Thymosin beta4 and corneal wound healing: visions of the future.

Persistent corneal epithelial defects and inflammation within the central cornea can directly distort visual acuity and may lead to permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents such as corticosteroids would result in improved clinical outcome and would represent a major advance in the field. Despite much progress in the areas of corneal wound healing research, clinically available pharmacological therapies that can promote repair and limit the visual complications from persistent corneal wounds are severely limited and remains a major deficiency in the field. Prior studies from our laboratory have demonstrated the potent wound healing and anti-inflammatory effects of thymosin beta4 (Tbeta(4); Tbeta4) in numerous models of corneal injury. We are studying the mechanisms by which Tbeta(4) suppresses inflammation and promotes repair. Herein, we discuss some of our new basic scientific directions that may lead to the use of Tbeta(4) as a novel corneal wound healing and anti-inflammatory therapy.

Thymosin beta-4 and the eye: I can see clearly now the pain is gone.

The cornea epithelium responds to injury by synthesizing several cytokines, growth factors, and tissue remodeling molecules. Proinflammatory cytokines have been implicated in the inflammation that follows corneal epithelial injury and cytokine-mediated processes play a significant role in corneal epithelial wound healing. Poorly regulated corneal inflammatory reactions that occur after injury can retard healing. In turn, persistent corneal epithelial defects and inflammation may lead to ocular morbidity and permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents, such as corticosteroids, would result in improved clinical outcome and would represent a major advance in the field. Evidence is mounting to support the idea that thymosin beta-4 (Tbeta-4) has multiple, seemingly diverse, cellular functions. In the cornea, as in other tissues, Tbeta-4 promotes cell migration and wound healing, has anti-inflammatory properties, and suppresses apoptosis. Prior studies from our laboratory have demonstrated the potent wound healing and anti-inflammatory effects of Tbeta-4 in numerous models of corneal injury. Recently, we demonstrated that Tbeta-4 suppresses the activation of the transcription factor, nuclear factor-kappa b (NF-kappaB) in TNF-alpha-stimulated cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NF-kappaB, thus both are known mediators of the inflammatory process. These results have important clinical implications for the potential role of Tbeta-4 as a corneal anti-inflammatory and wound-healing agent.

Matrix metalloproteinase activity is necessary for thymosin beta 4 promotion of epithelial cell migration.

Studies from our laboratory provide substantial evidence that thymosin beta 4, (Tbeta(4)), an actin-sequestering protein, promotes corneal wound healing through its ability to stimulate epithelial cell migration. Matrix metalloproteinases (MMPs), which are expressed in a wide variety of tissues including the cornea, also play a key role in epithelial cell migration and wound healing. In this study we investigated the role of MMPs in Tbeta(4)-stimulated corneal epithelial cell migration. In Boyden chamber assays, XG076, an inhibitor of the conversion of pro- to active MMPs, had no effect on epithelial cell migration stimulated by exogenous activated MMP-1. However, in in vitro migration assays where the activation of pro-MMPs was blocked, XG076 significantly inhibited cell migration and wound healing in the presence or absence of Tbeta(4). GM6001, a broad-spectrum inhibitor of active MMPs and selective MMP inhibitors, also suppressed Tbeta(4)-stimulated cell migration. Tbeta(4) upregulated MMP-1 gene and protein expression in primary human corneal epithelial cells and in transformed human corneal epithelial cells following scrape wounding. From these results we conclude that MMP catalytic activity is necessary for Tbeta(4) promotion of epithelial cell migration. These novel findings are the first to demonstrate a functional link between the two.

Thymosin beta 4: A novel corneal wound healing and anti-inflammatory agent.

Thymosin beta 4 (Tbeta(4)) is a low molecular weight protein present in all cells except erythrocytes. Although Tbeta(4) is the major monomeric actin-sequestering peptide in cells and can depolymerize F-actin, evidence is mounting to support the idea that it has multiple, seemingly diverse, cellular functions. In cornea, as in other tissues, Tbeta(4) promotes cell migration and wound healing, has anti-inflammatory properties, and suppresses apoptosis. In this review we discuss the current state of knowledge regarding the effects of Tbeta(4) in maintaining the healthy, functional cornea. The clinical implications of the use of Tbeta(4) as a wound healing and anti-inflammatory agent are discussed.

