Novel atypical PKC inhibitors prevent vascular endothelial growth factor-induced blood-retinal barrier dysfunction

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.     

Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha

We tested the hypothesis that protein kinase C-alpha (PKC-alpha) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM lysates were analyzed for PKC-alpha mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cytoskeletal-associated PKC-alpha, 3) PKC-alpha activity, and 4) permeability to albumin. The TNF-alpha-induced increase in PKC-alpha protein, PKC-alpha activity, and permeability was prevented by a 4-h pretreatment with PKC-alpha antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-alpha-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha/beta, 100 microM) and calphostin (an inhibitor of the classic and novel PKC isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-alpha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatment. The data indicate that prolonged activation of PKC-alpha, maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha-induced increases in endothelial permeability in PEM.     

Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly

Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565-L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly.     

Protein phosphatase 2A in stretch-induced endothelial cell proliferation

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459–466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.     

ROCK mediates thrombin's endothelial barrier dysfunction

Thrombin-induced endothelial monolayer hyperpermeability is thought toresult from increased F-actin stress fiber-related contractile tension,a process regulated by the small GTP-binding protein Rho. We testedwhether this process was dependent on the Rho-associated proteinkinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects ofY-27632 on thrombin-induced myosin light chain phosphorylation (MLCP)and tyrosine phosphorylation of p125 focal adhesion kinase(p125^FAK) and paxillin were measured by Western blotting.F-actin organization and content were analyzed by digital imaging, andendothelial monolayer permeability was measured in bovine pulmonaryartery endothelial cell (EC) monolayers using a size-selectivepermeability assay. Y-27632 enhanced EC monolayer barrier function dueto a decline in small-pore number that was associated with increased ECsurface area, reduced F-actin content, and reorganization of F-actin to-catenin-containing cell-cell adherens junctions. Although Y-27632prevented thrombin-induced MLCP, stress fiber formation, and theincreased phosphotyrosine content of paxillin and p125^FAK,it attenuated but did not prevent the thrombin-induced formation oflarge paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to bepartially ROCK dependent.

     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

Protein phosphatase activity is controlled by an inhibitor phosphoprotein in tick salivary glands

Protein phosphatase activity in tick salivary glands was inhibited by heat-stable protein(s) from tick salivary glands as well as by an inhibitor protein from rabbit skeletal muscle. Inhibitor activity was increased after phosphorylation of inhibitor proteins with the catalytic subunit (C) of cyclic AMP-dependent protein kinase and ATP. C inhibited protein phosphatase activity of the partially purified enzyme, while purified cyclic AMP-dependent protein kinase inhibitor protein prevented inhibition of tick salivary gland protein phosphatase by C suggesting that the inhibitor phosphoprotein coelutes with the partially purified enzyme. A soluble heat-stable protein with a molecular weight of approx. 26 kDa was phosphorylated by C, suggesting that a protein phosphatase inhibitor protein similar to inhibitor-1 in mammalian tissue, is present in tick salivary glands.     

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction.

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.     

Bryostatin-1 attenuates TNF-induced epithelial barrier dysfunction: role of novel PKC isozymes

Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [(3)H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-epsilon. In addition, after bryostatin-1 treatment, PKC-delta and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.     

Peroxynitrite mediates TNF-alpha-induced endothelial barrier dysfunction and nitration of actin

We tested the hypothesis that tumor necrosis factor (TNF)-alpha induces a peroxynitrite (ONOO(-))-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated beta-actin (NO(2)-beta-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. beta-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted beta-actin was quantified in terms of its nitrotyrosine/beta-actin ratio with anti-nitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO(2)-beta-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with beta-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/beta-actin ratio at 0.5 h with minimal association of the NO(2)-beta-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/beta-actin ratio and permeability were prevented by the anti-ONOO(-) agent Urate. The data indicate that TNF induces an ONOO(-)-dependent barrier dysfunction, which is associated with the generation of NO(2)-beta-actin.     

Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.     

Protein tyrosine phosphatase activity inLeishmania donovani

L. Donovani promastigotes were grown to late-log and 3-day stationary phase to determine the level of protein tyrosine phosphatase activity in crude extracts and in fractions following gel filtration column chromatography. Over 90% of the activity was soluble in a low salt extraction buffer in both phases of growth. Several peaks of activity were resolved following gel filtration of the crude extracts indicating that multiple tyrosine phosphatases are present in these cells. Tyrosine phosphatase activity was lower in 3-day stationary than in late log-phase cells and a reduction in the major peak of activity, eluting in a gel fraction corresponding to an M _r of approximately 168kDa, was observed.In vivo tyrosine phosphorylation was revealed by Western blot analysis. The degree of phosphorylation of at least two proteins differed in cells obtained from late log phase cultures as compared with 3-day stationary phase cultures. These observations indicate that changes in the balance between tyrosine phosphorylation and dephosphorylation occur with increasing culture age.Abbreviations MBP myelin basic protein - PMSF phenyl-methanesulfonylfluoride - PTP protein tyrosine phosphatase - RCML reduced, carboxyamidomethylated, maleylated lysozyme - YINAS Tyr-Ile-Asn-Ala-Ser     

Protein tyrosine phosphatase 1B inhibitory activity of triterpenes isolated from Astilbe koreana

Bioassay-guided fractionation of a MeOH extract of the rhizomes of Astilbe koreana (Saxifragaceae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory assay, resulted in the isolation of a new triterpene, 3alpha,24-dihydroxyolean-12-en-27-oic acid (4), along with four triterpenes, 3-oxoolean-12-en-27-oic acid (1), 3beta-hydroxyolean-12-en-27-oic acid (beta-peltoboykinolic acid; 2), 3beta-hydroxyurs-12-en-27-oic acid (3), and 3beta,6beta-dihydroxyolean-12-en-27-oic acid (astilbic acid; 5). Compounds 1-5 inhibited PTP1B with IC50 values of 6.8+/-0.5, 5.2+/-0.5, 4.9+/-0.4, 11.7+/-0.9, and 12.8+/-1.1 microM, respectively. Our results indicate that 3-hydroxyl group and a carboxyl group in this type of triterpenes may be required for the activity, while addition of one more hydroxyl group at C-6 or C-24 may be responsible for a loss of activity. Thus, compounds 2 and 3 which possess only one hydroxyl group at C-3 and a carboxyl group at C-27 could be potential PTP1B inhibitors.     

Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-alpha with Rho. Activated PKC-alpha but not PKC-epsilon induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.