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.

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.

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.

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.

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.

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.

The EPAC-Rap1 pathway prevents and reverses cytokine-induced retinal vascular permeability.

Increased retinal vascular permeability contributes to macular edema, a leading cause of vision loss in eye pathologies such as diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusions. Pathological changes in vascular permeability are driven by growth factors such as VEGF and pro-inflammatory cytokines such as TNF-α. Identifying the pro-barrier mechanisms that block vascular permeability and restore the blood-retinal barrier (BRB) may lead to new therapies. The cAMP-dependent guanine nucleotide exchange factor (EPAC) exchange-protein directly activated by cAMP promotes exchange of GTP in the small GTPase Rap1. Rap1 enhances barrier properties in human umbilical endothelial cells by promoting adherens junction assembly. We hypothesized that the EPAC-Rap1 signaling pathway may regulate the tight junction complex of the BRB and may restore barrier properties after cytokine-induced permeability. Here, we show that stimulating EPAC or Rap1 activation can prevent or reverse VEGF- or TNF-α-induced permeability in cell culture and in vivo Moreover, EPAC activation inhibited VEGF receptor (VEGFR) signaling through the Ras/MEK/ERK pathway. We also found that Rap1B knockdown or an EPAC antagonist increases endothelial permeability and that VEGF has no additive effect, suggesting a common pathway. Furthermore, GTP-bound Rap1 promoted tight junction assembly, and loss of Rap1B led to loss of junctional border organization. Collectively, our results indicate that the EPAC-Rap1 pathway helps maintain basal barrier properties in the retinal vascular endothelium and activation of the EPAC-Rap1 pathway may therefore represent a potential therapeutic strategy to restore the BRB.

Inhibition of Atypical Protein Kinase C Reduces Inflammation-Induced Retinal Vascular Permeability.

Changes in permeability of retinal blood vessels contribute to macular edema and the pathophysiology of numerous ocular diseases, including diabetic retinopathy, retinal vein occlusions, and macular degeneration. Vascular endothelial growth factor (VEGF) induces retinal permeability and macular thickening in these diseases. However, inflammatory agents, such as tumor necrosis factor-α (TNF-α), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-α induce permeability through distinct mechanisms; however, both require the activation of atypical protein kinase C (aPKC). We provide evidence, using genetic mouse models and therapeutic intervention with small molecules, that inhibition of aPKC prevented or reduced vascular permeability in animal models of retinal inflammation. Expression of a kinase-dead aPKC transgene, driven by a vascular and hematopoietic restricted promoter, reduced retinal vascular permeability in an ischemia-reperfusion model of retinal injury. This effect was recapitulated with a small-molecule inhibitor of aPKC. Expression of the kinase-dead aPKC transgene dramatically reduced the expression of inflammatory factors and blocked the attraction of inflammatory monocytes and granulocytes after ischemic injury. Coinjection of VEGF with TNF-α was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-α-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.

Pathophysiology of Diabetic Retinopathy: Contribution and Limitations of Laboratory Research.

Preclinical models of diabetic retinopathy are indispensable in the drug discovery and development of new therapies. They are, however, imperfect facsimiles of diabetic retinopathy in humans. This chapter discusses the advantages, limitations, and physiological and pathological relevance of preclinical models of diabetic retinopathy. The judicious interpretation and extrapolation of data derived from these models to humans and a correspondingly greater emphasis placed on translational medical research in early-stage clinical trials are essential to more successfully inhibit the development and progression of diabetic retinopathy in the future.

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.

Occludin S490 Phosphorylation Regulates Vascular Endothelial Growth Factor-Induced Retinal Neovascularization.

Occludin is a transmembrane tight junction protein that contributes to diverse cellular functions, including control of barrier properties, cell migration, and proliferation. Vascular endothelial growth factor (VEGF) induces phosphorylation of occludin at S490, which is required for VEGF-induced endothelial permeability. Herein, we demonstrate that occludin S490 phosphorylation also regulates VEGF-induced retinal endothelial cell proliferation and neovascularization. Using a specific antibody, phospho-occludin was located in centrosomes in endothelial cell cultures, animal models, and human surgical samples of retinal neovessels. Occludin S490 phosphorylation was found to increase with endothelial tube formation in vitro and in vivo during retinal neovascularization after induction of VEGF expression. More important, expression of occludin mutated at S490 to Ala, completely inhibited angiogenesis in cell culture models and in vivo. Collectively, these data suggest a novel role for occludin in regulation of endothelial proliferation and angiogenesis in a phosphorylation-dependent manner. These findings may lead to methods of regulating pathological neovascularization by specifically targeting endothelial cell proliferation.

