Coordinated Regulation of Palladin and α-Smooth Muscle Actin by Transforming Growth Factor-ß in Human Corneal Fibroblasts.

PURPOSE: To investigate the role of palladin in the cornea, we examined expression of this actin assembly-related protein in normal, diseased, or injured corneal tissue as well as in cultured corneal fibroblasts. METHODS: Expression of palladin and α-smooth muscle actin (α-SMA) in the rat cornea with an incision wound, in the normal and diseased human cornea, and in cultured human corneal fibroblasts was examined by immunofluorescence or immunoblot analysis. RESULTS: The expression of both palladin and α-SMA was detected at the lesion site during wound healing in the rat cornea. Whereas neither palladin nor α-SMA was detected in the normal human cornea, the colocalization of both proteins was detected in diseased human corneas with underlying conditions characterized by the presence of fibrosis. The expression of both palladin and α-SMA in cultured human corneal fibroblasts was increased by transforming growth factor-ß (TGF-ß) in a manner sensitive to inhibition by blockers of Smad or mitogen-activated protein kinase (MAPK) signaling. Finally, RNA interference-mediated depletion of palladin attenuated the TGF-ß-induced upregulation of α-SMA expression in human corneal fibroblasts as well as TGF-ß-induced collagen gel contraction mediated by these cells. CONCLUSIONS: Palladin is expressed in the rat and human cornea in association with scar formation. Expression of palladin in human corneal fibroblasts is increased by TGF-ß in a manner dependent on Smad and MAPK signaling and is required for the TGF-ß-induced upregulation of α-SMA.

Inhibition by all-trans retinoic acid of collagen degradation mediated by corneal fibroblasts.

BACKGROUND: We examined the effect of all-trans retinoic acid on collagen degradation mediated by corneal fibroblasts. METHODS: Rabbit corneal fibroblasts were cultured with or without all-trans retinoic acid in a three-dimensional collagen gel, and the extent of collagen degradation was determined by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Matrix metalloproteinase expression was examined by immunoblot analysis and gelatin zymography. The abundance and phosphorylation state of the endogenous nuclear factor-kappaB inhibitor IκB-α were examined by immunoblot analysis. Corneal ulceration was induced by injection of lipopolysaccharide into the central corneal stroma of rabbits and was assessed by observation with a slitlamp microscope. RESULTS: All-trans retinoic acid inhibited interleukin-1ß-induced collagen degradation by corneal fibroblasts in a concentration- and time-dependent manner. It also attenuated the release and activation of matrix metalloproteinases as well as the phosphorylation and degradation of IκB-α induced by interleukin-1ß in these cells. Topical application of all-trans retinoic acid suppressed corneal ulceration induced by injection of lipopolysaccharide into the corneal stroma. CONCLUSIONS: All-trans retinoic acid inhibited collagen degradation mediated by corneal fibroblasts exposed to interleukin-1ß, with this effect being accompanied by suppression of nuclear factor-kappaB signalling as well as of matrix metalloproteinase release and activation in these cells. All-trans retinoic acid also attenuated lipopolysaccharide-induced corneal ulceration in vivo. Our results therefore suggest that all-trans retinoic acid might prove effective for the treatment of patients with corneal ulceration.

Functional role of PPARδ in corneal epithelial wound healing.

The peroxisome proliferator-activated receptor (PPAR) δ is involved in tissue repair. In this study, we investigated the functional role of PPARδ in corneal epithelial wound healing. In an in vivo corneal wound-healing model, the changes of PPARδ expression in corneal epithelia were examined by immunofluorescence microscopy, and the effect of topical administrations of a PPARδ agonist on corneal wound healing was also evaluated. The inhibitory effect of a PPARδ agonist on the cytokine-induced death of human corneal epithelial cells was evaluated using a DNA fragmentation assay kit. The changes of PPARδ expression and epithelial cell death were also investigated using human corneoscleral tissues ex vivo. Our findings showed that PPARδ expression was temporally up-regulated in corneal epithelial cells during experimental wound healing and that topical administration of a PPARδ agonist significantly promoted the healing of experimental corneal epithelial wounds. In human corneal epithelial cells, up-regulation of PPARδ and DNA fragmentation was demonstrated by stimulation with cytokines, and the DNA fragmentation was significantly inhibited by pretreatment with a PPARδ agonist. By using human corneoscleral tissues ex vivo, PPARδ was up-regulated in both healthy corneal epithelia (during re-epithelialization) and diseased corneal epithelia. Inflammatory stimulation-induced corneal epithelial cell death was inhibited by pretreatment with a PPARδ agonist. These results strongly suggest that PPARδ is involved in the corneal epithelial wound healing.

