Microtubule stabilization opposes the (TNF-α)-induced loss in the barrier integrity of corneal endothelium

Microtubule disassembly breaks down the barrier integrity in a number of epithelial and endothelial monolayers. This study has investigated effects of TNF-α, which is implicated in corneal allograft rejection, on microtubules and barrier integrity in cultured bovine corneal endothelial cells. Exposure to TNF-α led to disassembly of the microtubules, and also caused disruption of the perijunctional actomyosin ring (PAMR). As a measure of barrier integrity, trans-endothelial electrical resistance (TER) was determined based on electrical cell-substrate impedance sensing in realtime. Exposure to TNF-α caused a slow decline in TER for > 20 h, and a similar exposure to cells grown on porous culture inserts led to a significant increase in permeability to FITC dextran. These changes, indicating a loss of barrier integrity, were also reflected by dislocation of ZO-1 at the cell border and disassembly of cadherins. These effects of TNF-α were inhibited upon stabilization of microtubules by pre-treatment with paclitaxel or epothilone B. Microtubule stabilization may be a useful strategy to overcome (TNF-α)-induced loss of the barrier integrity of corneal endothelium during inflammation associated with transplant rejection and uveitis.     

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells

PURPOSE: Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. METHODS: Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. CONCLUSIONS: Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.     

Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells

PURPOSE: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC). METHODS: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis and immunoblotting. Elevation of cAMP in response to agonists of A2b receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity. RESULTS: Exposure to ADO (200 microm; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 microm; 30 min), known agonists of A2b receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 microm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 microm ADO. CGS-21680, a selective A2a agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 microM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm2. CONCLUSIONS: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity.     

Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells

Increased contractility of the actin cytoskeleton by phosphorylation of the regulatory myosin light chain (MLC) results in a loss of barrier integrity in corneal endothelial cells. This study has investigated the effect of extracellular ATP, which may influence both Ca2+ and cAMP signalling, on MLC phosphorylation and barrier integrity in cultured bovine corneal endothelial cells (BCEC) known to express A2B and P2Y purinergic receptors, and ecto-nucleotidases. Extracellular ATP (100 microM) promoted MLC dephosphorylation (pMLC=61.8% at 18 min; n=9). Pre-exposure to ARL-67156, an ecto-nucleotidase inhibitor, prevented ATP-induced dephosphorylation. Other P2Y agonists, UTP and ATPgammaS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. Thrombin (2 U/ml), which activate Rho kinase through PAR-1 receptors in the endothelium, induced MLC phosphorylation (pMLC=129.2%; n=14). This phosphorylation was completely abolished by concomitant exposure to ATP. When cells were pretreated with adenosine (100 microM; A2B agonist) or forskolin (10 microM), thrombin-induced phosphorylation was suppressed. ATP also led to a significant increase in cAMP (> 3-fold compared to 10 microM adenosine). Thrombin-induced increase in trans-endothelial flux of horseradish peroxidase (44 kDa) and disruption of the cortical actin were suppressed by ATP. These findings indicate that in BCEC (1) ATP induces elevated cAMP through its metabolite adenosine leading to MLC dephosphorylation, (2) Stimulation of P2Y2 receptors also leads to activation of MLCP since UTP- and ATPgammaS caused MLC dephosphorylation, and (3) ATP is antagonistic to thrombin since the latter inhibits MLCP through increased activity of Rho kinase. These findings further emphasize the role of contractility of the actin cytoskeleton in regulating the barrier integrity of corneal endothelium.     

