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Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a β-catenin-dependent pathway. Here, we have explored the contribution of disheveled-1 (DVL1) in norrin-induced blood-retinal barrier (BRB) restoration. We provide evidence that in addition to canonical signaling, DVL1 promotes tight junction (TJ) stabilization through a novel, non-canonical signaling pathway involving direct claudin-5 (CLDN5) binding. Immunofluorescence staining of rat retinal cross-sections showed enriched expression of DVL1 and 3 at endothelial capillaries and co-localization with CLDN5 and ZO-1 at the TJ complex in primary bovine retinal endothelial cells (BRECs). Barrier properties of BRECs were determined via measurements of trans-endothelial electrical resistance (TEER) or permeability to 70 kDa RITC-dextran. These studies demonstrated that norrin restoration of barrier properties after VEGF treatment required DVL1 as an siRNA knockdown of Dvl1 but not Dvl2 or Dvl3, reduced basal barrier properties and ablated norrin-induced barrier restoration. However, loss of Dvl1 did not decrease β-catenin signaling activity as measured by Axin2 mRNA expression, suggesting the contribution of a non-canonical pathway. DVL and TJ protein interactions were analyzed via co-immunoprecipitation of endogenous protein in BRECs, which demonstrated that DVL1 interacts with both CLDN5 and ZO-1, while DVL3 interacts only with ZO-1. These interactions were most abundant after inducing BRB restoration by treating BRECs with VEGF and norrin. DVL has previously been shown to form intramolecular bindings between the C-terminal PDZ-binding motif (PDZ-BM) with an internal PDZ domain. Co-transfection of HEK293 cells with DVL1 and CLDN5 or relevant mutants revealed that DVL1 interacts with CLDN5 through the DVL PDZ domain binding, CLDN5 PDZ-BM, in competition with DVL1 PDZ-BM, since DVL/CLDN5 interaction increases with deletion of the DVL1 PDZ-BM and decreases by co-expressing the C-terminal fragment of DVL1 containing the PDZ-BM or through deletion of CLDN5 PDZ-BM. In BREC cells, transfection of the C-terminal fragment of DVL1 downregulates the expression of CLDN5 but does not affect the expression of other proteins of the TJs, including ZO-1, occludin, CLDN1 or VE-cadherin. Blocking DVL1/CLDN5 interaction increased basal permeability and prevented norrin induction of barrier properties after VEGF. Combined with previous data, these results demonstrate that norrin signals through both a canonical β-catenin pathway and a non-canonical signaling pathway by which DVL1 directly binds to CLDN5 to promote barrier properties.

The tightly structured neural retina has a unique vascular network comprised of three interconnected plexuses in the inner retina (and choroid for outer retina), which provide oxygen and nutrients to neurons to maintain normal function. Clinical and experimental evidence suggests that neuronal metabolic needs control both normal retinal vascular development and pathological aberrant vascular growth. Particularly, photoreceptors, with the highest density of mitochondria in the body, regulate retinal vascular development by modulating angiogenic and inflammatory factors. Photoreceptor metabolic dysfunction, oxidative stress, and inflammation may cause adaptive but ultimately pathological retinal vascular responses, leading to blindness. Here we focus on the factors involved in neurovascular interactions, which are potential therapeutic targets to decrease energy demand and/or to increase energy production for neovascular retinal disorders.

Tight junction (TJ) proteins form a continuous intercellular network creating a barrier with selective regulation of water, ion, and solutes across endothelial, epithelial, and glial tissues. TJ proteins include the claudin family that confers barrier properties, members of the MARVEL family that contribute to barrier regulation, and JAM molecules, which regulate junction organization and diapedesis. In addition, the membrane-associated proteins such as MAGUK family members, i.e., zonula occludens, form the scaffold linking the transmembrane proteins to both cell signaling molecules and the cytoskeleton. Most studies of TJ have focused on the contribution to cell-cell adhesion and tissue barrier properties. However, recent studies reveal that, similar to adherens junction proteins, TJ proteins contribute to the control of cell proliferation. In this review, we will summarize and discuss the specific role of TJ proteins in the control of epithelial and endothelial cell proliferation. In some cases, the TJ proteins act as a reservoir of critical cell cycle modulators, by binding and regulating their nuclear access, while in other cases, junctional proteins are located at cellular organelles, regulating transcription and proliferation. Collectively, these studies reveal that TJ proteins contribute to the control of cell proliferation and differentiation required for forming and maintaining a tissue barrier.

Diabetic retinopathy is the leading cause of blindness in working age adults, and is projected to be a significant future health concern due to the rising incidence of diabetes. The recent advent of anti-vascular endothelial growth factor (VEGF) antibodies has revolutionized the treatment of diabetic retinopathy but a significant subset of patients fail to respond to treatment. Accumulating evidence indicates that inflammatory cytokines and chemokines other than VEGF may contribute to the disease process. The current review examines the presence of non-VEGF cytokines in the eyes of patients with diabetic retinopathy and highlights mechanistic pathways in relevant animal models. Finally, novel drug targets including components of the kinin-kallikrein system and emerging treatments such as anti-HPTP (human protein tyrosine phosphatase) β antibodies are discussed. Recognition of non-VEGF contributions to disease pathogenesis may lead to novel therapeutics to enhance existing treatments for patients who do not respond to anti-VEGF therapies., Competing Interests: David A. Antonetti has grant support from NovoNordisk and Unity Bioscience Mark T. Bolinger declares no conflict of interest. These funding sources had no role in the development or writing of this review.