Thymosin beta4 inhibits benzalkonium chloride-mediated apoptosis in corneal and conjunctival epithelial cells in vitro.

Thymosin beta-4 (Tbeta(4)) is known to promote ocular wound healing, to decrease ocular inflammation, and to have anti-apoptotic effects on corneal epithelium. In this study, the effect of Tbeta(4) on the survival of human ocular surface epithelial cells exposed to benzalkonium chloride (BAK) was measured. Human conjunctival epithelial cells (HC0597) or human corneal epithelial cells (HCET) were treated with 0%, 0.001%, 0.01%, or 0.1% BAK for 15 min. After 3 or 24h of recovery in culture medium containing 1 microg/ml Tbeta(4), a dosage that has been demonstrated effective in several published studies, DNA synthesis was measured using a colorimetric BrdU incorporation assay. Both conjunctival and corneal epithelial DNA synthesis was inhibited by BAK in a dose-dependent manner. Tbeta(4) did not protect the epithelial cells from BAK-induced inhibition of proliferation. To assess the ability of Tbeta(4) to prevent apoptosis, epithelial cells were treated with 0.01% BAK+Tbeta(4) and cell death was measured using a colorimetric assay. BAK-induced apoptosis increased throughout the duration of the assay, which was carried out to 5 days in culture. Treatment of HC0597 cells with Tbeta(4) significantly inhibited the apoptosis shown to be initiated by BAK. Treatment of non-transformed human corneal epithelial cells (HCEC) with Tbeta(4) also significantly inhibited the apoptosis shown to be initiated by BAK at later times in culture. Ocular solutions containing BAK as a preservative are typically used for extended periods of time. This study suggests that Tbeta(4) may be able to overcome the apoptotic side effect of BAK, and may be a useful additive to solutions containing this preservative.

Plasminogen activator inhibitor-1 (PAI-1) stimulates human corneal epithelial cell adhesion and migration in vitro.

In addition to its role as an inhibitor of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) is hypothesized to regulate epithelial cell adhesion and migration. We have previously reported that PAI-1 may be an important regulatory factor of the uPA system in cornea. The purpose of this study was to extend those observations by determining the effect of exogenous PAI-1 on the migration and adhesion of human corneal epithelial cells (HCEC) in vitro. The expression of PAI-1 in non-transformed early passage HCEC was confirmed by immunofluorescence microscopy and Western blot analysis. Colorimetric assays coupled with function-inhibiting antibody studies using the matrix assembled in situ by cultured cells demonstrate that immobilized PAI-1 serves as an efficient substrate for HCEC adhesion. HCEC attachment to PAI-1 is comparable to that of laminin-10, a known strong adhesion protein for epithelial cells. In addition to serving as an adhesion substrate, PAI-1 also functions as a chemotactic agent for corneal epithelium. Additionally it promotes the random migration of HCEC, from an initial cell cluster, along a culture substrate. Our results in corneal epithelium are consistent with reports from other investigators showing that PAI-1 facilitates both epithelial adhesion and migration. From our studies we conclude that PAI-1 may play a dual role in corneal wound healing. Initially PAI-1 may function to stimulate migration and facilitate the reepithelialization of the wound bed. Post-reepithelization, PAI-1 may ensure corneal epithelial cell adhesion to matrix to promote successful wound healing.

Thymosin-beta4 inhibits corneal epithelial cell apoptosis after ethanol exposure in vitro.

PURPOSE: The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta(4)) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tbeta(4) in preventing mitochondrial disruption and in inhibiting caspase-mediated apoptosis was examined. METHODS: Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and Tbeta(4). The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS: Tbeta(4) treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium Tbeta(4) treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tbeta(4) treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS: Tbeta(4) plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tbeta(4) may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.

Thymosin beta 4 stimulates laminin-5 production independent of TGF-beta.

