Therapeutic Aqueous Humor Concentrations of Latanoprost Attained in Rats by Administration in a Very-High-Molecular-Weight Hyaluronic Acid Eye Drop.

The temporal change in concentration of a novel medicine, Latanoprost (LP), was evaluated in the aqueous humor of rats (6-8-week-old Jcl:Wister rats) when delivered in a very-high-molecular-weight hyaluronic acid (vHiHA) eye drop. Animals were randomly assigned to three treatment groups (LP + vHiHA (LPvHiHA), commercial LP (cLP), and diluted LP (dLP)) and after instilling the eye drops, the aqueous humor (AH) was collected at 0.5, 1, 2, 4, and 6 h to measure the LP concentration using an enzyme-linked immunosorbent assay (ELISA). Although the LP concentration in the LPvHiHA eye drop formulation was 3.57 times lower than in the commercial eye drops used (cLP), the LP concentration in the AH following LPvHiHA administration reached a value close to that of cLP. The cLP was diluted to the same concentration of LP as in the LPvHiHA eye drops for the dLP group, but the LP concentration in the AH of these animals was lower than that of the LPvHiHA rats at all time points. The higher LP concentration in the AH of the LPvHiHA rats suggests that vHiHA may aid the transport of LP across the ocular surface epithelium.

Human corneal limbal organoids maintaining limbal stem cell niche function.

Corneal epithelial stem cells reside in the limbal area between the cornea and conjunctiva. We examined the potential use of limbal organoids as a source of transplantable limbal stem cells. After treating tissue with collagenase, limbal cells were seeded onto Matrigel and cultivated using limbal phenotype maintenance medium. After 1-month, approximately 500 organoids were formed from one donor cornea. Organoids derived from vertical sites (superior and inferior limbus) showed large colony forming efficiency, a higher ratio of slow cycling cells and N-cadherin-expressing epithelial cells compared to horizontal sites. The progenitor markers Keratin (K) 15 and p63 were expressed in epithelial sheets engineered form a single organoid. Organoids transplanted in the limbus of a rabbit limbal deficiency model confirmed the presence of organoid-derived cells extending on to host corneas by immunohistochemistry. Our data show that limbal organoids with a limbal phenotype can be maintained for up to 1 month in vitro which can each give rise to a fully stratified corneal epithelium complete with basal progenitor cells. Limbal organoids were successfully engrafted in vivo to provide epithelial cells in a rabbit limbal deficiency model, suggesting that organoids may be an efficient cell source for clinical use.

Effects of Amniotic Membrane-Derived Fibroblast Supernatant on Corneal Epithelium.

Purpose: To evaluate the effects of human amniotic membrane-derived fibroblast (AMF) cell supernatant (AMF-sup) on corneal epithelium. Methods: The phenotype of AMF cells was analyzed by flow cytometry using cell-surface markers. AMF cells were also induced to form osteoblasts and neural cells, and cell phenotypes were observed by staining and RT-PCR. Cultivated human corneal limbal epithelial sheets generated using AMF-sup were analyzed using immunohistochemistry and colony-forming efficiency, and the wound healing of epithelial defects was observed using a tissue-punch method. The effects of instillation of each supernatant in a rabbit corneal epithelial wound healing model were compared. Results: Mesenchymal stem cell (CD29, CD44, CD73, and CD90) and neural crest (CD49d and CD56) markers were expressed on the AMF cell surface. Following induction of differentiation, isolated AMF cells showed characteristics of osteoblasts and neural cells. Application of AMF-sup resulted in maintenance of the limbal epithelial phenotype and immature state, and significantly promoted wound healing in cultivated human corneal limbal epithelial sheets (P < 0.05) and rabbit corneal epithelium (P < 0.05) compared with the control. Conclusions: These data suggest that AMF cells have multi-differentiation potential, and that AMF-sup is effective in maintaining the limbal epithelial phenotype and promoting corneal epithelial wound healing, which may be of value in ocular surface reconstruction.

Cytokine profiling in the sub-silicone oil fluid after vitrectomy surgeries for refractory retinal diseases.

Silicone oil (SO) is an intraocular surgical adjuvant that reduces the surgical complications in refractory retinal diseases, although membrane and cellular proliferation is often seen even in SO-filled eyes. We hypothesised that the fluid in the space between the SO and the retina, named the "sub-silicone oil fluid (SOF)", enhances these biological responses. We proposed a safe method for SOF extraction. We also analysed inflammatory cytokine expressions and SOF osmotic pressures from eyes with rhegmatogenous retinal detachment (RRD), proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and macular hole-associated retinal detachment (MHRD). Interleukin (IL)-10, IL-12p40, IL-6, monocyte chemotactic protein-1, and vascular endothelial growth factor (VEGF) in the SOF with PVR were significantly higher than in those with RRD or MHRD. Fibroblast growth factor-2, IL-10, IL-12p40, IL-8, VEGF, and transforming growth factor beta 1 levels in eyes with exacerbated PDR indicated a significantly higher expression than those with simple PDR. IL-6 and tumour necrosis factor alpha in eyes with exacerbated PVR demonstrated a significantly higher expression than in those with simple PVR. However, there was no difference in SOF osmotic pressure between group of each disease. These studies indicate that disease-specific SOF is a significant reflection of disease status.

Nuclear Factor (Erythroid-Derived)-Related Factor 2-Associated Retinal Pigment Epithelial Cell Protection under Blue Light-Induced Oxidative Stress.

Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage. Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type and Nrf2 knockout (Nrf2-/-) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed on NRF2 mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells. Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure induced NRF2 mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells from Nrf2-/- mice than from wild-type mice. Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress.

Malondialdehyde induces autophagy dysfunction and VEGF secretion in the retinal pigment epithelium in age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness in developed countries and is closely related to oxidative stress, which leads to lipid peroxidation. Malondialdehyde (MDA) is a major byproduct of polyunsaturated fatty acid (PUFA) peroxidation. Increased levels of MDA have been reported in eyes of AMD patients. However, little is known about the direct relationship between MDA and AMD. Here we show the biological importance of MDA in AMD pathogenesis. We first confirmed that MDA levels were significantly increased in eyes of AMD patients. In ARPE-19 cells, a human retinal pigment epithelial cell line, MDA treatment induced vascular endothelial growth factor (VEGF) expression alternation, cell junction disruption, and autophagy dysfunction that was also observed in eyes of AMD patients. The MDA-induced VEGF increase was inhibited by autophagy-lysosomal inhibitors. Intravitreal MDA injection in mice increased laser-induced choroidal neovascularization (laser-CNV) volumes. In a mouse model fed a high-linoleic acid diet for 3 months, we found a significant increase in MDA levels, autophagic activity, and laser-CNV volumes. Our study revealed an important role of MDA, which acts not only as a marker but also as a causative factor of AMD pathogenesis-related autophagy dysfunction. Furthermore, higher dietary intake of linoleic acid promoted CNV progression in mice with increased MDA levels.