Cultured Corneal Fibroblasts Research Articles

Rose Bengal Photodynamic Therapy (RB-PDT) Modulates the Inflammatory Response in LPS-Stimulated Human Corneal Fibroblasts By Influencing NF-κB and p38 MAPK Signaling Pathways

Purpose To investigate the effect of rose bengal photodynamic therapy on lipopolysaccharide-induced inflammation in human corneal fibroblasts. Furthermore, to analyze potential involvement of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways in this process. Methods Human corneal fibroblast cultures underwent 0–2.0 µg/mL lipopolysaccharide treatment, and 24 h later rose bengal photodynamic therapy (0.001% RB, 565 nm wavelength illumination, 0.17 J/cm2 fluence). Interleukin-6, interleukin-8, intercellular adhesion molecule-1, interferon regulatory factor-3, interferon α2, and interferon β1 gene expressions were determined by quantitative PCR. Interleukin-6, interleukin-8, and C-C motif chemokine ligand-4 concentrations in the cell culture supernatant were measured by enzyme-linked immunosorbent assays and intercellular adhesion molecule-1 protein level in human corneal fibroblasts by western blot. In addition, the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways were investigated by quantitative PCR and phosphorylation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase by western blot. Results Rose bengal photodynamic therapy in 2.0 µg/mL lipopolysaccharide-stimulated human corneal fibroblasts triggered interleukin-6 and interleukin-8 mRNA (p < .0001) and interleukin-6 protein increase (p < .0001), and downregulated intercellular adhesion molecule-1 expression (p < .001). C-C motif chemokine ligand-4, interferon regulatory factor-3, interferon α2, and interferon β1 expressions remained unchanged (p ≥ .2). Rose bengal photodynamic therapy increased IκB kinase subunit beta, nuclear factor kappa B p65, extracellular signal-regulated kinases-2, c-Jun amino terminal kinase, and p38 transcription (p ≤ .01), and triggered nuclear factor kappa B p65 and p38 mitogen-activated protein kinase phosphorylation (p ≤ .04) in lipopolysaccharide treated human corneal fibroblasts. Conclusion Rose bengal photodynamic therapy of lipopolysaccharide-stimulated human corneal fibroblasts can modify the inflammatory response by inducing interleukin-6 and interleukin-8 expression, and decreasing intercellular adhesion molecule-1 production. C-C motif chemokine ligand-4, interferon regulatory factor-3, and interferon α and β expressions are not affected by rose bengal photodynamic therapy in these cells. The underlying mechanisms may be associated with nuclear factor kappa B and p38 mitogen-activated protein kinase pathway activation.

Substance P promotes transforming growth factor-β-induced collagen synthesis in human corneal fibroblasts.

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-β (TGF-β) and interleukin-1β (IL-1β), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-β on collagen type I synthesis without affecting that of IL-1β on the expression of matrix metalloproteinase-1. This effect of SP on TGF-β-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-β-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy.NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-β on collagen type I synthesis without affecting that of interleukin-1β on the expression of matrix metalloproteinase-1.

Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 μg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α–smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

Corneal scarring is a major cause of blindness worldwide with few effective therapeutic options. Finding a treatment would be of tremendous public health benefit, but requires a thorough understanding of the complex interactions that underlie this phenomenon. Here, we tested the hypothesis that the large increase in expression of Semaphorin 3A (SEMA3A) in corneal wounds contributes to the development of stromal fibrosis. We first verified this increased expression in vivo, in a cat model of photorefractive keratectomy-induced corneal wounding. We then examined the impact of adding exogenous SEMA3A to cultured corneal fibroblasts, and assessed how this affected the ability of transforming growth factor-beta1 (TGF-β1) to induce their differentiation into myofibroblasts. Finally, we examined how siRNA knockdown of endogenous SEMA3A affected these same phenomena. We found exogenous SEMA3A to significantly potentiate TGF-β1’s profibrotic effects, with only a minimal contribution from cell-intrinsic SEMA3A. Our results suggest a previously unrecognized interaction between SEMA3A and TGF-β1 in the wounded cornea, and a possible contribution of SEMA3A to the regulation of tissue fibrosis and remodeling in this transparent organ.

KCa3.1 ion channel: A novel therapeutic target for corneal fibrosis.

