Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization

Purpose: To investigate the transcriptional factors associated with epithelial-mesenchymal transition (EMT) in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). Methods: Paraffin sections of CNV obtained from patients with AMD (n=12) were stained for transcriptional factors related to EMT, i.e., Snail, Slug, SIP1, and Twist. As a control, postmortem sections of ocular normal tissue were used. Furthermore, using a human retinal pigment epithelial (RPE) cell line (ARPE-19), reverse transcription–polymerase chain reaction (RT–PCR) and immunofluorescence microscopy were performed to explore the cellular localization and expression levels of EMT-associated transcriptional factors upon cytokine stimulation. Results: Of 12 specimens, 11 CNV tissues (91.6%) showed staining for Snail localized in cellular nuclei, particularly in those of RPE cells. Snail was strongly co-localized with α-smooth muscle antigen (SMA) in RPE cells. In contrast, postmortem human retina showed no Snail staining in RPE cells. Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina. In ARPE-19 cells, RT–PCR and immunofluorescence microscopy showed that Snail mRNA was upregulated by transforming growth factor (TGF)-β and VEGF stimulation. Furthermore, TGF-β induced relocalization of Snail to the nucleus in RPE cells. Conclusions: The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV

Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca2+ efflux by activating the calmodulin-dependent Ca2+-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15–24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers – ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion