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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 C max ), 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 β-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/β (GSK-3α/β) 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 β-catenin-dependent manner., (© 2020 Díaz-Coránguez et al.)

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., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Diabetic retinopathy is the leading cause of visual impairment and preventable blindness, and represents a significant socioeconomic cost for health care systems worldwide. Therefore, new approaches beyond current standards of diabetes care are needed. Based on the crucial pathogenic role of vascular endothelial growth factor (VEGF) in the development of diabetic macular edema (DME), intravitreal anti-VEGF agents have emerged as new treatments. To provide an understanding of the rationale for use and clinical efficacy of anti-VEGF treatment, we examine this topic in a two-part Bench to Clinic narrative. In the Bench narrative, we provide an overview of the role of VEGF in the pathogenesis of diabetic retinopathy, the molecular characteristics of anti-VEGF agents currently used, and future perspectives and challenges in this area. In the Clinic narrative that follows our contribution, Cheung et al. provide an overview of the current evidence from clinical trials on anti-VEGF therapy for diabetic retinopathy.

Purpose: To determine the utility of polychromatic angiography (PCA) in the assessment of VEGF-induced blood retinal barrier (BRB) dysfunction in rabbits., Methods: Twenty-six eyes of 24 Dutch Belted rabbits were injected intravitreally with 1.25 μg (group A, n = 5), 10 μg (group C, n = 7), or 4 μg (group B, n = 6; group D, n = 4; and group E, n = 4) of VEGF on day 0. Groups D and E were also injected intravitreally with 1.25 μg and 12.5 μg bevacizumab, respectively, on day 2. On days 0, 2, 4, 7, 11, and 14, PCA was performed using a contrast agent mixture composed of fluorescein sodium, indocyanine green, PCM102, and PCM107 and imaged with a modified fundus camera. PCA scores were based on detected leaking fluorophores., Results: On day 7, there was a statistically significant difference between PCA scores of group A (0.6 ± 0.89) and both groups B (2.67 ± 1.37, P = 0.0154) and C (3.33 ± 0.52, P = 0.00085). There was also a statistically significant difference between groups B and E (PCA score 0.75 ± 0.96, P = 0.032) on day 7. On day 11, there was statistically significant difference between group C (1.80 ± 1.1) and both groups A (0, P = 0.021) and B (0.33 ± 0.52, P = 0.037)., Conclusions: A differential response to both increasing VEGF dose and administration of bevacizumab could be discerned using the PCA. PCA allowed stratification of VEGF-induced BRB dysfunction and inhibitory effects of bevacizumab therapy in the rabbit retina.

Therapies targeting vascular endothelial growth factor (VEGF) are revolutionizing the treatment of diabetic retinopathy (DR) and diabetic macular edema (DME). In August 2012, ranibizumab, a monoclonal antibody fragment targeting VEGF designed for ocular use, became the first and only U.S. Food and Drug Administration-approved medical therapy for DME and the first approved treatment in over 25 years. This approval was based on strong preclinical data followed by numerous clinical trials that demonstrate an essential role of VEGF in vascular permeability and angiogenesis in both normal physiology and disease pathology. In this Perspective, we will examine the experimental studies and scientific data that aided in the success of the development of therapies targeting VEGF and consider how these approaches may inform the development of future therapeutics for diabetic eye disease. A multipoint model is proposed, based on well-established drug development principles, with the goal of improving the success of clinical drug development. This model suggests that to provide a validated preclinical target, investigators should demonstrate the following: the role of the target in normal physiology, a causal link to disease pathogenesis, correlation to human disease, and the ability to elicit clinically relevant improvements of disease phenotypes in animal models with multiple, chemically diverse interventions. This model will provide a framework to validate the current preclinical targets and identify novel targets to improve drug development success for DR.

Pro-inflammatory cytokines and growth factors such as VEGF (vascular endothelial growth factor) contribute to the loss of the BRB (blood-retinal barrier) and subsequent macular oedema in various retinal pathologies. VEGF signalling requires PKCβ [conventional PKC (protein kinase C)] activity; however, PKCβ inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability, suggesting the involvement of alternative signalling pathways. In the present study, we provide evidence for the involvement of aPKC (atypical PKC) signalling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small-molecule inhibitors, and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small-molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. The results of the present study suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis, and the BBB (blood-brain barrier) in the presence of brain tumours.