Thymosin beta 4 (Tbeta(4)) stimulates epithelial cell migration and promotes laminin-5 (LM-5) expression. Using gene expression analysis with human corneal epithelial cells treated with Tbeta(4), we find that both LM-5 gamma2 chain and transforming growth factor beta 1 (TGFbeta-1) are increased by more than 2-fold over untreated cells. These findings were confirmed by RT-PCR and at the protein level. Although TGFbeta-1 increases LM-5 synthesis in a dose-dependent manner, it does not appear to be the mechanism by which Tbeta(4) acts on LM-5 gamma2 chain synthesis based on three independent experiments. In a time-course analysis, Tbeta(4) increases LM-5 gamma2 chain expression at 2 h and peaks at 6 h, while TGFbeta-1 increases LM-5 gamma2 chain expression only at 4 h and peaks at 8 h. When Tbeta(4)-induced LM-5 gamma2 chain expression is blocked with neutralizing antibodies to TGFbeta-1, LM-5 gamma2 chain expression is increased. Finally, in TGFbeta-1 knock-out mice, Tbeta(4) increases LM-5 gamma2 chain expression to levels higher than that observed in wild-type mice treated with Tbeta(4). These findings demonstrate that Tbeta(4) induces both TGFbeta-1 and LM-5 gamma2 chain expression in corneal epithelial cells. Tbeta(4) and TGFbeta-1 increase LM-5 gamma2 chain expression by independent pathways. Suppression of TGFbeta-1 further increases LM-5 gamma2 chain expression.

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity.

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.

Pseudomonas aeruginosa binds to extracellular matrix deposited by human corneal epithelial cells.

PURPOSE: To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding. METHODS: Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2). P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2). The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2). Binding in the presence of antibodies directed against laminin-5 was also measured. RESULTS: Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. CONCLUSIONS: The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.

Thymosin beta4 promotes human conjunctival epithelial cell migration.

PURPOSE: In this study the effects of thymosin beta4 (Tbeta4) on migration and production of laminin-5 in the human conjunctival cell line HC0597 was analyzed. METHODS: Boyden chamber assays assessed the ability of Tbeta4 to stimulate in vitro cell migration. Control or Tbeta4-treated cells were processed for immunofluorescence microscopy using antibodies to vinculin or laminin-5. Cell lysates were processed for Western blot and densitometric analysis using antibodies to laminin-5 alpha3 or gamma2 chains. RESULTS: Tbeta4 stimulated migration in a dose-dependent manner. Focal adhesions present in Tbeta4-treated cells were smaller and more rounded compared to the "streaks" characteristic of controls. Western blot analysis and densitometry revealed that Tbeta4-treated cells expressed more laminin-5 alpha3 and gamma2 chain protein. CONCLUSION: Tbeta4 stimulates in vitro conjunctival epithelial cell migration, and results in altered focal adhesion formation and increased extracellular laminin-5 deposition. The increased migration may be correlated with increased production of laminin-5.

Laminin alpha5 chain adhesion and signaling in conjunctival epithelial cells.

PURPOSE: To identify peptides of the LG4 module of the laminin alpha5 chain that mediate human conjunctival epithelial cell adhesion to the laminin-10 isoform. METHODS: A peptide corresponding to a major heparin- and cell-binding domain of the LG4 module of the laminin alpha5 chain was analyzed. The attachment of conjunctival epithelial cells to the peptide compared with laminin-10 was determined by colorimetric adhesion assay. The role of glycosaminoglycans in mediating adhesion to the peptide was determined by altering their function at the cell surface and by blocking adhesion with exogenous glycosaminoglycans. The role of syndecan-4 in cell adhesion to the peptide was examined by adhesion assay. The role of the peptide in cell signaling was examined by phosphotyrosine Western blot analysis. RESULTS: The peptide facilitated the adhesion of conjunctival epithelium, although not as efficiently as laminin-10. Heparinase had no effect on adhesion to the peptide. In contrast, adhesion to the peptide decreased in glycosaminoglycan-deficient cells, heparatinase-treated cells, cells blocked with exogenous heparan sulfate proteoglycan, and cells treated with antibodies to the ectodomain of syndecan-4. Cell adhesion to the peptide for 90 or 120 minutes resulted in a significant increase in focal adhesion kinase (FAK) tyrosine phosphorylation, compared with the nonadherent control. CONCLUSIONS: Human conjunctival epithelial cells use a heparin- and cell-binding peptide of the LG4 module of laminin alpha5 chain in adhesion to laminin-10. Syndecan-4 is one mechanism by which the peptide facilitates adhesion. In addition to adhesion, the peptide may function in cell-signaling events.