Imaging Modalities for Assessing the Vascular Component of Diabetic Retinal Disease: Review and Consensus for an Updated Staging System.

Purpose: To review the evidence for imaging modalities in assessing the vascular component of diabetic retinal disease (DRD), to inform updates to the DRD staging system. Design: Standardized narrative review of the literature by an international expert workgroup, as part of the DRD Staging System Update Effort, a project of the Mary Tyler Moore Vision Initiative. Overall, there were 6 workgroups: Vascular Retina, Neural Retina, Systemic Health, Basic and Cellular Mechanisms, Visual Function, and Quality of Life. Participants: The Vascular Retina workgroup, including 16 participants from 4 countries. Methods: Literature review was conducted using standardized evidence grids for 5 modalities: standard color fundus photography (CFP), widefield color photography (WFCP), standard fluorescein angiography (FA), widefield FA (WFFA), and OCT angiography (OCTA). Summary levels of evidence were determined on a validated scale from I (highest) to V (lowest). Five virtual workshops were held for discussion and consensus. Main Outcome Measures: Level of evidence for each modality. Results: Levels of evidence for standard CFP, WFCP, standard FA, WFFA, and OCTA were I, II, I, I, and II respectively. Traditional vascular lesions on standard CFP should continue to be included in an updated staging system, but more studies are required before they can be used in posttreatment eyes. Widefield color photographs can be used for severity grading within the area covered by standard CFPs, although these gradings may not be directly interchangeable with each other. Evaluation of the peripheral retina on WFCP can be considered, but the method of grading needs to be clarified and validated. Standard FA and WFFA provide independent prognostic value, but the need for dye administration should be considered. OCT angiography has significant potential for inclusion in the DRD staging system, but various barriers need to be addressed first. Conclusions: This study provides evidence-based recommendations on the utility of various imaging modalities for assessment of the vascular component of DRD, which can inform future updates to the DRD staging system. Although new imaging modalities offer a wealth of information, there are still major gaps and unmet research needs that need to be addressed before this potential can be realized. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Disheveled-1 Interacts with Claudin-5 and Contributes to Norrin-Induced Endothelial Barrier Restoration.

Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a ß-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 ß-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 ß-catenin pathway and a non-canonical signaling pathway by which DVL1 directly binds to CLDN5 to promote barrier properties.

Delineating effects of angiopoietin-2 inhibition on vascular permeability and inflammation in models of retinal neovascularization and ischemia/reperfusion.

Introduction: Clinical trials demonstrated that co-targeting angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF-A) with faricimab controls anatomic outcomes and maintains vision improvements, with strong durability, through 2 years in patients with neovascular age-related macular degeneration and diabetic macular edema. The mechanism(s) underlying these findings is incompletely understood and the specific role that Ang-2 inhibition plays requires further investigation. Methods: We examined the effects of single and dual Ang-2/VEGF-A inhibition in diseased vasculatures of JR5558 mice with spontaneous choroidal neovascularization (CNV) and in mice with retinal ischemia/reperfusion (I/R) injuries. Results: In JR5558 mice, Ang-2, VEGF-A, and dual Ang-2/VEGF-A inhibition reduced CNV area after 1 week; only dual Ang-2/VEGF-A inhibition decreased neovascular leakage. Only Ang-2 and dual Ang-2/VEGF-A inhibition maintained reductions after 5 weeks. Dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 1 week. Both Ang-2 and dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 5 weeks. In the retinal I/R injury model, dual Ang-2/VEGF-A inhibition was statistically significantly more effective than Ang-2 or VEGF-A inhibition alone in preventing retinal vascular leakage and neurodegeneration. Discussion: These data highlight the role of Ang-2 in dual Ang-2/VEGF-A inhibition and indicate that dual inhibition has complementary anti-inflammatory and neuroprotective effects, suggesting a mechanism for the durability and efficacy of faricimab in clinical trials.

Transgenic animal models to explore and modulate the blood brain and blood retinal barriers of the CNS.

The unique environment of the brain and retina is tightly regulated by blood-brain barrier and the blood-retinal barrier, respectively, to ensure proper neuronal function. Endothelial cells within these tissues possess distinct properties that allow for controlled passage of solutes and fluids. Pericytes, glia cells and neurons signal to endothelial cells (ECs) to form and maintain the barriers and control blood flow, helping to create the neurovascular unit. This barrier is lost in a wide range of diseases affecting the central nervous system (CNS) and retina such as brain tumors, stroke, dementia, and in the eye, diabetic retinopathy, retinal vein occlusions and age-related macular degeneration to name prominent examples. Recent studies directly link barrier changes to promotion of disease pathology and degradation of neuronal function. Understanding how these barriers form and how to restore these barriers in disease provides an important point for therapeutic intervention. This review aims to describe the fundamentals of the blood-tissue barriers of the CNS and how the use of transgenic animal models led to our current understanding of the molecular framework of these barriers. The review also highlights examples of targeting barrier properties to protect neuronal function in disease states.

Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCß phosphorylation of occludin.

The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cß (PKCß) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA-/- mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCß-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCß, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCß after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCß phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCß inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.

Structure and function of the retina of low-density lipoprotein receptor-related protein 5 (Lrp5)-deficient rats.

Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by measurement of permeability to Evans blue dye and staining for claudin-5. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed and are correlated with increased Evans blue permeability and absence of claudin-5 expression in superficial vessels. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.

Correlation of Retinal Structure and Visual Function Assessments in Mouse Diabetes Models.

Purpose: Diabetic retinopathy results in vision loss with changes to both retinal blood vessels and neural retina. Recent studies have revealed that animal models of diabetes demonstrate early loss of visual function. We explored the time course of retinal change in three different mouse models of diabetes in a longitudinal study using in vivo measures of retinal structure (optical coherence tomography [OCT]) and visual function (optomotor and pupillary responses). Methods: OCT analysis of retinal microstructure, optokinetic response as a measure of visual acuity, and pupillary response to light stimulation were compared among the db/db, Ins2Akita, and streptozotocin (STZ)-induced mouse models of diabetes at 1.5, 3, 6, and 9 months of diabetes. Results: The db/db, Ins2Akita, and STZ-induced models of diabetes all exhibited vision loss and retinal thinning as disease progressed. Both structural changes and functional measures were significantly correlated with the blood glucose levels. Despite this, vision loss and retinal thinning were not consistently correlated, except for the inner retinal layer thickness at 6 months of diabetes. Conclusions: This longitudinal study compiled structural measures and functional outcome data for type 1 and 2 diabetes mouse models commonly used for diabetes studies and demonstrated an overall decline in retinal-related health in conjunction with weight change and blood glucose alterations. The relationship between the structural change and functional outcome could be correlative but is not necessarily causative, as retinal thinning was not sufficient to explain visual acuity decline.

Regulation of Adrenergic, Serotonin, and Dopamine Receptors to Inhibit Diabetic Retinopathy: Monotherapies versus Combination Therapies.

We compared monotherapies and combinations of therapies that regulate G-protein-coupled receptors (GPCRs) with respect to their abilities to inhibit early stages of diabetic retinopathy (DR) in streptozotocin-diabetic mice. Metoprolol (MTP; 0.04-1.0 mg/kg b.wt./day), bromocriptine (BRM; 0.01-0.1 mg/kg b.wt./day), doxazosin (DOX; 0.01-1.0 mg/kg b.wt./day), or tamsulosin (TAM; 0.05-0.25 mg/kg b.wt./day) were injected individually daily for 2 months in dose-response studies to assess their effects on the diabetes-induced increases in retinal superoxide and leukocyte-mediated cytotoxicity against vascular endothelial cells, both of which abnormalities have been implicated in the development of DR. Each of the individual drugs inhibited the diabetes-induced increase in retinal superoxide at the higher concentrations tested, but the inhibition was lost at lower doses. To determine whether combination therapies had superior effects over individual drugs, we intentionally selected for each drug a low dose that had little or no effect on the diabetes-induced retinal superoxide for use separately or in combinations in 8-month studies of retinal function, vascular permeability, and capillary degeneration in diabetes. At the low doses used, combinations of the drugs generally were more effective than individual drugs, but the low-dose MTP alone totally inhibited diabetes-induced reduction in a vision task, BRM or DOX alone totally inhibited the vascular permeability defect, and DOX alone totally inhibited diabetes-induced degeneration of retinal capillaries. Although low-dose MTP, BRM, DOX, or TAM individually had beneficial effects on some endpoints, combination of the therapies better inhibited the spectrum of DR lesions evaluated. SIGNIFICANCE STATEMENT: The pathogenesis of early stages of diabetic retinopathy remains incompletely understood, but multiple different cell types are believed to be involved in the pathogenic process. We have compared the effects of monotherapies to those of combinations of drugs that regulate GPCR signaling pathways with respect to their relative abilities to inhibit the development of early diabetic retinopathy.

Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury.

BACKGROUND: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. METHODS: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. RESULTS: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1ß, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. CONCLUSIONS: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.

Vascular Expression of Permeability-Resistant Occludin Mutant Preserves Visual Function in Diabetes.

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive preclinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation-resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced vascular endothelial growth factor (VEGF)-induced permeability and edema formation after intraocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial-specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation-dependent manner and that loss of inner blood-retinal barrier integrity induced by diabetes contributes to vision loss.

Current understanding of the molecular and cellular pathology of diabetic retinopathy.