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

Antifibrotic Actions of Peroxisome Proliferator-Activated Receptor γ Ligands in Corneal Fibroblasts Are Mediated by β-Catenin–Regulated Pathways

Wound healing after corneal injury typically involves fibrosis, with transforming growth factor β1 (TGF-β1) as one of its strongest mediators. A class of small molecules-peroxisome proliferator-activated receptor γ (PPARγ) ligands-exert potent antifibrotic effects in the cornea by blocking phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, why this blocks fibrosis remains unknown. Herein, we show that PPARγ ligands (rosiglitazone, troglitazone, and 15-deoxy-Δ12,14-prostaglandin J2) decrease levels of β-catenin. We also show that β-catenin siRNA and the Wingless/integrated (Wnt) inhibitor pyrvinium block the ability of corneal fibroblasts to up-regulate synthesis of α-smooth muscle actin (α-SMA), collagen 1 (COL1), and fibronectin (FN) in response to TGF-β1. Activation of TGF-β receptors and p38 MAPK increased glycogen synthase kinase 3β (GSK3β) phosphorylation, whereas a chemical inhibitor of p38 MAPK (SB203580) reduced the phosphorylation of GSK3β, decreasing active β-catenin levels in both cytoplasmic and nuclear fractions. Finally, lithium chloride, a GSK3 inhibitor, also attenuated the TGF-β1-induced increase in α-SMA, COL1, and FN expression. All in all, our results suggest that TGF-β1 stimulation increases active β-catenin concentration in cultured corneal fibroblasts through p38 MAPK regulation of canonical Wnt/β-catenin signaling, increasing α-SMA, COL1, and FN synthesis. Thus, PPARγ ligands, by blocking TGF-β1-induced p38 MAPK phosphorylation, prevent increases in both total and active β-catenin through p38 MAPK-GSK3β signaling.

Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro

BackgroundWe aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay.MethodsBone marrow aspirates and eyes were collected from normal, euthanized horses with subsequent isolation and culture of BM-MSCs and corneal stromal cells. Corneal stromal cells were culture-expanded in the culture well of transwell plates and then treated with an autologous BM-MSC suspension (dose: 2.5 × 105/100 μL media with the BM-MSCs contained within the insert well), MSC-Sp solution, or naive culture media (control) for 72 h. A linear defect in confluent cell cultures was created (i.e., corneal scratch assay) to assess the cellular closure (“healing”) over time. Three representative areas of the scratch in each culture were photographed at each time point and the scratch area was quantitated using image analysis software (ImageJ). Media from the scratches were analyzed for various growth factors using human enzyme-linked immunosorbent assay (ELISA) kits that crossreact with the horse.ResultsThere was a significant percentage decrease in the scratch area remaining in the BM-MSC and MSC-Sp groups compared to the control group. There was also a significant percentage decrease in the scratch area remaining in the BM-MSC group compared to the MSC-Sp group at 36 h post-scratch and all time points thereafter. The concentration of transforming growth factor (TGF)-β1 in the media was significantly higher in the BM-MSC group compared to the control group.ConclusionsThe significant decrease in scratch area in equine corneal fibroblast cultures treated with autologous BM-MSCs compared to MSC-Sp or control treatments suggests that BM-MSCs may substantially improve corneal wound healing in horses. MSC-Sp may also improve corneal wound healing given the significant decrease in scratch area compared to control treatments, and would be an immediately available and cost-effective treatment option.

Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

Galectin-3 Inhibition by a Small-Molecule Inhibitor Reduces Both Pathological Corneal Neovascularization and Fibrosis.

PurposeCorneal neovascularization and scarring commonly lead to significant vision loss. This study was designed to determine whether a small-molecule inhibitor of galectin-3 can inhibit both corneal angiogenesis and fibrosis in experimental mouse models.MethodsAnimal models of silver nitrate cautery and alkaline burn were used to induce mouse corneal angiogenesis and fibrosis, respectively. Corneas were treated with the galectin-3 inhibitor, 33DFTG, or vehicle alone and were processed for whole-mount immunofluorescence staining and Western blot analysis to quantify the density of blood vessels and markers of fibrosis. In addition, human umbilical vein endothelial cells (HUVECs) and primary human corneal fibroblasts were used to analyze the role of galectin-3 in the process of angiogenesis and fibrosis in vitro.ResultsRobust angiogenesis was observed in silver nitrate–cauterized corneas on day 5 post injury, and markedly increased corneal opacification was demonstrated in alkaline burn–injured corneas on days 7 and 14 post injury. Treatment with the inhibitor substantially reduced corneal angiogenesis and opacification with a concomitant decrease in α-smooth muscle actin (α-SMA) expression and distribution. In vitro studies revealed that 33DFTG inhibited VEGF-A–induced HUVEC migration and sprouting without cytotoxic effects. The addition of exogenous galectin-3 to corneal fibroblasts in culture induced the expression of fibrosis-related proteins, including α-SMA and connective tissue growth factor.ConclusionsOur data provide proof of concept that targeting galectin-3 by the novel, small-molecule inhibitor, 33DFTG, ameliorates pathological corneal angiogenesis as well as fibrosis. These findings suggest a potential new therapeutic strategy for treating ocular disorders related to pathological angiogenesis and fibrosis.

