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

ABSTRACT: 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-? (TNF-?), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-? 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-? was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-?-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.

SUBMITTER: Lin CM

PROVIDER: S-EPMC6180272 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Lin Cheng-Mao CM Titchenell Paul M PM Keil Jason M JM Garcia-Ocaña Adolfo A Bolinger Mark T MT Abcouwer Steven F SF Antonetti David A DA

The American journal of pathology 20180913 10

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. Recen ...[more]

PMID: 30220554

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Project description: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.

Project description:Aims/hypothesisBreakdown of the inner blood-retinal barrier (BRB) is an early event in the pathogenesis of diabetic macular oedema, that eventually leads to vision loss. We have previously shown that diabetes causes an imbalance of nerve growth factor (NGF) isoforms resulting in accumulation of its precursor proNGF and upregulation of the p75 neurotrophin receptor (p75NTR), with consequent increases in the activation of Ras homologue gene family, member A (RhoA). We also showed that genetic deletion of p75NTR in diabetes preserved the BRB and prevented inflammatory mediators in retinas. This study aims to examine the therapeutic potential of LM11A-31, a small-molecule p75NTR modulator and proNGF antagonist, in preventing diabetes-induced BRB breakdown. The study also examined the role of p75NTR/RhoA downstream signalling in mediating cell permeability.MethodsMale C57BL/6 J mice were rendered diabetic using streptozotocin injection. After 2 weeks of diabetes, mice received oral gavage of LM11A-31 (50 mg kg-1 day-1) or saline (NaCl 154 mmol/l) for an additional 4 weeks. BRB breakdown was assessed by extravasation of BSA-AlexaFluor-488. Direct effects of proNGF were examined in human retinal endothelial (HRE) cells in the presence or absence of LM11A-31 or the Rho kinase inhibitor Y-27632.ResultsDiabetes triggered BRB breakdown and caused significant increases in circulatory and retinal TNF-α and IL-1β levels. These effects coincided with significant decreases in retinal NGF and increases in vascular endothelial growth factor and proNGF expression, as well as activation of RhoA. Interventional modulation of p75NTR activity through treatment of mouse models of diabetes with LM11A-31 significantly mitigated proNGF accumulation and preserved BRB integrity. In HRE cells, treatment with mutant proNGF (10 ng/ml) triggered increased cell permeability with marked reduction of expression of tight junction proteins, zona occludens-1 (ZO-1) and claudin-5, compared with control, independent of inflammatory mediators or cell death. Modulating p75NTR significantly inhibited proNGF-mediated RhoA activation, occludin phosphorylation (at serine 490) and cell permeability. ProNGF induced redistribution of ZO-1 in the cell wall and formation of F-actin stress fibres; these effects were mitigated by LM11A-31.Conclusions/interpretationTargeting p75NTR signalling using LM11A-31, an orally bioavailable receptor modulator, may offer an effective, safe and non-invasive therapeutic strategy for treating macular oedema, a major cause of blindness in diabetes.

Project description:IntroductionPropionibacterium acnes, a ubiquitous skin bacterium, stimulates keratinocytes to produce a number of proinflammatory cytokines and may contribute to inflammatory acne. The aim of the study was to investigate whether P. acnes-induced proinflammatory cytokine release is mediated by P. acnes-induced activation of p38 mitogen-activated protein kinase (p38 MAPK or p38) in human keratinocytes.MethodsImmunohistochemistry was used to evaluate p38 phosphorylation in human skin samples with or without acne. Primary human keratinocytes and epidermal skin equivalents were exposed to viable P. acnes. Phosphorylation of MAPKs without or with p38 inhibitors was examined by Western blot and cytokine secretion was detected by Enzyme-Linked Immunosorbent Assay (ELISA).ResultsIncreased levels of phospho-p38 were observed in human acne lesions, predominantly in follicular and perifollicular keratinocytes. Exposure of cultured human keratinocytes to viable P. acnes resulted in phosphorylation of multiple members of the MAPK family, including rapid and transient activation of p38 and extracellular signal-related kinase (ERK1/2) and relatively slow but sustained activation of c-Jun N-terminal kinases (JNK1/2). Viable P. acnes induced the secretion of interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and IL-8 from human keratinocytes. The phosphorylation of p38 (phospho-p38) and the secretion of cytokines induced by P. acnes in cultured keratinocytes were inhibited by SB203580, a p38α/β inhibitor. Furthermore, SCIO-469, a selective inhibitor of p38α, showed similar effects in cultured keratinocytes. Topical treatment of SCIO-469 inhibited the P. acnes-induced phospho-p38 and cytokine secretion in human epidermal equivalents.ConclusionThe data demonstrate that P. acnes induces p38-dependent inflammatory responses in keratinocytes, and suggest that p38 may play an important role in the pathogenesis of inflammatory acne.FundingJohnson & Johnson.

Project description:BackgroundMany retinal diseases are associated with vascular dysfunction accompanied by neuroinflammation. We examined the ability of minocycline (Mino), a tetracycline derivative with anti-inflammatory and neuroprotective properties, to prevent vascular permeability and inflammation following retinal ischemia-reperfusion (IR) injury, a model of retinal neurodegeneration with breakdown of the blood-retinal barrier (BRB).MethodsMale Sprague-Dawley rats were subjected to 45 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Rats were treated with Mino prior to and following IR. At 48 h after reperfusion, retinal gene expression, cellular inflammation, Evan's blue dye leakage, tight junction protein organization, caspase-3 activation, and DNA fragmentation were measured. Cellular inflammation was quantified by flow-cytometric evaluation of retinal tissue using the myeloid marker CD11b and leukocyte common antigen CD45 to differentiate and quantify CD11b+/CD45low microglia, CD11b+/CD45hi myeloid leukocytes and CD11bneg/CD45hi lymphocytes. Major histocompatibility complex class II (MHCII) immunoreactivity was used to determine the inflammatory state of these cells.ResultsMino treatment significantly inhibited IR-induced retinal vascular permeability and disruption of tight junction organization. Retinal IR injury significantly altered mRNA expression for 21 of 25 inflammation- and gliosis-related genes examined. Of these, Mino treatment effectively attenuated IR-induced expression of lipocalin 2 (LCN2), serpin peptidase inhibitor clade A member 3 N (SERPINA3N), TNF receptor superfamily member 12A (TNFRSF12A), monocyte chemoattractant-1 (MCP-1, CCL2) and intercellular adhesion molecule-1 (ICAM-1). A marked increase in leukostasis of both myeloid leukocytes and lymphocytes was observed following IR. Mino treatment significantly reduced retinal leukocyte numbers following IR and was particularly effective in decreasing the appearance of MHCII+ inflammatory leukocytes. Surprisingly, Mino did not significantly inhibit retinal cell death in this model.ConclusionsIR induces a retinal neuroinflammation within hours of reperfusion characterized by inflammatory gene expression, leukocyte adhesion and invasion, and vascular permeability. Despite Mino significantly inhibiting these responses, it failed to block neurodegeneration.

Dataset Information

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

Publications

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

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