Cell signaling in regulation of the barrier integrity of the corneal endothelium

The barrier integrity of the corneal endothelium, which is conferred by its tight and adherens junctions, is critical for the maintenance of deturgescence of the corneal stroma. Although characteristically leaky, the barrier integrity restricts fluid leakage into the stroma such that the rate of leak does not exceed the rate of the endothelial active fluid transport directed toward the aqueous humor. At a molecular level, the barrier integrity is influenced by the actin cytoskeleton and microtubules, which are coupled to tight and adherens junctions via a variety of linker proteins. Since the cytoskeleton is affected by Rho family small GTPases and p38 MAP kinase, among others, many pathophysiological stimuli induce plasticity to the cytoskeleton and thereby elicit dynamic regulation of the barrier integrity. This review presents an overview of the impact of several bioactive factors on the barrier integrity of the corneal endothelium through altered actin cytoskeleton and/or disassembly of microtubules. The main focus is on the effect of TNF-α (tumor necrosis factor-α) which is a pro-inflammatory molecule found in the intraocular milieu during allograft rejection and anterior uveitis. This cytokine elicits acute activation of p38 MAP kinase, induces disassembly of microtubules, disrupts the peri-junctional actomyosin ring, and concomitantly breaks down the barrier integrity. These effects of TNF-α could be inhibited by stabilizing the microtubules, co-treating with a selective p38 MAP kinase inhibitor, and elevating intracellular cAMP via A2B receptors or direct exposure to forskolin. Overall, the corneal edema following a potential breakdown of the endothelial barrier integrity can be rescued pharmacologically by inhibiting specific cell-signaling mechanisms.     

Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells

PURPOSE: To investigate histamine-induced changes in the phosphorylation of myosin light chain (MLC) and its influence on the barrier integrity of corneal endothelial cells through altered contractility of the actin cytoskeleton. METHODS: Experiments were performed in cultured bovine corneal endothelial cells (BCECs). Phosphorylation of MLC, which increases contractility of the actin cytoskeleton through actomyosin interaction, was assessed by urea-glycerol gel electrophoresis and Western blot analysis. Immunocytochemistry was used to locate phosphorylated MLC in relation to tight junctions. Phosphorylation of the 17-kDa PKC-potentiated inhibitory protein of type 1 protein phosphatase (CPI-17), which inhibits MLC phosphatase, was studied using Western blot analysis. The cortical actin cytoskeleton was visualized by staining with Texas-red phalloidin. Barrier integrity was determined by quantifying horseradish peroxidase (HRP; 44 kDa) flux across cells grown on porous filters. RESULTS: RT-PCR and Western blot analysis confirmed the expression of Galphaq/11-coupled H1 receptors in BCECs. Exposure to histamine (100 microM; 10 minutes) led to phosphorylation of MLC (134% relative to untreated cells) and of CPI-17. Histamine also increased the flux of HRP by sevenfold and disrupted the assembly of the dense cortical actin found in resting cells. PKC activation by phorbol 12-myristate 13-acetate (PMA; 100 nM; 30 minutes) caused phosphorylation of both MLC and CPI-17. The histamine-induced MLC phosphorylation was reduced by pre-exposure to either ML-7 (50 microM), an MLCK (MLC kinase) inhibitor, or chelerythrine (10 microM), an inhibitor of PKC. Cotreatment with agents that elevate cAMP in BCECs prevented the histamine-induced MLC phosphorylation and the disruption of the actin cytoskeleton, and increased HRP flux. Phosphorylated MLC in response to histamine or PMA was found in a punctate form in close proximity to ZO-1, a marker of the tight junctional complex. CONCLUSIONS: Histamine induces MLC phosphorylation by activating MLCK and partly inhibiting MLC phosphatase. The latter is facilitated by the phosphorylation of CPI-17. Localization of phosphorylated MLC in proximity to ZO-1 suggests increased contractility of the cortical actin at the tight junctional complex. This contractility oppose the tethering forces and lead to a breakdown of the barrier integrity. Last, elevated cAMP prevents histamine-induced loss of the barrier integrity, not only by blocking inactivation of MLC phosphatase but also by inactivating MLCK.     

Benzalkonium chloride induces dephosphorylation of Myosin light chain in cultured corneal epithelial cells

