PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2-STAT3 signaling pathway

Purpose: Platelet-derived growth factor (PDGF) is associated with corneal fibroblast migration and proliferation and plays an important role in corneal wound healing. However, the intracellular mechanisms of PDGF-mediated functions in corneal fibroblasts are poorly understood. We tested the hypothesis that PDGF functional activities in the cornea involve the Janus kinase-2/signal transducers and activators of transcription-3 (JAK2-STAT3) signaling pathway and whether PDGF induces the expression of suppressors of cytokine signaling 3 (SOCS3), belonging to the novel family of feedback regulators of cytokine and growth factor activities. Methods: Human corneal fibroblast (HSF) cultures were used as an in vitro model for functional analysis. Real-time polymerase chain reactions were performed to quantify gene expression. Immunoprecipitation and immunoblotting techniques were used to measure protein expression. Cell growth, migration, and ELISA assays were used for functional validation. Results: Low endogenous levels of STAT3 and SOCS3 mRNA and protein expression were noted in HSFs. PDGF treatment of HSF significantly induced SOCS3 mRNA (3.0–4.5 fold) and protein (1.5–2.5 fold) expression in a timedependent manner. Similarly, PDGF treatment of HSF significantly increased STAT3 protein expression at two tested time points (2.5–2.96 fold). Cultures exposed to vehicle (control) did not show any change in SOCS3 and STAT3 mRNA or protein expression. An addition of AG-490, a selective inhibitor of the JAK2-STAT3 pathway, significantly inhibited PDGF-mediated STAT3 induction and cell growth and migration in HSF. We also observed that PDGF induced interleukin-8 (IL8) chemokine secretion (2 fold) and AG-490 inhibited IL8 secretion. Conclusions: Our data showed that PDGF induced STAT3, SOCS3, and IL8 chemokine secretion in human corneal fibroblasts. Further, PDGF-induced cell growth, migration, and IL8 secretion in corneal fibroblast involve the JAK2- STAT3 signaling pathway

Trichostatin A Inhibits Corneal Haze \u3cem\u3ein vitro\u3c/em\u3e and \u3cem\u3ein vivo\u3c/em\u3e

PURPOSE. Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to suppress TGF- –induced fibrogenesis in many nonocular tissues. The authors evaluated TSA cytotoxicity and its antifibrogenic activity on TGF- –driven fibrosis in the cornea with the use of in vitro and in vivo models. METHODS. Human corneal fibroblasts (HSFs) were used for in vitro studies, and New Zealand White rabbits were used for in vivo studies. Haze in the rabbit cornea was produced with photorefractive keratectomy (PRK) using excimer laser. Trypan blue exclusion and MTT assays evaluated TSA cytotoxicity to the cornea. Density of haze in the rabbit eye was graded with slit lamp biomicroscopy. Real-time PCR, immunoblotting, or immunocytochemistry was used to measure -smooth muscle actin (SMA), fibronectin, and collagen type IV mRNA or protein levels. TUNEL assay was used to detect cell death. RESULTS. TSA concentrations of 250 nM or less were noncytotoxic and did not alter normal HSF morphology or proliferation. TGF- 1 treatment of HSF significantly increased mRNA and protein levels of SMA (9-fold), fibronectin (2.5-fold), and collagen type IV (2-fold). TSA treatment showed 60% to 75% decreases in TGF- 1–induced SMA and fibronectin mRNA levels and 1.5- to 3.0-fold decreases in protein levels but had no effect on collagen type IV mRNA or protein levels in vitro. Two-minute topical treatment of TSA on rabbit corneas subjected to 9 D PRK significantly decreased corneal haze in vivo. CONCLUSIONS. TSA inhibits TGF- 1–induced accumulation of extracellular matrix and myofibroblast formation in the human cornea in vitro and markedly decreases haze in rabbit cornea in vivo

Stromal Haze, Myofibroblasts, and Surface Irregularity after PRK

The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups[.] Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin α4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2 ± 0.2 cells/400× field) and group V (1.8 ± 0.4), with significantly more in groups II (3.5 ± 1.8), III (6.8 ± 1.6), VII (7.9 ± 3.8), IV (12.4 ± 4.2) and VI (14.6 ± 5.1). The screened groups were significantly different from each other (p In conclusion, these results demonstrated a relationship between the level of corneal haze formation after PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane regeneration and increased epithelium-derived TGFβ signalling to the stroma that increases myofibroblast generation. Late apoptosis appears to have a role in the disappearance of myofibroblasts and haze over time

Effect of Prophylactic and Therapeutic Mitomycin C on Corneal Apoptosis, Cellular Proliferation, Haze, and Long-Term Keratocyte Density in Rabbits