Detection of ICP0 protein in tear fluid of individuals with active herpetic epithelial keratitis.

PURPOSE: Infected cell protein 0 (ICP0) has been detected in the tear fluid of rabbits with herpetic keratitis. Here, we investigated whether ICP0 of herpes simplex virus 1 is detectable in the tear fluid of patients with herpetic epithelial keratitis. METHODS: Seven patients with herpetic epithelial keratitis (age range, 51-76 years) and 11 healthy volunteers (age range, 48-85 years) were enrolled in the study. Tear fluid was collected with the use of Schirmer test strips and subjected to immunoblot analysis with antibodies to ICP0. RESULTS: ICP0 was not detected in the tear fluid of the healthy controls, whereas it was detected in 4 of the 7 patients with herpetic keratitis. The tear fluid of the 4 ICP0-positive patients was collected within 3 days of the onset of keratitis, whereas that of the ICP0-negative patients was obtained at least 7 days after disease onset. CONCLUSIONS: ICP0 was detected in the tear fluid of patients in the early phase of herpetic epithelial keratitis, but not in that of patients in the later stages of the disease nor in that of the healthy controls. ICP0 expression may thus be dependent on the disease phase.

Integrin-mediated inhibition of interleukin-8 secretion from human neutrophils by collagen type I.

The function of neutrophils in the inflammatory response is modulated by contact with ECM proteins. We have now investigated the effect of collagen type I on secretion of the cytokine IL-8 by human neutrophils in vitro. Collagen type I inhibited the secretion of IL-8 from neutrophils maintained under basal conditions or stimulated with fMLF. This effect was accompanied by down-regulation of IL-8 mRNA, and it appeared to be specific to collagen type I among ECM proteins, in that it was not observed with fibronectin or laminin. The inhibitory effect of collagen type I on IL-8 secretion was dependent on collagen concentration and cell density. It was also abolished in the presence of antibodies to integrin alpha2beta1 but was not affected by antibodies to integrin alpha5beta1 or beta4. Our results thus suggest that collagen type I inhibits the secretion of IL-8 by human neutrophils in a selective manner and that this effect is mediated by the interaction of collagen with integrin alpha2beta1.

Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts.

PURPOSE: The production of proinflammatory cytokines and chemokines as well as the surface expression of intercellular adhesion molecule (ICAM)-1 by corneal fibroblasts contribute to corneal inflammation. The effects of triptolide on the expression of these proteins induced by lipopolysaccharide (LPS) in human corneal fibroblasts were examined in comparison with those of dexamethasone. METHODS: The release of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1beta, and IL-8 from cultured corneal fibroblasts was measured with assay kits. Surface expression of ICAM-1 on the cultured cells was measured with a whole-cell enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide (LPS) induced the release of the proinflammatory cytokine IL-6 and that of the chemokines G-CSF, MCP-1, MIP-1beta, and IL-8 as well as surface expression of ICAM-1 by corneal fibroblasts, whereas IL-1beta, TNF-alpha, and GM-CSF were not detected in the culture supernatants of cells incubated with or without LPS. Triptolide and dexamethasone each inhibited in a concentration-dependent manner the LPS-induced release of IL-6, G-CSF, MCP-1, and IL-8 by corneal fibroblasts. Whereas the inhibitory effect of dexamethasone on LPS-induced IL-6 release was greater than that of triptolide, the inhibitory effect of triptolide on LPS-induced G-CSF release was more pronounced than was that of dexamethasone. Dexamethasone also inhibited LPS-induced MIP-1beta release, whereas triptolide did not. Both compounds inhibited the LPS-induced surface expression of ICAM-1. CONCLUSIONS: Triptolide inhibits the LPS-induced expression of IL-6, chemokines (G-CSF, MCP-1, IL-8), and ICAM-1 in cultured human corneal fibroblasts. This compound might thus be expected to limit the infiltration of immune cells into the cornea.