PURPOSE: Phosphorylation of myosin light chain (MLC) is essential for the contractility of the actin cytoskeleton, which regulates barrier integrity, adhesion, and migration. This study was conducted to investigate the effect of benzalkonium chloride (BAK), a preservative in topical ophthalmic formulations, on MLC phosphorylation in primary cultures of bovine corneal epithelial cells (BCECs). METHODS: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by Western blot analysis. Activation of RhoA, which inhibits MLC phosphatase through Rho kinase, was examined by immunoprecipitation. The release of adenosine triphosphate (ATP) was measured by the luciferase-luciferin bioluminescence technique. RESULTS: Positive expression of MLC kinase (MLCK) was found at the mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Exposure to BAK for 10 to 20 minutes at concentrations of 0.0005%, 0.001%, and 0.003% reduced MLC phosphorylation by more than 30%. In addition, BAK led to thinning of the cortical actin and a decrease in cell adhesion. However, RhoA activity was found to increase with BAK treatment. Similar to BAK, ATP-depletion (induced by both antimycin-A and hypoxia) led to MLC dephosphorylation. BAK exposure also showed acute ATP release. CONCLUSIONS: BAK induces acute ATP release and concomitant MLC dephosphorylation in bovine corneal epithelial cells. The dephosphorylation, presumably due to ATP loss, is indicative of a loss of contractility of the actin cytoskeleton that could affect cellular functions contributing to the maintenance of epithelial barrier integrity.     

Delayed disruption of barrier function in cultured human corneal epithelial cells induced by tumor necrosis factor-alpha in a manner dependent on NF-kappaB

PURPOSE: The corneal epithelium provides a barrier that is both important for corneal homeostasis and dependent on tight junctions (TJs) between adjacent epithelial cells. The authors examined the effects of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, on barrier function and the expression of TJ proteins in simian virus 40-transformed human corneal epithelial (HCE) cells. METHODS: The barrier function of cultured HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular distribution of the TJ proteins zonula occludens-1 (ZO-1) and occludin and that of the p65 subunit of nuclear factor-kappaB (NF-kappaB) were determined by immunofluorescence staining. The expression of ZO-1 and occludin and the phosphorylation and degradation of the NF-kappaB inhibitory protein IkappaB-alpha were examined by immunoblot analysis. RESULTS: TNF-alpha induced a decrease in the TER of HCE cells in a concentration- and time-dependent manner. It also induced the disappearance of ZO-1 from the interfaces of neighboring HCE cells without affecting the localization of occludin. The abundance of neither ZO-1 nor occludin was affected by TNF-alpha. TNF-alpha induced the phosphorylation and downregulation of IkappaB-alpha and the translocation of the p65 subunit of NF-kappaB to the nucleus. The NF-kappaB inhibitor curcumin blocked the effects of TNF-alpha on TER and the subcellular localization of ZO-1 at late phase. CONCLUSIONS: TNF-alpha disrupted the barrier function of HCE cells, apparently by affecting the localization of ZO-1 at TJs in a manner dependent on NF-kappaB at late phase. This action of TNF-alpha may contribute to the loss of corneal epithelial barrier function associated with ocular inflammation.     

Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells

PURPOSE: Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility. METHODS: Experiments were performed using cultured bovine TM cells. Phosphorylated MLC (pMLC), expressed as the % of untreated cells, was assessed by urea-glycerol gel electrophoresis and Western blotting. RhoA activity was determined by affinity precipitation of RhoA-GTP to RhoA binding domain of an effector of RhoA. Intracellular cAMP levels were measured by ELISA. RESULTS: Exposure to LPA (lysophosphatidic acid) led to increased MLC phosphorylation (LPA: pMLC=133%) and activation of RhoA. These responses of LPA were suppressed by co-treatment with forskolin (LPA+forskolin: pMLC=88%). Similarly, ET-1 and nocodazole-induced MLC phosphorylation (ET-1: pMLC=145%; nocodazole: pMLC=145%) as well as RhoA activation were suppressed by co-treatment with forskolin (ET-1+forskolin: pMLC=99%; nocodazole+forskolin: pMLC=107%). Exposure to forskolin alone led to MLC dephosphorylation (pMLC=68%). Forskolin alone led to a 4-fold increase in cAMP levels. This increase was not affected when co-treated with LPA or ET-1. CONCLUSIONS: Forskolin prevents MLC phosphorylation induced by LPA, ET-1, and nocodazole through inhibition of RhoA-Rho kinase axis. MLC dephosphorylation and consequent relaxation of actin cytoskeleton in TM cells presumably underlies the increased outflow facility reported in response to forskolin.     

