains unknown. However, it has been established that both up-regulation of VEGF and down-regulation of PEDF at the outermost layer of the retina, in the so-called retinal pigment epithelium – choroid complex, is the milestone of this pathological process. The regulatory mechanisms that ultimately fail to inhibit this pro-angiogenic environment are unknown and current treatments are not completely effective preventing blindness due to this disease. Nevertheless, the transcriptional factor hypoxia induced factor -1 (HIF-1) has been implicated in the pathogenesis of choroidal neovascularization (CNV). The action of HIF-1 upon hypoxia mediated-stabilization is to activate the transcription of specific hypoxia adaptive genes, including VEGF. Curcumin is a natural diphenol extracted from Curcuma longa, and displays a wide range of anti-cancer activities. Among them, inhibition of HIF-1 by yet undefined pathways has been observed in tumor cells. Hence, we aimed to determine the effect of curcumin on hypoxia mediated-choroidal neovascularization. To this end, we used a bi cellular model, including retinal-pigmented epithelium (RPE) cells and coroidal vascular endothelium (CVE) cells. Firstly, we determined RPE response to hypoxia. Secondly, we explored the response of CVE cells to RPE hypoxia, and the effect that curcumin may elicit in this signaling. Finally, we used an in vivo model to further confirm our in vitro findings. As expected, we observed an angiogenic activity in CVE cells in response to RPE hypoxia through soluble factors contained in the conditioned medium. Curcumin was able to inhibit this effect, preventing CVE cell proliferation, migration and hypoxia-induced experimental CNV. The production of VEGF in RPE under hypoxia was also decreased upon curcumin treatment. In an inverse fashion, PEDF expression was upregulated with curcumin treatment. Finally, curcumin was able to inhibit hypoxia RPE-induced angiogenesis in vivo. These data suggest that curcumin could eventually be considered as a possible therapeutic molecule to prevent or treat choroidal neovascular disease and thus, reduce blindness. AMD: Age Macular Degeneration. Macular degeneration, often age-related macular degeneration (AMD or ARMD), is a medical condition that usually affects older adults and results in a loss of vision in the center of the visual field (the macula) produced by damage to the retina. It is a major cause of blindness and visual impairment in older adults (>50 years). It occurs in "dry" and "wet" forms. In the wet (exudative and age-related) form, which is more severe, blood vessels grow up from the choroid behind the retina (known as choroidal neovascularization), and the retina can also become detached. The dry form does not produce choroidal neovascularization and rarely leads to blindness. ARPE-19: ARPE-19 is a spontaneously arising retinal pigment epithelial (RPE) cell line derived in 1986 by Amy Aotaki-Keen from the normal eyes of a 19-year-old male who died from head trauma in a motor vehicle accident. These cells form stable monolayers, which exhibit morphological and functional polarity. ARPE-19 expresses the RPE-specific markers CRALBP and RPE-65. CNV: Choroidal neovascularization. Consists in the creation of new blood vessels in the choroid layer of the eye. This is a common sign of the wet type of AMD. CVE: Choroid Vascular Endothelium. Is the endothelium that underlies capillaries of the choroid, which is the vascular layer of the eye. The coroid also contains connective tissue, and lies between the retina and the sclera. The choroid provides oxygen and nourishment to the outer layers of the retina. DR: Diabetic Retinopathy. Is a disease of the retina caused as complication of Diabetes, as result of microvascular retinal changes and hypoxia. This disease can also establish a pro angiogenic environment in the retina, leading to retinal neovascularization and blindness. FGF: Fibroblast Growth Factor. A family of small polypeptide growth factors that share several common features, including a strong affinity for HEPARIN, and a central barrelshaped core region of 140 amino acids that is highly homologous between family members. Although originally studied as proteins that stimulate the growth of fibroblasts, this distinction is no longer a requirement for membership in the fibroblast growth factor family. HIF-1: Hypoxia-induced Factor 1. A basic helix-loop-helix transcription factor that plays a role in apoptosis. It is composed of two subunits: aryl hydrocarbon receptor nuclear translocator and hypoxia-inducible factor 1, alpha subunit. HRE: Hypoxia Responsive Element. Binding site for HIF-1α in the promoter region of specific hypoxia-regulated target genes. PBS: Phosphate Buffer Saline. RPE: Retinal Pigment Epithelium. Pigmented monolayer of cuboidal cells with phagocytic function, responsible for maintaining the subretinal space and the external hemato-retinal barrier. It also degrades the discs of the outer segments of the photoreceptors. shRNA: "Short hairpin RNA". Corresponds to small RNA fragments used to attach and degrade specific mRNA via proteasome. This means that a "silenced" gene is achieved, leading to a decrease in the final functional protein. TER: Trans Epithelial Resistance. The electrical resistance of an epithelium, which means resistance to the passage of an electric current through that epithelium. The inverse quantity is electrical conductance, the ease with which an electric current passes. The unit of electrical resistance is the ohm (Ω). VEGF-A: Vascular Endothelium Growth Factor-A. The original member of the family of endothelial cell growth factors referred to as VASCULAR ENDOTHELIAL GROWTH FACTORS. Vascular endothelial growth factor-A was originally isolated from tumor cells and referred to as "tumor angiogenesis factor" and "vascular permeability factor". Although expressed at high levels in certain tumor-derived cells, it is produced by a wide variety of cell types, including RPE cells. In addition to stimulating vascular growth and vascular permeability, it may play a role in stimulating vasodilation. Alternative splicing of the mRNA for vascular endothelial growth factor-A originates several isoforms of the protein.