Oxidative stress-mediated TXNIP loss causes RPE dysfunction.

The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood-retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.

Real-time estimation of paracellular permeability of cerebral endothelial cells by capacitance sensor array.

Vascular integrity is important in maintaining homeostasis of brain microenvironments. In various brain diseases including Alzheimer's disease, stroke, and multiple sclerosis, increased paracellular permeability due to breakdown of blood-brain barrier is linked with initiation and progression of pathological conditions. We developed a capacitance sensor array to monitor dielectric responses of cerebral endothelial cell monolayer, which could be utilized to evaluate the integrity of brain microvasculature. Our system measured real-time capacitance values which demonstrated frequency- and time-dependent variations. With the measurement of capacitance at the frequency of 100 Hz, we could differentiate the effects of vascular endothelial growth factor (VEGF), a representative permeability-inducing factor, on endothelial cells and quantitatively analyse the normalized values. Interestingly, we showed differential capacitance values according to the status of endothelial cell monolayer, confluent or sparse, evidencing that the integrity of monolayer was associated with capacitance values. Another notable feature was that we could evaluate the expression of molecules in samples in our system with the reference of real-time capacitance values. We suggest that this dielectric spectroscopy system could be successfully implanted as a novel in vitro assay in the investigation of the roles of paracellular permeability in various brain diseases.

Investigation of barrier characteristics in the hyaloid-retinal vessel of zebrafish.

The blood-retinal barrier (BRB) is essential for the physiological integrity of the retinal vessels. In particular, ocular pathologies of retinal neovascularization could be causally related to the BRB breakdown. Zebrafish have emerged as an advantageous model for studying vascular development and characteristics. Here we investigated for the first time the barrier characteristics of the hyaloid-retinal vessel using fli1-EGFP transgenic zebrafish. By 7 dpf, the hyaloid-retinal vessel was formed between lens and retina, where intercellular junctional complexes were already present between endothelial cells. Interestingly, NG-2 expression, but not GFAP, was colocalized with EGFP-positive cells of the hyaloid-retinal vessel. Among endothelial tight junction proteins, claudin-5 was expressed on EGFP-positive cells of the hyaloid-retinal vessel, whereas occludin and ZO-1 were not observed on the vessel. In addition, the hyaloid-retinal vessel was so leaky that a mixture of fluorescein tracers (2,000-kDa FITC-dextran, 10-kDa rhodamine-dextran, and 350-Da DAPI) diffusely infiltrated into all retinal layers. Our results suggest that, unlike retinal vessels of higher vertebrates, the hyaloid-retinal vessel of zebrafish shows insufficient characteristics to meet a functional endothelium-based CNS barrier. Therefore, it might be not suitable to use the hyaloid-retinal vessel of zebrafish for studying BRB biogenesis.