Synergic effects of EP2 and FP receptors co-activation on Blood-Retinal Barrier and Microglia

Abstract

Macular edema (ME) is caused with disruption of the blood-retinal barrier (BRB) followed by fluid accumulation in the subretinal space. Main components of the outer and inner BRB are retinal pigment epithelial (RPE) cells and retinal microvascular endothelial cells, respectively. In addition, glial cells also participate in the functional regulation of the BRB as the member of 'neurovascular unit'. Under various stresses, cells in neurovascular units secrete inflammatory cytokines. Neuroinflammation induced by these cytokines can cause BRB dysfunction by degrading barrier-related proteins and contribute to the pathophysiology of ME. Prostaglandins (PGs) are crucial lipid mediators involved in neuroinflammation. Among PGs, a novel EP2 agonist, omidenepag (OMD) acts on not only the uveoscleral pathway but also the conventional pathway, unlike F prostanoid (FP) receptor agonists. Moreover, the combination use of the EP and the FP agonist is not recommended because of the risk of inflammation. In this study, we investigated effects of OMD and latanoprost acid (LTA), a FP agonist, on BRB and microglia in vitro and in vivo. To investigate the function of outer/inner BRB and microglia, in vitro, ARPE-19 cells, human retinal microvascular endothelial cells (HRMECs), and MG5 cells were used. Cell viability, inflammatory cytokines mRNA and protein levels, barrier morphology/function, and microglial activation were evaluated using proliferation assays, qRT-PCR, ELISA, immunocytochemistry, trans-epithelial electrical resistance, and permeability assay. Moreover, after vitreous injection into the mouse, outer BRB morphology, glial activation, and cytokine expression were assessed. Each OMD and LTA alone did not affect the viability or cytokines expression of the three types of cells. In ARPE-19 cells, the co-stimulation of OMD and LTA increased the mRNA and protein levels of inflammatory cytokines (IL-6, TNF-α, and VEGF-A) and decreased the barrier function and the junction-related protein (ZO-1 and β-catenin). By contrast in HRMECs, the co-stimulation affected significant differences in the mRNA levels of some cytokine (IL-6 and TNF-α) but enhanced the barrier function. In MG5 cells, the cytokines mRNA and size of Iba1-expressed cell were increased. A non-steroidal anti-inflammatory inhibited the barrier dysfunction and the junction-related protein downregulation in ARPE-19 cells and activation of MG5 cells. Also in vivo, the co-stimulation induced outer BRB disruption, cytokine increase, and retinal glial activation. Therefore, the co-stimulation of EP2 and FP induced the inflammatory cytokine-mediated outer BRB disruption, the enhanced inner BRB function, and the microglial activation. The BRB imbalance and the intrinsic prostaglandin production may be involved in OMD-related inflammation.

Keywords: Blood-retinal barrier; EP2 agonist; FP agonist; Macular edema; Microglia; Neuroinflammation; Neurovascular unit; Omidenepag.