Differential Effects of Cytopathic Hypoxia on Human Retinal Endothelial Cellular Behavior: Implication for Ischemic Retinopathies

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 (CoCl(2)). Furthermore, data were deconvoluted to test the effect of cytopathic hypoxia on the three key components of barrier integrity; R(b) (paracellular resistance between HRECs), α (basolateral adhesion between HRECs and the extracellular matrix), and C(m) (HREC membrane capacitance). Our results showed that CoCl(2) decreased the Z of HRECs dose-dependently. Specifically, the R(b) 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 R(b) began to fall to its minimum, other parameters of the HREC barrier, including α and C(m), were unaffected. Interestingly, the compromised effect of cytopathic hypoxia on R(b) 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.