FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro.

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-ß1 and -ß3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-ß1, TGF-ß3, or TGF-ß1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-ß1 or TGF-ß3 impart distinct effects on genes involved in wound healing and fibrosis-ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-ß1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-ß3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-ß1 + FAKi attenuated TGF-ß1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-ß1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Initiation of fibrosis in the integrin Αvß6 knockout mice.

We previously demonstrated that ß6 knockout mice showed impaired wound repair in corneal debridement and keratectomy wounds. In the current investigation, we continued our examination of integrin αvß6 in order to determine if it was required for the initiation of wound healing in a corneal wound model that normally heals in a fibrotic manner. A full-thickness corneal incision was made in C57BL/6 J wild type (WT) and C57BL/6-Itgb6 KO (ß6-/-) mice. The mice were observed at 3, 7, 14, and 28 days post-incision. The morphology of corneal restoration was observed in tissue sections stained with hemotoxilin and eosin (H&E). In addition, indirect-immunofluorescence (IF) was performed on sections and/or whole mounts to evaluate the immunolocalization of α-smooth muscle actin (SMA) and thrombospondin-1 (TSP-1). H&E staining revealed that the corneas in ß6-/- mice healed slower than those in WT mice, with an obvious delay in the restoration of the stromal matrix and epithelium. In sections at 3 and 7 days, SMA and TSP-1 were greatly reduced in the ß6-/- mice as compared to WT, but peaked at 28 days after incision. Whole mount SMA IF results were consistent with those from sections. Therefore, the initiation of fibrosis was inhibited by the lack of αvß6; however, there appeared to be an alternate mechanism that initiated fibrosis 7-14 days later. Localization of TSP-1 correlated with expression of SMA whether wound healing was delayed or initiated immediately after wounding.

Inhibition of Human Corneal Myofibroblast Formation.

Purpose: Transforming growth factor-beta (TGF-ß) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-ß/Smad- or TGF-ß/p38-signaling pathway in human corneal fibroblasts (HCF). Methods: In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-ß/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-ß/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results: We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions: HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.

Development of wound healing models to study TGFß3's effect on SMA.

The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFß3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFß1 or ß3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy.

PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.

Purpose: The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor ß3 (TGF-ß3) and TGF-ß1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-ß3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-ß1 or -ß3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-ß3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-ß1, while in HCF-P, both TGF-ß1 and -ß3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-ß3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-ß3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-ß3 treatment. Understanding the role of PDGFRα in TGF-ß3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-ß1 and TGF-ß3 in corneal wound healing.

Connective tissue growth factor is not necessary for haze formation in excimer laser wounded mouse corneas.

We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy (PTK) and the corneas were allowed to heal. The extent of scaring was observed in non-induced mice, heterozygotes, and full homozygous knockout mice and quantified by macrophotography. The eyes from these mice were collected after euthanization for re-genotyping to control for possible Cre-mosaicism. Primary corneal fibroblasts from CTGF knockout corneas were established in a gel plug assay. The plug was removed, simulating an injury, and the rate of hole closure and the capacity for these cells to form light reflecting cells in response to CTGF and platelet-derived growth factor B (PDGF-B) were tested and compared to wild-type cells. We found that independent of genotype, each group of mice was still capable of forming light reflecting haze in the cornea after laser ablation (p = 0.40). Results from the gel plug closure rate in primary cell cultures of knockout cells were not statistically different from serum starved wild-type cells, independent of treatment. Compared to the serum starved wild-type cells, stimulation with PDGF-BB significantly increased the KO cell culture's light reflection (p = 0.03). Most interestingly, both reflective cultures were positive for α-SMA, but the cellular morphology and levels of α-SMA were distinct and not in proportion to the light reflection seen. This new work demonstrates that corneas without CTGF can still form sub-epithelial haze, and that the light reflecting phenotype can be reproduced in culture. These data support the possibilities of growth factor redundancy and that multiple pro-haze pathways exist.

Cornea As a Model for Testing CTGF-Based Antiscarring Drugs.

Scarring remains a serious complication of the wound healing process that can lead to the formation of excessive fibrous connective tissue in an organ or tissue leading to pain and loss of function. This process is mainly regulated by Transforming growth factor ß1 (TGF-ß1), which binds to receptors and induces its downstream mediator, Connective tissue growth factor (CTGF). The number of drugs targeting CTGF for treating scars has been on the rise in the past few years. The purpose of this article is to suggest the possibility of using cornea as a model for testing anti-CTGF therapies for scarring.

Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells.

AIM: To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling. METHODS: The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-ß1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid ß-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor ß (PDGFRß) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRß and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. RESULTS: In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid ß-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls (P < 0.01). Cells containing the cystine knot of BMP-4 expressed the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-ß1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of ß-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRß receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRß and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2. CONCLUSION: CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-ß. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

Assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas.

The effects of a triple combination of siRNAs targeting key scarring genes were assessed using an ex vivo organ culture model of excimer ablated rabbit corneas. The central 6 mm diameter region of fresh rabbit globes was ablated to a depth of 155 microns with an excimer laser. Corneas were excised, cultured at the air-liquid interface in defined culture medium supplemented with transforming growth factor beta 1 (TGFB1), and treated with either 1% prednisolone acetate or with 22.5 µM cationic nanoparticles complexed with a triple combination of siRNAs (NP-siRNA) targeting TGFB1, TGFB Receptor (TGFBR2) and connective tissue growth factor (CTGF). Scar formation was measured using image analysis of digital images and levels of smooth muscle actin (SMA) were assessed in ablated region of corneas using qRT-PCR and immunostaining. Ex vivo cultured corneas developed intense haze-like scar in the wounded areas and levels of mRNAs for pro-fibrotic genes were significantly elevated 3-8 fold in wounded tissue compared to unablated corneas. Treatment with NP-siRNA or steroid significantly reduced quantitative haze levels by 55% and 68%, respectively, and reduced SMA mRNA and immunohistostaining. This ex vivo corneal culture system reproduced key molecular patterns of corneal scarring and haze formation generated in rabbits. Treatment with NP-siRNAs targeting key scarring genes or an anti-inflammatory steroid reduced corneal haze and SMA mRNA and protein.

Conditional knockout of CTGF affects corneal wound healing.

PURPOSE: This study aimed to elucidate the role of connective tissue growth factor (CTGF) in healthy eyes and wounded corneas of mice and rabbits. Conditional knockout mice were used to determine the role of CTGF in corneal healing. METHODS: CTGF expression was determined using transgenic mice carrying CTGF promoter driven-eGFP, quantitative RT-PCR, and immunofluorescent staining. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were used to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining. RESULTS: Connective tissue growth factor expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate upregulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P < 0.01) in the cornea re-epithelialization rate in knockout mice compared with wild-type mice. CONCLUSIONS: Connective tissue growth factor is expressed in the naïve cornea, lens, iris, and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.

Triple combination of siRNAs targeting TGFß1, TGFßR2, and CTGF enhances reduction of collagen I and smooth muscle actin in corneal fibroblasts.

PURPOSE: Transforming growth factor ß1 (TGFß1), TGFß receptor (TGFßR2), 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: Simultaneous targeting of TGFß1, TGFßR2, 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.

MicroRNA signature in wound healing following excimer laser ablation: role of miR-133b on TGFß1, CTGF, SMA, and COL1A1 expression levels in rabbit corneal fibroblasts.

PURPOSE: The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes. METHODS: Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3' untranslated region (UTR) of transforming growth factor ß1 (TGFß1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration. RESULTS: At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at -14.33. Pre-miR-133b targeted the 3' UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor ß1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFß1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF. CONCLUSIONS: Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.