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

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. 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. 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-β (TGF-β) 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-β-induced upregulation of α-SMA expression in human corneal fibroblasts as well as TGF-β-induced collagen gel contraction mediated by these cells. 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-β in a manner dependent on Smad and MAPK signaling and is required for the TGF-β-induced upregulation of α-SMA.

Inhibitory effects of PPARγ ligands on TGF-β1-induced CTGF expression in cat corneal fibroblasts

Ligands of Peroxisome Proliferator Activated Receptor gamma (PPARγ) possess strong anti-fibrotic properties in the cornea and several other body tissues. In the cornea, we recently showed this class of molecules to prevent stromal myofibroblast differentiation partially by blocking the actions of p38 mitogen-activated protein kinase (MAPK). However, given the important role assigned to connective tissue growth factor (CTGF) in mediating corneal fibrosis, here we asked whether PPARγ ligands also act by affecting transforming growth factor-β (TGF-β) 1-induced expression of CTGF in cultured corneal fibroblasts. Corneal keratocytes were isolated from young, adult cats and early passage cells were exposed to TGF-β1 with or without the PPARγ ligands Rosiglitazone, Troglitazone and 15d-PGJ2. Western blots were used to assay levels of CTGF and alpha smooth muscle actin (αSMA), a marker of myofibroblast differentiation. CTGF siRNA demonstrated a critical role for CTGF in TGF-β1-mediated myofibroblast differentiation, while exogenously applied CTGF potentiated the pro-fibrogenic effects of TGF-β1. TGF-β1-mediated increases in CTGF and αSMA expression were strongly inhibited by all three PPARγ ligands tested, and by a c-jun N-terminal kinase (JNK) inhibitor. However, while extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (AKT) and p38 MAPK inhibitors also blocked TGF-β1-induced αSMA induction, they did not dampen TGF-β1-induced increases in levels of CTGF. Thus, we conclude that PPARγ ligands block TGF-β1-induced increases in CTGF levels in cat corneal fibroblasts. They appear to do this in addition to their anti-fibrotic effect on p38 MAPK, providing a second intracellular pathway by which PPARγ ligands block αSMA induction.

Diversity of heparan sulfate and HSV entry: basic understanding and treatment strategies.

A modified form of heparan sulfate (HS) known as 3-O-sulfated heparan sulfate (3-OS HS) generates fusion receptor for herpes simplex virus (HSV) entry and spread. Primary cultures of corneal fibroblasts derived from human eye donors have shown the clinical significance of this receptor during HSV corneal infection. 3-OS HS- is a product of a rare enzymatic modification at C3 position of glucosamine residue which is catalyzed by 3-O-sulfotransferases (3-OSTs) enzymes. From humans to zebrafish, the 3-OST enzymes are highly conserved and widely expressed in cells and tissues. There are multiple forms of 3-OSTs each producing unique subset of sulfated HS making it chemically diverse and heterogeneous. HSV infection of cells or zebrafish can be used as a unique tool to understand the structural-functional activities of HS and 3-OS HS and likewise, the infection can be used as a functional assay to screen phage display libraries for identifying HS and 3-OS HS binding peptides or small molecule inhibitors. Using this approach over 200 unique 12-mer HS and 3-OS HS recognizing peptides were isolated and characterized against HSV corneal infection where 3-OS HS is known to be a key receptor. In this review we discuss emerging role of 3-OS HS based therapeutic strategies in preventing viral infection and tissue damage.

Evidence for the Involvement of MMP14 in MMP2 Processing and Recruitment in Exosomes of Corneal Fibroblasts.

Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis. We isolated MMP14-containing exosomes from corneal fibroblasts by sucrose density gradient and evaluated exosome content and purity by Western blot analysis. We then investigated exosome transport in vitro from corneal fibroblasts to two populations of vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAECs). Western blot analysis and gelatin zymography were used to determine levels of MMP14 and MMP2, respectively, in exosomal fractions derived from cultured wild-type, MMP14 enzymatic domain-deficient (MMP14Δexon4), and MMP14-null corneal fibroblasts. Matrix metalloproteinase 14-containing exosomes isolated from corneal fibroblasts were readily taken up in vitro by HUVECs and CPAECs. We found that MMP14 was enriched in exosomal fractions of cultured corneal fibroblasts. Moreover, loss of the MMP14 enzymatic domain resulted in accumulation of pro-MMP2 protein in exosomes, whereas MMP2 was nearly undetectable in exosomes of MMP14-null fibroblasts. Our results indicate that exosomes secreted by corneal fibroblasts can transport proteins, including MMP14, to vascular endothelial cells. In addition, recruitment of MMP2 into corneal fibroblast exosomes is an active process that depends, at least in part, on the presence of MMP14. The role of exosomal MMP14 transport in corneal angiogenesis has important implications for therapeutic applications targeting angiogenic processes in the cornea.

Aloe vera: an in vitro study of effects on corneal wound closure and collagenase activity

To evaluate the in vitro effects of an aloe vera solution on (i) the viability and wound healing response of corneal cells and (ii) the ability to alter collagenase and gelatinase activities. Primary cultures of corneal epithelial cells and fibroblasts were prepared from grossly normal enucleated canine globes and treated with an aloe solution (doses ranging from 0.0-2 mg/mL). Cellular viability was evaluated using a colorimetric assay. A corneal wound healing model was used to quantify cellular ingrowth across a defect made on the confluent surface. Anticollagenase and antigelatinase activities were evaluated by incubating a bacterial collagenase/gelatinase with aloe solution (doses ranging from 0.0-500 μg/mL) and comparing outcome measures to a general metalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum (doses ranging from 0.0-100%). None of the concentrations of aloe solution tested significantly affected the viability of corneal epithelial cells or fibroblasts. Concentrations ≤175 μg/mL slightly accelerated corneal epithelial cell wound closure; this change was not significant. Concentrations ≥175 μg/mL significantly (P ≤ 0.001) slowed the rate of corneal fibroblast wound closure, while aloe concentrations <175 μg/mL did not significantly alter fibroblast wound closure. Aloe solution did not alter the ability for collagenase to degrade gelatin or collagen Type I but increased the ability for collagenase to degrade Type IV collagen. Although additional experiments are required, lower concentrations of aloe solution may be beneficial in healing of superficial corneal wounds to help decrease fibrosis and speed epithelialization. An increase in collagenase activity with aloe vera warrants further testing before considering in vivo studies.

Triple Combination of siRNAs Targeting TGFβ1, TGFβR2, and CTGF Enhances Reduction of Collagen I and Smooth Muscle Actin in Corneal Fibroblasts

Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA). Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry. Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability. Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.

Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity.

To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α-smooth muscle actin (αSMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFβ1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in αSMA mRNA and 86-88% (P < 0.001) decrease in αSMA+ immunocytochemical staining. SAHA treatment also resulted in 4.5- to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 μm dose; 0.5-fold decrease with 10 μm dose). No change in fibroblast viability was observed with a 5 μm SAHA dose, whereas a 10 μm dose resulted in a moderate 17% decrease in cell viability. Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFβ-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.

Topical rosiglitazone is an effective anti-scarring agent in the cornea.

Corneal scarring remains a major cause of blindness world-wide, with limited treatment options, all of which have side-effects. Here, we tested the hypothesis that topical application of Rosiglitazone, a Thiazolidinedione and ligand of peroxisome proliferator activated receptor gamma (PPARγ), can effectively block scar formation in a cat model of corneal damage. Adult cats underwent bilateral epithelial debridement followed by excimer laser ablation of the central corneal stroma to a depth of ∼160 µm as a means of experimentally inducing a reproducible wound. Eyes were then left untreated, or received 50 µl of either 10 µM Rosiglitazone in DMSO/Celluvisc, DMSO/Celluvisc vehicle or Celluvisc vehicle twice daily for 2 weeks. Cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and post-mortem immunohistochemistry for alpha smooth muscle actin (αSMA). Impacts of the wound and treatments on optical quality were assessed using wavefront sensing and optical coherence tomography at 2, 4, 8 and 12 weeks post-operatively. In parallel, cat corneal fibroblasts were cultured to assess the effects of Rosiglitazone on TGFβ-induced αSMA expression. Topical application of Rosiglitazone to cat eyes after injury decreased αSMA expression and haze, as well as the induction of lower-order and residual, higher-order wavefront aberrations compared to vehicle-treated eyes. Rosiglitazone also inhibited TGFβ-induced αSMA expression in cultured corneal fibroblasts. In conclusion, Rosiglitazone effectively controlled corneal fibrosis in vivo and in vitro, while restoring corneal thickness and optics. Its topical application may represent an effective, new avenue for the prevention of corneal scarring with distinct advantages for pathologically thin corneas.

