Breakdown of the blood-retinal barrier (BRB), as occurs in diabetic retinopathy and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing vision loss. Vasoinhibins are N-terminal fragments of prolactin that prevent BRB breakdown during diabetes. They modulate the expression of some transient receptor potential (TRP) family members, yet their role in regulating the TRP vanilloid subtype 4 (TRPV4) remains unknown. TRPV4 is a calcium-permeable channel involved in barrier permeability, which blockade has been shown to prevent and resolve pulmonary edema. We found TRPV4 expression in the endothelium and retinal pigment epithelium (RPE) components of the BRB, and that TRPV4-selective antagonists (RN-1734 and GSK2193874) resolve BRB breakdown in diabetic rats. Using human RPE (ARPE-19) cell monolayers and endothelial cell systems, we further observed that (i) GSK2193874 does not seem to contribute to the regulation of BRB and RPE permeability by vasoinhibins under diabetic or hyperglycemic-mimicking conditions, but that (ii) vasoinhibins can block TRPV4 to maintain BRB and endothelial permeability. Our results provide important insights into the pathogenesis of diabetic retinopathy that will further guide us toward rationally-guided new therapies: synergistic combination of selective TRPV4 blockers and vasoinhibins can be proposed to mitigate diabetes-evoked BRB breakdown.

SummaryThe identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca2+ rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr−/−) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

A better understanding of the molecular and cellular mechanisms involved in retinal hydro-mineral homeostasis imbalance during diabetic macular edema (DME) is needed to gain insights into retinal (patho-)physiology that will help elaborate innovative therapies with lower health care costs. Transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4) plays an intricate role in homeostatic processes that needs to be deciphered in normal and diabetic retina. Based on previous findings showing that TRPV4 antagonists resolve blood-retina barrier (BRB) breakdown in diabetic rats, we evaluated whether TRPV4 channel inhibition prevents and reverts retinal edema in streptozotocin(STZ)-induced diabetic mice. We assessed retinal edema using common metrics, including retinal morphology/thickness (histology) and BRB integrity (albumin-associated tracer), and also by quantifying water mobility through apparent diffusion coefficient (ADC) measures. ADC was measured by diffusion-weighted magnetic resonance imaging (DW-MRI), acquired ex vivo at 4 weeks after STZ injection in diabetes and control groups. DWI images were also used to assess retinal thickness. TRPV4 was genetically ablated or pharmacologically inhibited as follows: left eyes were used as vehicle control and right eyes were intravitreally injected with TRPV4-selective antagonist GSK2193874, 24 h before the end of the 4 weeks of diabetes. Histological data show that retinal thickness was similar in nondiabetic and diabetic wt groups but increased in diabetic Trpv4 -/- mice. In contrast, DWI shows retinal thinning in diabetic wt mice that was absent in diabetic Trpv4 -/- mice. Disorganized outer nuclear layer was observed in diabetic wt but not in diabetic Trpv4 -/- retinas. We further demonstrate increased water diffusion, increased distances between photoreceptor nuclei, reduced nuclear area in all nuclear layers, and BRB hyperpermeability, in diabetic wt mice, effects that were absent in diabetic Trpv4 -/- mice. Retinas of diabetic mice treated with PBS showed increased water diffusion that was not normalized by GSK2193874. ADC maps in nondiabetic Trpv4 -/- mouse retinas showed restricted diffusion. Our data provide evidence that water diffusion is increased in diabetic mouse retinas and that TRPV4 function contributes to retinal hydro-mineral homeostasis and structure under control conditions, and to the development of BRB breakdown and increased water diffusion in the retina under diabetes conditions. A single intravitreous injection of TRPV4 antagonist is however not sufficient to revert these alterations in diabetic mouse retinas.

Given the ever-increasing prevalence of type 2 diabetes and obesity, the pressure on global healthcare is expected to be colossal, especially in terms of blindness. Electroretinogram (ERG) has long been perceived as a first-use technique for diagnosing eye diseases, and some studies suggested its use for preventable risk factors of type 2 diabetes and thereby diabetic retinopathy (DR). Here, we show that in a non-evoked mode, ERG signals contain spontaneous oscillations that predict disease cases in rodent models of obesity and in people with overweight, obesity, and metabolic syndrome but not yet diabetes, using one single random forest-based model. Classification performance was both internally and externally validated, and correlation analysis showed that the spontaneous oscillations of the non-evoked ERG are altered before oscillatory potentials, which are the current gold-standard for early DR. Principal component and discriminant analysis suggested that the slow frequency (0.4–0.7 Hz) components are the main discriminators for our predictive model. In addition, we established that the optimal conditions to record these informative signals, are 5-minute duration recordings under daylight conditions, using any ERG sensors, including ones working with portative, non-mydriatic devices. Our study provides an early warning system with promising applications for prevention, monitoring and even the development of new therapies against type 2 diabetes.

Given the ever-increasing prevalence of type 2 diabetes and obesity, the pressure on global healthcare is expected to be colossal, especially in terms of blindness. Electroretinogram (ERG) has long been perceived as a first-use technique for diagnosing eye diseases, but existing methods are insufficient to screen early risk factors of diabetic retinopathy (DR). Here, we introduce non-evoked ERG as a simple, fast modality to record spontaneous activity, from which we developed a single random forest-based model that predicts disease cases in rodent models of obesity and in people with overweight, obesity, and metabolic syndrome. Classification performance was validated using a dataset from an independent eye center. Our algorithm can be coupled with different ERG sensors, including ones working with portative, non-mydriatic devices. Principal component and discriminant analysis suggest slow spontaneous ERG frequencies as main discriminators for our predictive model. Our study will facilitate the implementation of interventions for the prevention of overweight and obesity by providing a robust, quantitative, and non-invasive identification and follow-up approach, which should ultimately reduce DR incidence.