The role of protein tyrosine phosphorylation in the cell–cell junctions and intercellular permeability of post-confluent bovine corneal epithelial cells

PurposeTo evaluate the role of protein tyrosine phosphatase (PTP) in controlling the integrity of cell–cell junction and intercellular permeability in postconfluent bovine corneal epithelial cells.MethodsConfluent cultures of bovine corneal epithelial cells were treated with different concentrations of general phosphate inhibitors andsodium orthovanadate for varying periods. An MTS assay was used to confirm no cellular death under the treatment profile. Immunocytochemical (ICC) analysis was performed to demonstrate protein tyrosine phosphorylation after treatment with sodium orthovanadate, and the effect of sodium orthovanadate on junctional proteins such as p120, α-catenin, occludin, ZO-1, and ZO-2. Western blot analysis was used to analyze the changes in p120, α-catenin, occludin, ZO-1, and ZO-2 after treatment. Paracellular permeability was evaluated by transepithelial electrical resistance (TER).ResultsDuring the whole course of treatment, no significant cellular death was noticed. Dose- and time-dependent effects of sodium orthovanadate on protein tyrosine phosphorylation were confirmed by ICC. ICC also demonstrated the dose- and time-dependent effect of sodium orthovanadate on the disruption of p120, α-catenin, occludin, ZO-1, and ZO-2. However, results of Western blot analysis showed no change in the expression levels of p120, α-catenin, occludin, ZO-1, and ZO-2. Dose- and time-dependent increase of paracellular permeability was evaluated by TER.ConclusionInhibition of protein tyrosine phosphatase activity can increase protein tyrosine phosphorylation. A dose- and time-dependent release of cell–cell contacts and increased transepithelial permeability were found in postconfluent culture of bovine corneal epithelial cells.     

Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor

PURPOSE: To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer. METHODS: Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer. RESULTS: Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, and increased chemotactic migration of RPE cells from the monolayer. Primary cultures of confluent RPE cells showed a progressive decrease in TER. Western blot analysis showed rapid tyrosine phosphorylation of ZO-1, occludin, and beta-catenin within 20 minutes of stimulation. There was a marked loss of ZO-1 protein within 1 hour of HGF treatment. After 6 hours of treatment with HGF, occludin, claudin-1, and beta-catenin were redistributed from the membrane to the cytoplasm. CONCLUSIONS: Treatment of RPE explants with HGF results in rapid disassembly of tight and adherens junctions associated with loss or redistribution of junctional proteins, decreased TER, and increased migration of RPE cells from the monolayer.     

Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by keratinocyte growth factor

PURPOSE: The possible detrimental effect of hypoxia on the barrier function of corneal epithelial cells and whether keratinocyte growth factor (KGF) might protect against such an effect were investigated. METHODS: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured for 4 days to allow the establishment of barrier function. They were then deprived of serum for 24 hours before exposure to 1% (hypoxia) or 21% (normoxia) oxygen for 24 hours. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). The localization of ZO-1 and occludin was determined by immunofluorescence microscopy, and the expression of these tight junctional proteins as well as the phosphorylation of the mitogen-activated protein kinases ERK, p38, and JNK were examined by immunoblot analysis. RESULTS: Hypoxia induced a decrease in the TER of HCE cells compared with that of cells maintained under normoxia. The localization of ZO-1 at cell-cell borders was disrupted by hypoxia, whereas the distribution of occludin was not affected. Hypoxia also induced the downregulation of ZO-1 and a decrease in the phosphorylation of ERK without affecting the phosphorylation of p38 or JNK. All these effects of hypoxia were inhibited by KGF. The effects of KGF on TER and ZO-1 localization in cells exposed to hypoxia were inhibited by PD98059, an inhibitor of ERK signaling. Neither hypoxia nor KGF exhibited mitogenic or cytotoxic effects in HCE cells. CONCLUSIONS: Hypoxia induces disruption of the barrier function of HCE cells by eliciting the redistribution and degradation of ZO-1, and this effect is inhibited by KGF in a manner dependent on ERK activation.     

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 microm thick type I collagen matrices. After 24h, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24h of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. Following 24h exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho-kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture.     