PURPOSE—To determine the mechanism through which topical mitomycin C prevents and treats corneal haze after photorefractive keratectomy (PRK) and to examine the effects of dosage and duration of exposure. METHODS—In 224 New Zealand rabbits, −9.0 diopter PRK with mitomycin C or balanced salt solution was performed. Haze level was graded at the slit-lamp. Rabbits were sacrificed at 4 hours, 24 hours, 4 weeks, or 6 months after surgery and immunohistochemistry was performed with TUNEL assay, Ki67 and α-SMA. RESULTS—TUNEL-positive apoptotic cells marginally increased in all mitomycin C groups whereas Ki67-positive mitotic cells decreased significantly following mitomycin C application. A greater decrease in myofibroblasts was noted with prophylactic mitomycin C treatment than therapeutic mitomycin C treatment. There was, however, an anterior stromal acellular zone (approximately 20% of the total stroma) in eyes treated with mitomycin C, which persisted to the maximum follow-up of 6 months. CONCLUSIONS—Mitomycin C treatment induces apoptosis of keratocytes and myofibroblasts, but the predominate effect in inhibiting or treating haze appears to be at the level of blocked replication of keratocytes or other progenitor cells of myofibroblasts. Treatment with 0.002% mitomycin C for 12 seconds to 1 minute appears to be just as effective as higher concentrations for longer duration in the rabbit model. However, a persistent decrease in keratocyte density in the anterior stroma could be a warning sign for future complications and treatment should be reserved for patients with significant risk of developing haze after PRK

Corneal Injury Is Associated With Stromal and Vascular Alterations Within Cranial Dura Mater

The cornea and cranial dura mater share sensory innervation. This link raises the possibility that pathological impulses mediated by corneal injury may be transmitted to the cranial dura, trigger dural perivascular/connective tissue nociceptor responses, and induce vascular and stromal alterations affecting dura mater blood and lymphatic vessel functionality. In this study, using a mouse model, we demonstrate for the first time that two weeks after the initial insult, alkaline injury to the cornea leads to remote pathological changes within the coronal suture area of the dura mater. Specifically, we detected significant pro-fibrotic changes in the dural stroma, as well as vascular remodeling characterized by alterations in vascular smooth muscle cell (VSMC) morphology, reduced blood vessel VSMC coverage, endothelial cell expression of the fibroblast specific protein 1, and significant increase in the number of podoplanin-positive lymphatic sprouts. Intriguingly, the deficiency of a major extracellular matrix component, small leucine-rich proteoglycan decorin, modifies both the direction and the extent of these changes. As the dura mater is the most important route for the brain metabolic clearance, these results are of clinical relevance and provide a much-needed link explaining the association between ophthalmic conditions and the development of neurodegenerative diseases

Efficacious and Safe Tissue-Selective Controlled Gene Therapy Approaches for the Cornea

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients

PDGF-mediated STAT3 induction in human corneal fibroblasts involves JAK2-STAT3 pathway

Human corneal fibroblasts were induced with the mixture of PDGF (AA, AB, and BB) at a final concentration of 20 ng/ml for 1 h or 8 h in the presence or absence of 100 μM AG-490. Protein levels were measured by immunoblotting using a polyclonal STAT3 antibody. PDGF treatment significantly increased STAT3 protein expression by 2.5 fold (1 h) to 2.96 fold (8 h). β-Actin immunoblot shows the equal loading in each lane.<p><b>Copyright information:</b></p><p>Taken from "PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2-STAT3 signaling pathway"</p><p></p><p>Molecular Vision 2008;14():1020-1027.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408775.</p><p></p

PDGF-mediated SOCS3 induction in human corneal fibroblasts involves JAK2-STAT3 pathway

Human corneal fibroblasts were exposed to a mixture of PDGF (AA, AB, and BB) at a final concentration of 20 ng/ml with or without JAK2-STAT3 inhibitor, AG-490 (final concentration of 100 μM) for 1 h or 8 h. SOCS3 transcript levels were quantified by real-time PCR, and protein levels were determined by immunoprecipitation and immunoblotting using the SOCS3 polyclonal antibody. : The fold change in SOCS3 transcript levels compared to untreated control is shown as mean ±SEM. The PDGF treatment increases SOCS3 mRNA levels by threefold at 1 h and 4.5 fold at 8 h, and AG-490 treatment inhibits PDGF-induced mRNA levels significantly. The asterisk indicates a p<p><b>Copyright information:</b></p><p>Taken from "PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2-STAT3 signaling pathway"</p><p></p><p>Molecular Vision 2008;14():1020-1027.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408775.</p><p></p