Inhibitory effect of triptolide on chemokine expression induced by proinflammatory cytokines in human corneal fibroblasts.

PURPOSE: Synthesis of various chemokines, including interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, as well as the surface expression of intercellular adhesion molecule (ICAM)-1 in corneal fibroblasts contribute to corneal inflammation. The effects of triptolide, the major constituent of extracts of the herb Tripterygium wilfordii hook f, on the expression of these proteins in human corneal fibroblasts were examined in comparison with those of dexamethasone. METHODS: The release of IL-8 and MCP-1 from and the surface expression of ICAM-1 on cultured corneal fibroblasts were measured with enzyme-linked immunosorbent assays. The cellular abundance of the mRNAs for these proteins was determined by reverse transcription and real-time polymerase chain reaction analysis. The activities of the transcription factors NF-kappaB and AP-1 were assessed by cell transfection with secretory alkaline phosphatase reporter genes. RESULTS: Both triptolide and dexamethasone inhibited in a dose-dependent manner the expression of IL-8 and MCP-1 in corneal fibroblasts induced by the proinflammatory cytokines IL-1beta or tumor necrosis factor (TNF)-alpha. These inhibitory effects were apparent at both the mRNA and protein levels. Both compounds also had a lesser inhibitory effect on cytokine-induced ICAM-1 expression. The activation of NF-kappaB by IL-1beta was markedly inhibited by both triptolide and dexamethasone, whereas the activity of AP-1 was not affected by either agent. CONCLUSIONS: Like dexamethasone, triptolide inhibited IL-8 and MCP-1 expression in cultured human corneal fibroblasts exposed to proinflammatory cytokines, an action most likely mediated by inhibition of NF-kappaB activation. Similar effects of triptolide in vivo may be expected to limit the infiltration of neutrophils and monocytes into the cornea.

Novel 6-hydroxy-3-morpholinones as cornea permeable calpain inhibitors.

A novel series of 6-hydroxy-3-morpholinones, in which the functional aldehyde and the hydroxy group of P(2) site form a cyclic hemiacetal, was identified as calpain inhibitors. The placement of isobutyl group at the 2-position of the 3-morpholinone was the most effective modification for inhibiting micro- and m-calpains. Substitutions of benzyl at the 5-position in the S-configuration had virtually no effect on inhibitory activity. Several compounds showed appreciable selectivity for calpains over cathepsin B. NMR experiments demonstrated that the representative 6-hydroxy-3-morpholinone 10a (SNJ-1757) was more stable to nucleophilic attack than the corresponding aldehyde inhibitor 24. Furthermore, 6-hydroxy-3-morpholinone 10a proved to have better corneal permeability than aldehyde inhibitor 24 in an in vitro experiment.

Inhibition by triptolide of IL-1-induced collagen degradation by corneal fibroblasts.

PURPOSE: Extracts of the herb Tripterygium wilfordii hook f, the major component of which is triptolide, have been used in traditional Chinese medicine for the treatment of rheumatoid arthritis. Triptolide also exerts many other biological actions both in vitro and in vivo. The effect of this agent on collagen degradation by cultured corneal fibroblasts was examined. METHODS: Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of interleukin (IL)-1beta or triptolide. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of the culture supernatants. The activities of matrix metalloproteinase (MMP)-1 and plasmin were measured with the specific substrates thiopeptolide and S-2251, respectively. The release of MMPs into the culture supernatant was assessed by immunoblot analysis and gelatin zymography, and the abundance of MMP mRNAs in the cells was determined by reverse transcription and real-time polymerase chain reaction. RESULTS: Triptolide inhibited the IL-1beta-induced degradation of collagen by corneal fibroblasts in a dose- and time-dependent manner. Neither the activity of purified recombinant MMP-1 nor that of plasmin in culture supernatants was affected by triptolide. The IL-1beta-induced expression of MMP-1, -2, -3, and -9 by corneal fibroblasts was inhibited by triptolide at the protein or mRNA level. CONCLUSIONS: Triptolide inhibits collagen degradation by corneal fibroblasts by inducing downregulation of the production of MMPs, without directly affecting the collagenolytic activity of these enzymes.

