The role of bone morphogenetic protein 4 in the pathogenesis of choroidal neovascularization in age-related macular degeneration.

摘要

Recently our studies indicated that bone morphogenetic protein-4 (BMP4), one growth factor of the transforming growth factor--beta (TGF--beta) superfamily, may be involved in the molecular switch that determines which advanced form of AMD an individual develops. This work investigated how BMP4 was down-regulated in the context of CNV and utilized transgenic mice overexpressing BMP4 in RPE to address the functional role of BMP4 in ocular angiogenesis. Therefore, we have provided detailed molecular pathways of BMP4 regulation in two forms of advanced AMD and suggest that BMP4 is part of a molecular switch determining which phenotypic pathway is taken in the progression of AMD. We first demonstrated that our previous finding of decreased BMP4 expression in human CNV translated to decreased BMP4 expression in laser-induced CNV in mice. We showed for the first time that the pro-inflammatory factor, tumor necrosis factor (TNF) significantly down-regulated BMP4 expression in human RPE cells, in mouse eyecups, as well as in the laser-induced CNV mouse model. We identified two specificity protein 1 (Sp1) binding sites on the BMP4 promoter that are required for its basal expression and TNF's down-regulation. Gel shift assay confirmed the physical interaction of transcription factor Sp1 with the BMP4 promoter, which was also reduced by TNF. Through c-Jun NH 2-terminal kinase (JNK) activation, TNF modulated Sp1 phosphorylation, thus decreased its DNA affinity to BMP4 promoter. We also showed that TNF significantly reduced transepithelial resistance in highly differentiated, polarized RPE cell cultures. The down-regulation of BMP4 expression by TNF in CNV and mechanisms established may be useful for defining targets for AMD therapy.;As an important regulator in ocular development, BMP4 has been shown to inhibit RPE proliferation, induce RPE senescence and cause apoptosis of venous and capillary endothelial cells, indicating BMP4 as a negative regulator in the eye. While we showed the low levels of BMP4 and up-regulated TNF in CNV would be a permissive environment for angiogenesis, we also provided evidence for an anti-angiogenic role of BMP4 in laser-induced CNV. Our data showed that in transgenic mice overexpressing BMP4 in RPE cells, the lesion size and leakage of CNV at Day 7 post laser were less severe than those in the control mice. Both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) expression were down-regulated after laser in the transgenic mice, compared to the control mice. The in vivo findings were further verified by in vitro studies. VEGF secretion in RPE cells isolated from the transgenic mice was reduced and lost response to TNF stimulation. We found that BMP4 also reduced TNF-induced MMP-9 secretion in human RPE cells through a Smad-dependent pathway. In addition, BMP4 induced bovine choroidal endothelial cell (CEC) apoptosis in a dose-dependent way through caspase-3 activation, although BMP4's effects on bovine CEC proliferation, migration or tube formation were not significant in our test system. The finding that BMP is anti-angiogenic could explain why most patients have dry AMD without CNV. When oxidative stress increased BMP4 expression in the RPE cells, in addition to RPE senescence, the up-regulated BMP4 inhibits MMP-9 expression directly, and thus downregulates VEGF indirectly, creating an anti-angiogenic environment to prevent CNV formation in dry AMD. However, local inflammation in some cases can shift the microenvironment into a pro-angiogenic one.;In summary, this project has established how the differentially regulated BMP4 is involved in the molecular switch determining which phenotypic pathway is taken in the progression of AMD. Chronic oxidative stress can promote BMP4 expression in RPE and induce RPE senescence as seen in early AMD. If BMP4 expression is sustained, the senescent RPE cells eventually undergo apoptosis and lead to geographic atrophy with secondary degeneration of the neuroretina and the choroid. On the other hand, inflammatory mediator such as TNF can decrease BMP4 expression in RPE cells and promote angiogenesis by increasing VEGF and MMP-9. BMP4 may also participate in CNV regression by downregulating MMP-9 and inducing CEC apoptosis. Therefore, the development of two forms of AMD depends on the microenvironment and its impact on the senescent RPE cells. We elucidated the role of JNK activation and Sp1 in the regulation of BMP4 by TNF, therefore, interference with the JNK pathway or the transcription factor Sp1 could be a promising approach for CNV treatment. (Abstract shortened by UMI.)