VEGF modulation of retinal pigment epithelium resistance

Fluid accumulation into the subretinal space and the development of macular edema is a common condition in age-related macular degeneration, diabetic retinopathy, and following ocular surgery, or injury. Vascular endothelial growth factor (VEGF) and other cytokines have been implicated in the disruption of retinal pigment epithelium (RPE) barrier function and a reduction in the regulated removal of subretinal fluid; however, the cellular and molecular events linking these agents to the disruption of barrier function have not been established. In the current study, cultures of ARPE-19 and primary porcine retinal pigment epithelium (RPE) cells were utilized to investigate the effects of the VEGF-induced modifications to the barrier properties of the RPE. The barrier function was determined by transepithelial resistance (TER) measurements and morphology of the RPE monolayers. In both ARPE-19 and primary porcine RPE cells the administration of VEGF produced a significant drop in TER, and this response was only observed following apical administration. Maximum reduction in TER was reached 5h post VEGF administration. These responses were concentration-dependent with an EC(50) of 502pg/mL in ARPE-19 cells and 251pg/mL in primary porcine cells. In both ARPE-19 and primary RPE cells, the response to VEGF was blocked by pretreatment with the relatively selective VEGF-R2 antagonists, SU5416 or ZM323881, or the protein tyrosine kinase inhibitor, genistein. Administration of the relatively selective VEGF-R2 agonist, VEGF-E, also reduced TER in a concentration-dependent manner (EC(50) of 474pg/mL), while VEGF-R1 agonist, placental growth factor (PlGF), did not significantly alter the TER. Immunolocalization studies demonstrated that confluent monolayers exhibited continuous cell-to-cell ZO-1 protein contacts and apical localization of the VEGF-R2 receptors. These data provide evidence that the VEGF-induced breakdown of RPE barrier function is mediated by the activation of apically-oriented VEGF-R2 receptors. Thus, VEGF-mediated increases in RPE permeability are initiated by a rise in intraocular levels of VEGF.     

Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells

Activation of protein kinase C (PKC) by exposure of cultured human corneal epithelial cells to phorbol 12-myristate 13-acetate (PMA) resulted in an increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance (TER). A change in the membrane distribution of the tight junction protein ZO-1 was also observed in the PMA-treated cells. In contrast, when the cells were treated with PMA in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, all barrier characteristics were preserved, suggesting that PKC induces tight junction disruption through the activation of MAPK. The role of this signaling pathway in the regulation of epithelial permeability was further elucidated by the use of corneal epithelial-derived cell lines expressing constitutively activated (ca) or dominant-negative (dn) mutants of MAPK kinase-1 (MEK1). Transfectants of caMEK1, when compared to parental cells, had higher levels of phosphorylated extracellular regulated protein kinase (ERK), altered distribution of ZO-1 and occludin, and much reduced TER. On the other hand, dnMEK1 transfectants had lower but detectable levels of ERK phosphorylation, more flattened morphology, and, most importantly, significantly higher TER when compared to parental cells. Our study demonstrates that activation of PKC causes the disruption of tight junctions through activation of MAP kinase and that the MAP kinase signaling pathway plays a key role in the regulation of epithelial cell morphology and barrier function in the cornea.     

Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium

Recent evidence indicates that the anti-angiogenic peptide endostatin may modulate some of the vasomodulatory effects of vascular endothelial growth factor (VEGF) in the retina, including reduction of blood retinal barrier function although it remains uncertain how endostatin promotes endothelial barrier properties. The current study has sought to examine how physiological levels of endostatin alters VEGF-induced inner BRB function using an in vitro model system and evaluation of occludin and ZO-1 regulatory responses. In addition, the ability of exogenous endostatin to regulate VEGF-mediated retinal vascular permeability in vivo was investigated. Retinal microvascular endothelial cells (RMEC's) were exposed to various concentrations of endostatin. In parallel studies, RMEC monolayers were treated with vascular endothelial growth factor (VEGF165). Vasopermeability of RMEC monolayers and occludin expression were determined. Blood retinal barrier integrity was quantified in mouse retina using Evans Blue assay following intravitreal delivery of VEGF165, endostatin or a VEGF/endostatin combination. Endostatin increased the levels of expression of occludin whilst causing no significant change in FITC-dextran flux across the RMEC monolayer. Endostatin reversed the effects of VEGF165-enhanced permeability between microvascular endothelial cells and induced phosphorylation of occludin. Evans Blue leakage from retinas treated with VEGF was 2.0 fold higher than that of contra-lateral untreated eyes (P<0.05) while leakage of eyes from endostatin treated animals was unchanged. When eyes were injected with a combination of VEGF165 and endostatin there was a significant reduction in retinal vasopermeability when compared to VEGF-injected eyes (P<0.05). We conclude that endostatin can promote integrity of the retinal endothelial barrier, possibly by preventing VEGF-mediated alteration of tight junction integrity. This suggests that endostatin may be of clinical benefit in ocular disorders where significant retinal vasopermeability changes are present.     