Vascular endothelial growth factor (VEGF)-induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.

Objective: Vascular endothelial growth factor (VEGF) contributes to diabetic retinopathy, but control of its expression is not well understood. Here, we tested the hypothesis that hyperglycemia mediates induction of VEGF expression in a eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) 1 and 2 dependent manner., Research Design and Methods: The retina was harvested from control and type 1 diabetic rats and mice and analyzed for VEGF mRNA and protein expression as well as biomarkers of translational control mechanisms. Similar analyses were performed in Müller cell cultures exposed to hyperglycemic conditions. The effect of 4E-BP1 and 4E-BP2 gene deletion on VEGF expression was examined in mice and in mouse embryo fibroblasts (MEFs)., Results: Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes. Increases in expression of 4E-BP1 protein mirrored those of VEGF and expression of 4E-BP1 mRNA was unchanged. Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions. Importantly, the diabetes-induced increase in VEGF expression was not observed in mice deficient in 4E-BP1 and 4E-BP2, nor in MEFs lacking the two proteins., Conclusions: Hyperglycemia induces VEGF expression through cap-independent mRNA translation mediated by increased expression of 4E-BP1. Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites. These findings provide new insights into potential targets for treatment of diabetic retinopathy.

Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.

Purpose: VEGF is a potent permeabilizing factor that contributes to the pathogenesis of diabetic retinopathy and brain tumors. VEGF-induced vascular permeability in vivo and in cell culture requires PKC activity, but the mechanism by which PKC regulates barrier properties remains unknown. This study was conducted to examine how VEGF and diabetes alter occludin phosphorylation and endothelial cell permeability., Methods: Chemical PKC inhibitors and activators were used to treat primary retinal endothelial cells in culture. In vitro kinase assays and Western blot analysis of two-dimensional (2D) and one-dimensional (1D) gel retardation assays were used to analyze occludin phosphorylation. Endothelial cell permeability was determined by measuring the flux of 70-kDa dextran through a cell monolayer in culture. Exogenous expression of a dominant negative PKCbetaII mutant (S217A) was used to assess the PKC dependence of VEGF-induced occludin phosphorylation and endothelial permeability. Occludin phosphorylation was also determined in retinas of streptozotocin-induced diabetic rats., Results: VEGF stimulated the phosphorylation of occludin in primary retinal endothelial cells. Chemical inhibitors of PKC activity blocked the VEGF-induced increase in occludin phosphorylation, as assessed by 2D gel and gel retardation in Western blot analysis, and blocked part of the VEGF-induced monolayer permeability to 70-kDa dextran. Expression of a dominant negative PKCbetaII mutant blocked VEGF-induced occludin phosphorylation and endothelial permeability. Finally, elevated occludin phosphorylation was observed in the retina of diabetic animals., Conclusions: These results strongly suggest that VEGF-induced endothelial permeability requires PKC-dependent phosphorylation of occludin. Regulation of PKC activity and tight junction protein modifications may have therapeutic implications for treatment of diabetic retinopathy and brain tumors.

Vascular endothelial growth factor (VEGF) increases microvascular permeability and has been implicated in the development of numerous pathologies including diabetic retinopathy (DR), hypoxia/ischemia, and tumor biology. The transport pathways by which water and solutes cross the endothelium in response to VEGF, however, are not completely understood. We measured, in real time, bovine retinal endothelial cell (BREC) hydraulic conductivity (Lp), 70 kDa dextran permeability (Pe), and the solvent-drag reflection coefficient (sigma) before and after addition of 50 ng/ml VEGF. The diffusional permeability coefficient for dextran (Pd) was measured before pressure gradient application. The sudden application of a 10-cm H2O hydrostatic pressure gradient induced water and solute fluxes that decayed to steady-state values after approximately 2 h. Subsequently, the addition of VEGF significantly increased Lp and Pe by 4.3-fold +/- 0.7-fold and 3.0-fold +/- 0.3-fold, respectively, after 110 min; however, the reflection coefficient remained approximately constant throughout the experiment (approximately 0.8). These observations suggest that water and dextran utilize common paracellular channels across BREC monolayers. Furthermore, the addition of VEGF increases the number or availability of channels but does not alter the selectivity of the monolayer to 70 kDa dextran.

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.