Permissive effect of fibronectin on collagen gel contraction mediated by bovine trabecular meshwork cells.

PURPOSE: The effect of fibronectin on the contractility of trabecular meshwork (TM) cells was investigated. METHODS: The contractility of bovine TM cells was evaluated by culture of the cells in a collagen gel and measurement of the change in the diameter of the gel under various conditions. The formation of stress fibers and the localization of integrin alpha5 and beta1 chains (which together form a fibronectin receptor) in bovine TM cells were investigated by laser confocal microscopy of cells stained with phalloidin and antibodies to the integrin subunits. RESULTS: The addition of fibronectin to collagen gels containing bovine TM cells induced marked gel contraction in a time- and concentration-dependent manner. Cytochalasin D (an inhibitor of microfilament formation) and the peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), a fibronectin receptor antagonist, each inhibited this effect of fibronectin, whereas nocodazole (an inhibitor of microtubule polymerization) and the control peptide GRGESP (Gly-Arg-Gly-Glu-Ser-Pro) did not. Furthermore, fibronectin induced the spreading of cells, the formation of actin stress fibers, and the expression of integrin alpha5 in the collagen gel-embedded TM cells. CONCLUSIONS: Fibronectin promotes collagen gel contraction mediated by bovine TM cells. Moreover, the formation of actin stress fibers and upregulation of integrin alpha5 appear to contribute to this permissive effect of fibronectin. The interaction of fibronectin with TM cells may thus be a determinant of the contractility of TM tissue.

Enhancement by neutrophils of collagen degradation by corneal fibroblasts.

Activated corneal fibroblasts and infiltrated leukocytes are thought to contribute to corneal ulceration. The potential roles of neutrophil-fibroblast and cell-matrix interactions in the degradation of stromal collagen associated with corneal ulceration have now been investigated with the use of three-dimensional cultures of rabbit cells in collagen gels. Degradation of collagen fibrils during culture was measured by spectrophotometric determination of released hydroxyproline. Whereas corneal fibroblasts alone degraded collagen fibrils to a small extent, neutrophils did not. However, the addition of neutrophils or neutrophil-conditioned medium (CM) to cultures of corneal fibroblasts resulted in a marked increase in the amount of collagen degraded by the fibroblasts. The effect of CM from neutrophils cultured in collagen gels on collagen degradation by corneal fibroblasts was greater than that of medium conditioned by neutrophils in monolayer culture. Immunoblot as well as reverse transcription and real-time polymerase chain reaction analyses revealed that neutrophil-CM stimulated the synthesis of matrix metalloproteinase (MMP)-1 and MMP-3 by corneal fibroblasts. The stimulatory effect of neutrophils on collagen degradation by corneal fibroblasts was inhibited by the synthetic MMP inhibitor ilomastat and by interleukin-1 (IL-1) receptor antagonist. These results suggest that factors secreted by collagen-stimulated neutrophils augment collagen degradation by corneal fibroblasts through a stimulatory effect on MMP synthesis and that IL-1 released by neutrophils may contribute to this effect.

Differential influence of proteolysis by calpain 2 and Lp82 on in vitro precipitation of mouse lens crystallins.