Retinylamine Benefits Early Diabetic Retinopathy in Mice.

Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat(-/-)) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat(-/-) diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of early DR in diabetic mice. The visual cycle constitutes a novel target for inhibition of DR.

Adrenergic and serotonin receptors affect retinal superoxide generation in diabetic mice: relationship to capillary degeneration and permeability.

Reactive oxygen species play an important role in the pathogenesis of diabetic retinopathy. We studied the role of adrenergic and serotonin receptors in the generation of superoxide by retina and 661W retinal cells in high glucose and of the α1-adrenergic receptor (AR) on vascular lesions of the retinopathy in experimentally diabetic C57Bl/6J mice (and controls) after 2 and 8 months. Compared with 5 mM glucose, incubating cells or retinal explants in 30 mM glucose induced superoxide generation. This response was reduced or ablated by pharmacologic inhibition of the α1-AR (a Gq-coupled receptor) or Gs-coupled serotonin (5-HT2, 5-HT4, 5-HT6, and 5-HT7) receptors or by activation of the Gi-coupled α2-AR. In elevated glucose, the α1-AR produced superoxide via phospholipase C, inositol triphosphate-induced Ca(2+) release, and NADPH oxidase, and pharmacologic inhibition of these reactions prevented the superoxide increase. Generation of retinal superoxide, expression of proinflammatory proteins, and degeneration of retinal capillaries in diabetes all were significantly inhibited with daily doxazosin or apocynin (inhibitors of α1-AR and NADPH oxidase, respectively), but increased vascular permeability was not significantly affected. Adrenergic receptors, and perhaps other GPCRs, represent novel targets for inhibiting the development of important features of diabetic retinopathy.

Moving Past Anti-VEGF: Novel Therapies for Treating Diabetic Retinopathy.

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) ß 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.

The occludin and ZO-1 complex, defined by small angle X-ray scattering and NMR, has implications for modulating tight junction permeability.

Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468-475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin:ZO-1 complex.

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.

Occludin is a direct target of thyroid transcription factor-1 (TTF-1/NKX2-1).

The thyroid transcription factor 1 gene (TTF-1 or NKX2-1) is essential to lung development; however, it is also a critical factor in lung cancer. TTF-1 is amplified in lung cancers, suggesting that it is a gain-of-function lung oncogene. Conversely, TTF-1 counters epithelial to mesenchymal transition in cell-based studies and inhibits progression of primary lung adenocarcinomas to metastases in an animal model of lung adenocarcinomas. The unifying theory regarding TTF-1 is that it exhibits both pro-oncogenic and anti-metastatic function depending on the cellular context. Occludin is the first discovered constituent of the epithelial tight junction; in recent years, a functional role of occludin as a tumor suppressor has begun to emerge. Here, we demonstrate that TTF-1 transactivated the expression of the epithelial tight junction molecules occludin (OCLN) and claudin-1 (CLDN1). We show that transcriptional activation occurred through a direct interaction of TTF-1 with the OCLN and CLDN1 promoters. Furthermore, in cells that lack TTF-1, exogenous TTF-1 expression dampened the inhibitory effect of TGF-ß on occludin and claudin-1 content. Using cells derived from a genetically engineered mouse model of lung adenocarcinomas, we observed that silenced TTF-1 expression down-regulated occludin, which we supported with additional siRNA experiments. Finally, TTF-1 knockdown conferred human lung cancer cells resistance to anoikis, and expression of occludin restored cellular sensitivity to anoikis. Overexpression of occludin impeded migration and induced anoikis in lung carcinoma cells. Collectively, these data suggest that TTF-1 transcriptionally regulates occludin, which represents another avenue of TTF-1-mediated metastasis suppression.