JUN siRNA regulates matrix metalloproteinase-2 expression, microvascular endothelial growth and retinal neovascularisation.

Transcription factors link changes in the extracellular environment with alterations in gene expression. As such, these molecules serve as attractive targets for intervention in pathological settings. Since JUN has been linked with microvascular disease in humans, we hypothesised that small interfering RNA (siRNA) targeting this immediate-early gene may be useful agents that suppress endothelial growth and neovascularisation. Here we show that Jun siRNA inhibits Jun mRNA and protein expression in murine microvascular endothelial cells, blocks cell proliferation and suppresses migration in a scratch-wound assay. It also inhibits three-dimensional tubular formation on basement membrane extracts and reduces angiogenesis in mice bearing Matrigel plugs as subcutaneous implants. Single intravitreal administration of Jun siRNA reduces neovascularisation in a murine model of proliferative retinopathy, and suppresses endothelial JUN and matrix metalloproteinase-2 (MMP-2) immunoreactivity in retinal vessels, data supported by its repression of MMP-2 expression and gelatinolytic activity in vitro. Co-administration of TGFbeta with the siRNA reverses this neovascular inhibitory effect, which is in turn abrogated by cis-9-octadecenoyl-N-hydroxylamide, consistent with the involvement of a metalloproteinase such as MMP-2. Thus, JUN siRNA can serve as a specific inhibitor of aberrant endothelial and neovascular growth.

The role of ultraviolet irradiation and heparin-binding epidermal growth factor-like growth factor in the pathogenesis of pterygium.

Ultraviolet (UV) light is one of the major factors implicated in the pathogenesis of pterygium. The mechanism by which UV light induces this disease remains elusive. The aim of this study was to evaluate the effects of UVB irradiation on the expression of growth factors in cultured pterygium epithelial cells and to demonstrate their distribution within pterygium. We cultured pterygial epithelial cells from pterygium explants and these cells were exposed to 20 mJ/cm(2) of UVB. Total RNA was extracted at 0, 6, and 12 hours after irradiation. (32)P-labeled cDNA was synthesized and analyzed using microarray technology to determine the differential expression of 268 growth factor and cytokine related genes. Semiquantitative reverse transcriptase-polymerase chain reaction was used to corroborate this data. Conditioned media derived from cells exposed to UVB irradiation was analyzed for protein expression by enzyme-linked immunosorbent assay. Immunohistochemistry was used to evaluate the distribution of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pterygium tissue. Analysis of the hybridization signals revealed that the genes encoding HB-EGF, fibroblast growth factor 3, and cytotoxic trail ligand receptor were consistently elevated at 6 and 12 hours after UVB treatment. HB-EGF mRNA was elevated 6.8-fold at 6 hours after irradiation and was augmented in culture supernatants after the same treatment. Furthermore, HB-EGF reactivity was identified in the epithelium and vasculature of pterygium by immunohistochemistry. HB-EGF was present in normal limbal epithelium, although it was not induced in cultured limbal epithelial cells by UV irradiation. HB-EGF is a potent mitogen, localized in pterygium tissue, and significantly induced by UVB in pterygium-derived epithelial cells. We postulate that this growth factor is a major driving force in the development of pterygia and a means by which UV irradiation causes the pathogenesis of pterygium.