The purpose of the present study was to compare the susceptibility of crystallins proteolyzed by ubiquitous calpain 2 and by lens-specific calpain Lp82 to insolubilization. To test this, transgenic (TG) mice expressing a calpain 2, in which the active site cysteine 105 was mutated to alanine, were produced. Expression of mutated calpain 2 was driven in lens by coupling the mutated gene to the betaB1-crystallin promoter. Light scattering was measured in solutions of lens proteins after activation of endogenous calpain 2 and/or Lp82. Mass spectrometric analysis was performed to determine the cleavage sites and the calpain responsible for insolubilization of crystallins. Lens proteins from TG mice incubated in vitro with calcium showed higher light scattering compared to proteins from wild type (WT) mice. alphaA-crystallin from TG mice was proteolyzed by Lp82. In contrast, alphaA-crystallin in lenses from WT mice were proteolyzed by both calpain 2 and Lp82. These results suggested that Lp82-induced proteolysis of crystallins caused increased susceptibility of truncated crystallins to in vitro precipitation. Since Lp82 is highest in young animals, Lp82-induced proteolysis and precipitation may be one of the factors responsible for the cataract formation in young rodents.

Signaling mechanism of TGF-beta1-induced collagen contraction mediated by bovine trabecular meshwork cells.

PURPOSE: To characterize the intracellular signaling mechanism that underlies the contraction of trabecular meshwork (TM) tissue. METHODS: The contraction of collagen mediated by bovine TM cells was evaluated by measuring changes in the diameter of collagen gels in which the cells were embedded. Changes in the organization of the actin cytoskeleton were examined by laser-scanning confocal microscopy of cells stained with fluorescent phalloidin. Cell motility was monitored by time-lapse video microscopy. RESULTS: Transforming growth factor (TGF)-beta1 induced marked TM-cell-mediated contraction of collagen gels in a concentration- and time-dependent manner. Inhibitors of protein kinase C (PKC) blocked this effect of TGF-beta1, whereas an inhibitor of PKA and -G did not. An inhibitor of the small guanosine triphosphatase (GTPase) Rho also inhibited TGF-beta1-induced collagen contraction, whereas an activator of Rho promoted this effect of TGF-beta1. Furthermore, inhibition either of the release of Ca(2+) from internal stores or of the activation of myosin light-chain kinase (MLCK) prevented gel contraction in response to TGF-beta1. The effects of these various agents on TGF-beta1-induced contraction of collagen gels mediated by TM cells were mirrored by their effects on TGF-beta1-induced formation of actin stress fibers, cell spreading (the extension of cellular processes), and cell motility under conditions in which cell contraction was not possible. CONCLUSIONS: TGF-beta1 induces TM-cell-mediated collagen gel contraction through activation of Rho and the Ca(2+)-dependent enzymes PKC and MLCK. These same signaling molecules contribute to TGF-beta1-induced rearrangement of the actin cytoskeleton, cell spreading, and cell motility.

Active matrix metalloproteinases in the tear fluid of individuals with vernal keratoconjunctivitis.

Corneal epithelial lesions distinguish vernal keratoconjunctivitis (VKC) from other ocular allergic diseases. Such lesions result from degradation of the corneal epithelial basement membrane, which comprises mostly type IV collagen and laminin. Matrix metalloproteinase 2 (MMP-2) and MMP-9 catalyze the degradation of these 2 extracellular matrix proteins. The possible role of MMP-2 and MMP-9 in the pathogenesis of corneal lesions associated with VKC was investigated by assaying tear fluid for the presence of these enzymes. Tear fluid was collected from 6 eyes of 6 patients with active VKC, 14 eyes of 14 patients with active allergic conjunctivitis, and 6 eyes of 6 nonallergic healthy volunteers. Gelatin zymography revealed that the tear fluid of healthy volunteers contained inactive proforms of both MMP-2 and MMP-9 but not the active forms of these enzymes. Active forms of MMP-2 or MMP-9 were detected in a minority of patients with allergic conjunctivitis. However, with the exception of one individual for whom active MMP-9 was not detected, tear fluid from all patients with VKC contained both proforms and active forms of MMP-2 and MMP-9. These results implicate MMP-2 and MMP-9 in the pathogenesis of corneal epithelial disorders associated with VKC.