mTOR regulates TGF-β2-induced epithelial–mesenchymal transition in cultured human lens epithelial cells

Background

Post-cataract surgery fibrosis in the lens capsule is caused by epithelial to mesenchymal transition (EMT) of the lens epithelium. Mammalian target of rapamycin (mTOR) has been demonstrated to be a key regulator of EMT. The aim of this study was to investigate the role of mTOR in transforming growth factor β₂ (TGF-β₂)-induced EMT in human lens epithelial cells (HLECs).

Methods

Human lens epithelial B-3 (HLEB-3) cells were cultured with 10 ng/ml TGF-β₂ for different periods of time. The expression of E-cadherin, connexin 43, fibronectin and α-smooth muscle actin (α-SMA), and activation of mTOR were determined by Western blots. Cell migration was assessed by wound healing assay. An inhibition test was performed using two kinds of mTOR inhibitors.

Results

E-cadherin and connexin 43 expressions were suppressed, whereas fibronectin and α-SMA expressions were increased in HLEB-3 cells after treatment with TGF-β₂. mTOR was activated during the TGF-β₂-induced EMT in a time-dependent manner. Rapamycin or Ku-0063794 with 100 nM was able to inhibit the phosphorylation of mTOR and impaired EMT induced by TGF-β₂. Cell motility enhanced by TGF-β₂ for 24 h was attenuated by both rapamycin and Ku-0063794.

Conclusions

mTOR is activated during TGF-β₂-induced EMT in HLECs, suggesting that it is involved in the regulation of TGF-β₂-induced EMT and may contribute to the development of posterior capsule opacification.     

Simulation von Korneaepithelverletzungen mittels mechanischer und korrosiver Schädigung

Introduction

The present study describes simulation of corneal epithelial injury and its regeneration using an in-vitro model of immortalized human corneal epithelial cells (HCE-T) growing as monolayer cultures.

Material and methods

The epithelial model was damaged using defined strengths by mechanical injury or partial damage using chemical detergents (SDS and acidified medium) and subsequently the epithelium was further cultivated using serum-containing and serum-free medium supplemented with varying concentrations of calcium pantothenat. After mechanical injury wound healing was evaluated using a photomicroscope over a period of up to 48 h whereas after chemical injury a cell viability assay was used to detect the course of ATP levels in the cell layers as an indicator for the metabolic activity.

Results

Depending on the kind of injury pantothenat showed a regeneration enhancing effect in the concentration range from 0.001% to 0.01%. However, a concentration of 0.1% pantothenat appeared to be regeneration inhibiting. The combination of pantothenat and serum was more beneficial for wound healing than pantothenat alone, whereas serum partly levelled the effect of pantothenat.

Conclusion

The described model allowed simulation of corneal epithelial injury and its regeneration, whereby the influence of the serum content and the kind of injury could be determined.     

KGF, FGFb, VEGF, HGF and TGFβ1 secretion of human keratocytes following photodynamic inactivation (PDI) in vitro

Purpose

With increasing resistance of microorganisms to antibiotics, photodynamic inactivation (PDI) may be a potential treatment alternative for infectious keratitis. Growth factors have the function to regulate proliferation, apoptosis and motility of the cells, and thereby affect wound healing. The purpose of this study was to evaluate the possible impact of PDI on the secretion of keratinocyte growth factor (KGF), fibroblast growth factor beta (FGFb), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1) of human keratocytes, in vitro.

Methods

Primary human keratocytes were isolated by digestion in collagenase A (1.0 mg/ml) from human corneal buttons, and cultured in DMEM/Ham’s F12 medium supplemented with 10 % FCS. Keratocyte cell cultures underwent illumination using red (670 nm) light for 13 min following exposure to 100 nM concentration of the photosensitizer chlorin e6 (Ce6) in culture medium. Five hours and 24 hours after PDI, secretion of KGF, FGFb, VEGF, HGF and TGFβ1 was measured by enzyme-linked immunoabsorbent assay (ELISA).

Results

Compared to untreated controls, FGFb secretion of keratocytes increased (p?<?0.0001) and HGF expression decreased (p?<?0.01) significantly 5 hours after PDI, whereas KGF, VEGF, and TGFβ1 secretion remained unchanged. Twenty-four hours following PDI, KGF secretion decreased (p?<?0.0001) significantly, while FGFb, HGF, VEGF and TGFβ1 concentrations did not differ markedly from untreated controls.

Conclusions

Photodynamic inactivation triggers FGFb and inhibits HGF secretion of keratocytes transiently (5 hours) and inhibits KGF secretion 24 hours following treatment. In the short term, PDI does not have an impact on VEGF and TGFβ1 secretion of keratocytes, in vitro.     

Komplikationen der Vernetzung der Hornhaut

Background

More than 10 years after the clinical introduction of corneal cross-linking (CXL) the indications and contraindications are still not yet defined. Fundamental for such a list is the incidence of complications.