Transforming growth factor-β regulates the expression of anosmin (KAL-1) in human retinal pigment epithelial cells

In a microarray analysis of human retinal pigment epithelial cells (HRPE) treated with TGF-β, in addition to the alteration of a number of known Extracellular matrix (ECM)-related genes regulated by TGF-β, we found a significant increase in the expression of Kallmann Syndrome (KAL)-1 gene, that codes for the protein anosmin-1. Enhanced expression of KAL-1 by TGF-β was validated by real-time PCR analysis. In in vitro experiments, TGF-β receptor inhibitor abolished TGF-β-induced expression of KAL-1. Immunofluorescence staining showed increased presence of anosmin-1 in TGF-β treated HRPE cells, with distinct localization at the intercellular junctions. Treatment of HRPE cells with TGF-β enhanced secretion of anosmin-1 and the release of anosmin-1 was further augmented by heparin sulfate. Enhanced secretion of anosmin-1 in the presence of TGF-β and heparin was also observed in other ocular cells such as corneal epithelial and corneal fibroblast cultures. The role of anosmin-1, a protein with adhesion functions, in retinal structure, function and pathology has not been known and remains to be investigated.

Cytokine and Chemokine Secretion Induced by Poly(I:C) through NF-κB and Phosphoinositide 3-Kinase Signaling Pathways in Human Corneal Fibroblasts

Purpose/Aim: Viral infection of the cornea can result in inflammation and scarring and eventually lead to blindness. Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, induces the secretion of cytokines and chemokines from cultured corneal fibroblasts. We have now investigated the role of nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K) signaling pathways in poly(I:C)-induced cytokine and chemokine secretion from corneal fibroblasts.Materials and Methods: Human corneal fibroblasts were cultured with poly(I:C) in the absence or presence of IKK-2 inhibitor or LY294002, which are inhibitors of NF-κB and PI3K signaling, respectively. The release of the pro-inflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, IP-10, and RANTES from the cells was measured with an enzyme-linked immunosorbent assay.Results: Poly(I:C) induced the secretion of IL-6, IL-8, IP-10, and RANTES from corneal fibroblasts. Whereas the poly(I:C)-induced secretion of IL-6, IP-10, and RANTES was inhibited by both IKK-2 inhibitor and LY294002, that of IL-8 was blocked only by IKK-2 inhibitor.Conclusions: The poly(I:C)-induced secretion of IL-6, IP-10, and RANTES from human corneal fibroblasts is mediated by both NF-κB and PI3K signaling pathways, whereas that of IL-8 is mediated by the NF-κB pathway. These signaling pathways thus likely contribute to local inflammation in the corneal stroma induced by viral infection.

Up-regulation of matrix metalloproteinase-1 and interleukin-6 expression in cocultures of corneal fibroblasts and neural cells

The cornea is the most sensitive tissue in the human body, with the dense nerve endings of the cornea being derived from the first division of the ophthalmic nerve. The existence of such organized nerve fibers reflects the role of neural regulation in corneal homeostasis, with the proper distribution and function of these nerve fibers thus being required for maintenance of a healthy cornea. We recently established an in vitro model, based on the coculture of human corneal epithelial cells and fibroblasts on opposite sides of a collagen vitrigel membrane. We have now examined the role of neural cells in corneal homeostasis with the use of a similar coculture system. Reverse transcription–polymerase chain reaction and immunoblot analyses showed that the presence of neural cells (differentiated PC12 cells) increased the expression of matrix metalloproteinase-1 (MMP-1) in human corneal fibroblasts at both the mRNA and protein levels. The expression of MMP-2 and MMP-9 in corneal fibroblasts was not affected by PC12 cells. Furthermore, a multiplex assay showed that, among various cytokines assayed, only the release of interleukin-6 in cocultures of the two cell types was markedly greater than that in cultures of corneal fibroblasts alone. These results thus suggest that factors released from neural cells may play an important role in regulation of the function of corneal fibroblasts and thereby contribute to the maintenance of corneal structure and function.