PURPOSE: Oxidative stress has been implicated in a variety of diseases of the eye. In several other tissues, keratinocyte growth factor (KGF) has been shown to prevent negative cellular changes associated with oxidative insult, such as permeability increases and nuclear DNA damage. In this study, we looked at whether KGF provided these same protective effects to cultured human retinal pigmented epithelial (RPE) cells (ARPE-19). METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) using a published primer pair sequence followed by restriction endonuclease digestion with AvaI and HincII was used to look for the KGF receptor message in ARPE-19 cells. Cellular response to KGF was verified through proliferation assays and Western blot analysis for mitogen-activated protein kinase (MAPK). Single-cell gel electrophoresis was used to assess DNA damage, Western blot analysis was used to assay actin cytoskeletal changes, and electrical resistance and tracer experiments with Transwell tissue plates were used to assess permeability changes. Immunostaining was used to verify the existence of the tight junction protein occludin. RESULTS: It was verified through RT-PCR that the ARPE-19 cell line exhibited the message for FGFR2-IIIb, otherwise known as KGFR. KGF was also shown to increase cellular proliferation and activated the MAPK p44/p42 cascade. KGF ameliorated nuclear DNA damage and cytoskeletal rearrangement caused by oxidative stress through the addition of exogenous hydrogen peroxide but was unable to prevent permeability changes. CONCLUSIONS: KGF was shown to significantly reduce DNA damage and cytoskeletal rearrangement caused by oxidative stress in cultured ARPE-19 cells. This result may be useful in targeting future therapies to combat a multitude of diseases of the eye that result from increases in reactive oxygen species.
PURPOSE: Oxidative stress has been implicated in a variety of diseases of the eye. In several other tissues, keratinocyte growth factor (KGF) has been shown to prevent negative cellular changes associated with oxidative insult, such as permeability increases and nuclear DNA damage. In this study, we looked at whether KGF provided these same protective effects to cultured humanretinal pigmented epithelial (RPE) cells (ARPE-19). METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) using a published primer pair sequence followed by restriction endonuclease digestion with AvaI and HincII was used to look for the KGF receptor message in ARPE-19 cells. Cellular response to KGF was verified through proliferation assays and Western blot analysis for mitogen-activated protein kinase (MAPK). Single-cell gel electrophoresis was used to assess DNA damage, Western blot analysis was used to assay actin cytoskeletal changes, and electrical resistance and tracer experiments with Transwell tissue plates were used to assess permeability changes. Immunostaining was used to verify the existence of the tight junction protein occludin. RESULTS: It was verified through RT-PCR that the ARPE-19 cell line exhibited the message for FGFR2-IIIb, otherwise known as KGFR. KGF was also shown to increase cellular proliferation and activated the MAPK p44/p42 cascade. KGF ameliorated nuclear DNA damage and cytoskeletal rearrangement caused by oxidative stress through the addition of exogenous hydrogen peroxide but was unable to prevent permeability changes. CONCLUSIONS:KGF was shown to significantly reduce DNA damage and cytoskeletal rearrangement caused by oxidative stress in cultured ARPE-19 cells. This result may be useful in targeting future therapies to combat a multitude of diseases of the eye that result from increases in reactive oxygen species.