Effect of Heat on a Model of the Bruch’s Membrane Regeneration: Implications for the Prevention of Age-Related Macular Degeneration

Abstract

Purpose: : Bruch’s membrane (BrM) provides a physical barrier for the underlying choriocapillaris and contains anti-angiogenic factors including, endostatin, thrombospondin-1 (TSP-1), and pigment epithelium derived factor (PEDF). Early age-related macular degeneration (AMD) is associated with alterations in BrM such as fragmentation of the elastic layer and decreased levels of angiogenesis inhibitors. Previous reports have demonstrated that heat increases elastin expression in the human skin and that heat shock proteins increase the expression of endostatin and TSP-1 in tumors. We therefore investigated the effect of heat on an in vitro model of the BrM regeneration and in a murine model of choroidal neovascularization (CNV).

Methods: : ARPE-19 cells, a line of human retinal pigment epithelial cells (RPE), were differentiated on laminin-coated transwells for four weeks. For heat treatment, differentiated ARPE-19 cells were incubated at 43°C for 30 min. Changes in mRNA and protein levels of tropoelastin, endostatin, TSP-1, and PEDF were determined by real-time PCR and western blot analysis (n=3). For in vivo analysis, mouse retina, a distance three disc-diameters from the optic nerve were heated by a series of four infrared diode laser (TTT) treatments. Twenty-four hours later, photocoagulation (PC) was performed in the center of the TTT irradiated area to induce CNV. Seven days following PC, the area of neovascularization was quantified using Scion Image software (version 4.0.2; Scion Corp., Frederick, MD) (n=6).

Results: : In ARPE-19 cells heat treatment significantly increased the levels of mRNA for tropoelastin, TSP-1, PEDF, and MMP-7, which produces endostatin by cleaving the C-terminal of collagen XVIII (P<0.05). Heat treatment also led to increased level of cell-associated tropoelastin and TSP-1 protein, and secreted endostatin and PEDF (P<0.05). In vivo, pre-treatment of the retinas with TTT significantly reduced the neovascular area (P=0.0026).

Conclusions: : Heat treatment can increase the production of elastin and anti-angiogenic factors by human RPE in vitro and suppress experimental CNV in vivo. We speculate that inhibition of new vessel growth in vivo is mediated by heat-induced increase in elastin and anti-angiogenic agents. These findings suggest that treatment with TTT could be used to reduce the risk of choroidal neovascularization in AMD.

Keywords: age-related macular degeneration • Bruch's membrane • retinal pigment epithelium