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Meili Zhu, Yun-Zheng Le; Testing the effect of RPE barrier function regulators in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1770.
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Vascular leakage, a major pathological change in diabetic retinopathy (DR), is traditionally regarded as endothelial barrier dysfunction. However, the RPE barrier is responsible for approximately 80% of blood circulation to the retina. While a significant portion of cystoid diabetic macular edema (DME) patients have cellular lesions near the RPE, such as exudative subretinal detachment, the contribution of the RPE barrier breakdown in overall vascular leakage in DR is almost neglected. We recently developed an in vivo semi-quantitative imaging for RPE barrier-specific leakage and demonstrated a substantial contribution of the RPE barrier breakdown in diabetic rodents (IOVS 52: 2160, 2011). To assess the feasibility of our assay in screening/verifying therapeutic agents for diabetes-induced RPE barrier breakdown, we performed a proof-of-concept study and found that in vivo imaging analysis correlated well with in vitro RPE barrier function assay.
Blood-retina barrier (BRB) function regulators, such as vascular endothelial growth factor (VEGF) and extracellular-signal-regulated kinase (ERK) inhibitor, were used to measure the trans-epithelial resistance (TER) in ARPE-19 cells under various culture conditions. High molecular weight fluorescein isothiocyanate (FITC)-dextran was used to determine the effect of these BRB function regulators on RPE barrier in hypoxic mice generated with oxygen-induced retinopathy (OIR).
VEGF caused a significant reduction of TER in ARPE-19 cultures. This observation correlated with the increase of severe RPE barrier break points in OIR mice supplemented with VEGF intravitreally. Likewise, ERK inhibitor, which was suggested to play a role in downregulating VEGF, increased TER in hypoxic ARPE-19 cultures and reduced the frequency of severe RPE barrier break points in OIR mice. In vivo testing RPE barrier function for other BRB regulators is in progress.
We have demonstrated that our in vivo RPE barrier function analysis correlates well with traditional in vitro RPE barrier function assay. Therefore, a combination of in vivo and in vitro assays for RPE barrier function may be useful for mechanistic studies and for screening drugs with therapeutic potentials for RPE barrier breakdown in DME, a major vision loss in DR.
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