No abstract

Loss of barrier integrity of retinal endothelial cells (RECs) is an early feature of ischemic retinopathies (IRs), but the triggering mechanisms remain incompletely understood. Previous studies have reported mitochondrial dysfunction in several forms of IRs, which creates a cytopathic hypoxic environment where cells cannot use oxygen for energy production. Nonetheless, the contribution of cytopathic hypoxia to the REC barrier failure has not been fully explored. In this study, we dissect in-depth the role of cytopathic hypoxia in impairing the barrier function of REC. We employed the electric cell-substrate impedance sensing (ECIS) technology to monitor in real-time the impedance (Z) and hence the barrier functionality of human RECs (HRECs) under cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). Furthermore, data were deconvoluted to test the effect of cytopathic hypoxia on the three key components of barrier integrity; Rb (paracellular resistance between HRECs), α (basolateral adhesion between HRECs and the extracellular matrix), and Cm (HREC membrane capacitance). Our results showed that CoCl2 decreased the Z of HRECs dose-dependently. Specifically, the Rb parameter of the HREC barrier was the parameter that declined first and most significantly by the cytopathic hypoxia-inducing agent and in a dose-dependent manner. When Rb began to fall to its minimum, other parameters of the HREC barrier, including α and Cm, were unaffected. Interestingly, the compromised effect of cytopathic hypoxia on Rb was associated with mitochondrial dysfunction but not with cytotoxicity. In conclusion, our results demonstrate distinguishable dielectric properties of HRECs under cytopathic hypoxia in which the paracellular junction between adjacent HRECs is the most vulnerable target. Such selective behavior could be utilized to screen agents or genes that maintain and strengthen the assembly of HRECs tight junction complex.

Purpose: Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. Methods: Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell–cell interactions (Rb), cell–matrix interactions (α), and cell membrane permeability (Cm). Results: Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the Rb, α, and Cm parameters. Rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 h and increased Cm without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on Rb, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on Cm or α. Conclusion: Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.

PurposeDiabetes induced microvasculopathy (DIM) is a leading cause of atherosclerosis, heart failure, stroke, blindness, and renal failure. Current therapeutic interventions still heavily rely on controlling systemic hyperglycemia. Yet, many diabetic patients develop microvasculopathies despite having good glycemic control. Endothelial activation is a common underlying causative factor; however, the molecular basis of such activation remains a big gap in our knowledge. Aberrant glucose transport and metabolism by endothelial cells are attracting great interest as disease‐related mechanisms with little attention in the context of DIM. In this study we aimed to evaluate the pre‐clinical efficacy of the glucose transporter (GLUT)‐1 inhibitor, which is a derivative of Fluoro‐catechol ester of 3‐Hydroxy‐benzoic acid (FCEHBA), to halt microvascular dysfunctions induced by diabetes.MethodsHuman retinal endothelial cells (HRECs) were used and treated with high glucose (HG, 30 mM) or osmotic control with or without low oxygen (1%O2) in presence or absence of FCEHBA (10 mM) or Vehicle (DMSO). Barrier function was assessed by Electric Cell‐substrate Impedance Sensing (ECIS) and immunofluorescence organization of Zonula Occluden‐1 (ZO‐1). The angiogenic potential of HRECs was evaluated by migration; sprouting and tube formation. Group differences were evaluated by ANOVA followed by Tukey Posthoc test.Results(1) Increased glucose influx in HRECs through the increased expression of GLUT1 and its translocation from the cytosol to the plasma membrane have been observed in response to simultaneous insults of diabetes and hypoxia. (2) The increase in glucose influx via recruitment of GLUT1 to the plasma membrane was associated with (a) an accelerated breakdown of HREC barrier integrity, indicated by the significant drop in transendothelial electric resistance (TER); (b) a marked disruption of ZO‐1, a key tight junction protein (TJP) regulating barrier integrity in HRECs; (c) a dramatic enhancement of HRECs migration; and (d) an induction of profound HREC neovascularization and tube formation. (3) Importantly, FCEHBA was able to remarkably restore the drop in barrier function induced by both diabetes and hypoxia and to reverse their disruptive effects on ZO‐1 integrity. (4) FCEHBA was also able to effectively mitigate angiogenic effects of both diabetes and hypoxia on HRECs, including increased cell migration and tube formation, without affecting cell viability.ConclusionTo our knowledge, this study is the first to report that FCEHBA protects against High Glucose/Hypoxia‐induced microvasculopathy. This finding may lead to a new therapeutic approach that could be clinically translated to not only patients with diabetic retinopathy but also those with other related microvascular conditions including atherosclerosis, stroke, and cardiovascular diseases.Support or Funding InformationAmerican Heart Association Grant 18CDA34080403 (ASI) and the National Eye Institute grant R01EY023315 (MA).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.