Imbalanced IL-37/TNF-α/CTSS signaling disrupts corneal epithelial barrier in a dry eye model in vitro

PurposeTo explore novel role and molecular mechanism of a natural anti-inflammatory cytokine interleukin (IL) 37 in preventing corneal epithelial barrier disruption from hyperosmolar stress as can occur in dry eye disease.MethodsPrimary human corneal epithelial cells (HCECs) were cultured from fresh donor limbal explants. An in vitro dry eye model with hyperosmolar stress was established by switching HCECs from isosmolar (312mOsM) to hyperosmolar medium (350–500 mOsM), and some cells were treated with rhIL-37 or rhTNF-α, for different periods (2–48 h). The expression of cytokines and cathepsin S, and barrier protein integrity were evaluated by RT-qPCR, ELISA, and immunofluorescent staining with confocal microscopy.ResultsThe integrity of epithelial barrier was significantly disrupted in HCECs exposed to hyperosmolar medium, as shown by immunofluorescent images of tight junction (TJ, ZO-1, occludin and claudin-1) and adheren junction (E-cadherin) proteins. TNF-α accentuated hyperosmolar-induced disruption of TJ barrier functional integrity whereas exposure to IL-37 blunted or even reversed these changes. Cathepsin S, encoded by CTSS gene, was found to directly disrupt epithelial barrier integrity. Interestingly, CTSS expression was significantly induced by TNF-α and hyperosmolarity, while exogenous rhIL-37 inhibited TNF-α and CTSS expression at mRNA and protein levels following hyperosmolar stress. Furthermore, rhIL-37 restored barrier protein integrity, observed in 2D and 3D confocal immunofluorescent images, in HCECs under hyperosmolar stress.ConclusionOur findings demonstrate a novel signaling pathway by which anti-inflammatory cytokine IL-37 prevents corneal epithelial barrier disruption under hyperosmotic stress via suppressing TNF-α and CTSS expression. This study provides new insight into mechanisms protecting corneal barrier in dry eye disease.     

Effects of high glucose concentration on the barrier function and the expression of tight junction proteins in human retinal pigment epithelial cells

There is no information on the direct effect of high glucose concentrations on the barrier function of retinal pigment epithelium (RPE). The aim of this study was to explore the effect of high glucose concentrations on the permeability and the expression of tight junction proteins (occludin, zonula occludens-1 (ZO-1) and claudin-1) in a human RPE line (ARPE-19). For this purpose, ARPE-19 cells were cultured for 3 weeks in a medium containing 5.5 mM d-glucose (mimicking physiological conditions) and 25 mM d-glucose (mimicking hyperglycemia that occurs in diabetic patients). The permeability was evaluated by measuring transepithelial electrical resistance (TER) and apical–basolateral movements of dextran. The expression of tight junction proteins was evaluated by real-time PCR (RT-PCR) and Western blot. Cells grown at 25 mM of d-glucose showed a significant higher TER and a significant lower dextran diffusion than the ones maintained at 5.5 mM of d-glucose. Occludin and ZO-1 mRNA levels and protein content were similar in cultures maintained in 5.5 mM and 25 mM d-glucose. By contrast, high glucose concentrations induced a significant overexpression of claudin-1 (mRNA: 1.03 ± 0.48 vs 2.29 ± 0.7 RQ; p = 0.039, at 21 days. Protein levels: 0.92 ± 0.12 vs 1.14 ± 0.28 arbitrary units; p = 0.03, at 21 days). However, after blocking claudin-1 expression using siRNA no changes in TER and permeability were observed. We conclude that high glucose concentration results in a reduction of permeability in ARPE-19 cells. In addition, our results suggest that the overexpression of claudin-1 induced by high glucose concentrations is not involved in the mechanisms by which glucose increases the tight junction sealing function. Further studies addressed to unravel the complexity of permeability regulation in RPE are needed.