Diabetes mellitus has profound effects on multiple organ systems; however, the loss of vision caused by diabetic retinopathy might be one of the most impactful in a patient's life. The retina is a highly metabolically active tissue that requires a complex interaction of cells, spanning light sensing photoreceptors to neurons that transfer the electrochemical signal to the brain with support by glia and vascular tissue. Neuronal function depends on a complex inter-dependency of retinal cells that includes the formation of a blood-retinal barrier. This dynamic system is negatively affected by diabetes mellitus, which alters normal cell-cell interactions and leads to profound vascular abnormalities, loss of the blood-retinal barrier and impaired neuronal function. Understanding the normal cell signalling interactions and how they are altered by diabetes mellitus has already led to novel therapies that have improved visual outcomes in many patients. Research highlighted in this Review has led to a new understanding of retinal pathophysiology during diabetes mellitus and has uncovered potential new therapeutic avenues to treat this debilitating disease.

The neuroscience of diabetic retinopathy.

Diabetic retinopathy remains a leading cause of blindness despite recent advance in therapies. Traditionally, this complication of diabetes was viewed predominantly as a microvascular disease but research has pointed to alterations in ganglion cells, glia, microglia, and photoreceptors as well, often occurring without obvious vascular damage. In neural tissue, the microvasculature and neural tissue form an intimate relationship with the neural tissue providing signaling cues for the vessels to form a distinct barrier that helps to maintain the proper neuronal environment for synaptic signaling. This relationship has been termed the neurovascular unit (NVU). Research is now focused on understanding the cellular and molecular basis of the neurovascular unit and how diabetes alters the normal cellular communications and disrupts the cellular environment contributing to loss of vision in diabetes.

All-trans-Retinaldehyde Contributes to Retinal Vascular Permeability in Ischemia Reperfusion.

Purpose: Extracellular accumulation of all-trans-retinaldehyde (atRAL), a highly reactive visual cycle intermediate, is toxic to cells of the outer retina and contributes to retinal and macular degenerations. However, the contribution of atRAL to retinal capillary function has not been studied. We hypothesized that atRAL released from the outer retina can contribute to retinal vascular permeability. We, therefore, tested the contribution of atRAL to retinal ischemia-reperfusion (IR)-induced vascular permeability. Methods: IR was induced in mice by transient increase in intraocular pressure followed by natural reperfusion. The visual cycle was ablated in the Lrat-/- mice, reduced by dark adaptation or the use of the RPE65 inhibitor and atRAL scavenger emixustat. Accumulation of FITC-BSA was used to assess vascular permeability and DNA fragmentation quantified cell death after IR. Primary bovine retinal endothelial cell (BREC) culture was used to measure the direct effects of atRAL on endothelial permeability and cell death. Results: Inhibition of the visual cycle by Lrat-/-, dark adaptation, or with emixustat, all reduced approximately half of IR induced vascular permeability at 48 hours. An increase in BREC permeability with atRAL coincided with lactate dehydrogenase (LDH) release, a measure of cell death. Both permeability and toxicity were blocked by emixustat. Conclusions: Outer retinal pathology may contribute to vascular permeability by release of atRAL, which can act directly on vascular endothelial cells to alter barrier properties and induce cell death. These studies may have implications for a variety of blinding eye diseases that include outer retinal damage and retinal vascular permeability.

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidines as atypical protein kinase C inhibitors to control retinal vascular permeability and cytokine-induced edema.

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1-2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and Cmax), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.

Targeting Neurovascular Interaction in Retinal Disorders.

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.

Norrin restores blood-retinal barrier properties after vascular endothelial growth factor-induced permeability.

Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through ß-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/ß (GSK-3α/ß) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a ß-catenin-dependent manner.

Organ-On-A-Chip Technologies for Advanced Blood-Retinal Barrier Models.

The blood-retinal barrier (BRB) protects the retina by maintaining an adequate microenvironment for neuronal function. Alterations of the junctional complex of the BRB and consequent BRB breakdown in disease contribute to a loss of neuronal signaling and vision loss. As new therapeutics are being developed to prevent or restore barrier function, it is critical to implement physiologically relevant in vitro models that recapitulate the important features of barrier biology to improve disease modeling, target validation, and toxicity assessment. New directions in organ-on-a-chip technology are enabling more sophisticated 3-dimensional models with flow, multicellularity, and control over microenvironmental properties. By capturing additional biological complexity, organs-on-chip can help approach actual tissue organization and function and offer additional tools to model and study disease compared with traditional 2-dimensional cell culture. This review describes the current state of barrier biology and barrier function in ocular diseases, describes recent advances in organ-on-a-chip design for modeling the BRB, and discusses the potential of such models for ophthalmic drug discovery and development.

Tight Junctions in Cell Proliferation.

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.

Tight junction protein occludin regulates progenitor Self-Renewal and survival in developing cortex.

Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.