Methods

A PubMed search for complications of corneal crosslinking published up to March 2013 was carried out.

Results

The published complication rates ranged from 1 % to 10?% depending on the stage of keratoconus. Early postoperative complications were transient stromal haze, sterile infiltrates, endothelium decompensation, delayed epithelial healing and infectious keratitis. Stromal opacity can be a delayed postoperative event.

Conclusions

Complications after corneal cross-linking treatment for keratoconus are rare but the management of these complications may need keratoplasty.     

In vitro effects of three blood derivatives on human corneal epithelial cells

Purpose. We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. Methods. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. Results. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. Conclusions. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.     

The Ex Vivo Eye Irritation Test (EVEIT) in evaluation of artificial tears: Purite?-preserved versus unpreserved eye drops

Objective

Preservatives in artificial tears cause controversy. New developments such as the Purite? system have been introduced into the market, with the promise of little damage to the corneal surface. We wanted to give insight into the differences in the effect of preserved and unpreserved artifical tears on rabbit corneas cultured with the Ex Vivo Eye Irritation Test (EVEIT) system.

Materials

We compared the two artifical tears products Hylo Comod? and Optive? being dropped for 72?hours each hour one drop onto the corneal surface.

Methods

Each cornea was mechanically wounded with four epithelial defects on each cornea with a size of 3 to 4.5?mm². With n?=?4 corneas in the Hylo-Comod? and n?=?4 corneas in the Optive? group, we exposed the corneal surfaces to repeated doses of these artificial tears for 3?days. We observed healing of corneal erosions and surface epithelial integrity with sodium-fluoresceine staining under cobalt blue light illumination.

Results

We found nearly complete healing of epithelial defects with both artificial tears. The Hylo-Comod? group healed significantly faster. After 72?hours, the vast majority of epithelial defects were closed. All corneas exposed to Purite? showed superficial stippling, whereas the HyloComod? group did not show any stippling of the cornea; this difference was significant.

Discussion

Epithelial healing and recovery in the EVEIT system is observed in both groups, confirming the concept of artificial tears as a supporting factor of corneal health and healing. The superficial stippling of the corneal epithelium was observed only in the Optive? group. This effect is considered as a marker of dry eye syndrome, and should be prevented by the application of artificial tears. Preservative-free eye drops such as HyloComod? improve healing, and prevent symptoms of dry eye syndrome in the EVEITsystem. Compared to EVEIT results of former experiments with benzalconium chloride-preserved eye drops, Optive? promoted healing of corneal erosions.     

Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model

Purpose

To determine changes in expression of transforming growth factor β-2 (TGF-β2) and basic fibroblast growth factor (bFGF) in scleral desmocytes from anterior and posterior portions of experimentally-induced myopic eyes of guinea pigs.

Methods

Three groups (n?=?10) of 2-week-old guinea pigs were used to develop concave lens-induced myopia (LIM) in one eye via the out-of-focus method for 6, 15, or 30 days respectively, while the other eye in each guinea pig served as the self-control (SC). After myopia induction, lenses were removed, and scleral fibroblasts were cultured and passaged twice. TGF-β2 and bFGF expression levels of scleral desmocytes in LIM and SC groups were compared by immunocytochemistry, quantitative real-time PCR (qRT-PCR) and Western blot analyses.

Results

The TGF-β2 expression of the anterior portion of the sclera in the LIM group was significantly higher at 15 days, and at its highest at 30 days after myopia induction compared with the SC group (P?<?0.05). The TGF-β2 staining of the posterior sclera in the LIM group began to rise significantly at 6 days, peaked at 15 days and remained significantly higher than that of the anterior part, as well as the SC group, even at 30 days after myopia induction (P?<?0.05). BFGF levels in scleral desmocytes from the anterior and posterior regions in the LIM group were both significantly lower than those of the SC group at all time points after myopia induction (P?<?0.05). Furthermore, as the myopia progressed, bFGF expression in the anterior and posterior sclera in the LIM group gradually and statistically significantly decreased compared with the SC group (P?<?0.05); however, no significant differences were observed between the anterior and posterior parts in the LIM group at any time after myopia induction (P?>?0.05). All these results were consistent at the mRNA and protein levels.

Conclusions

During myopia development in lens-induced guinea pigs, the increase in TGF-β2 activity of scleral desmocytes initiated at the posterior pole. Along with the induction time, the TGF-β2 activity in all scleral desmocytes became elevated. By contrast, the bFGF activity showed a general decline in all scleral desmocytes, rather than mainly in the posterior pole. These results imply that expression of TGF-β2 in scleral desmocytes plays a direct role, while that of bFGF exerts an indirect role in myopia development.     

Increased intravitreal angiopoietin-2 levels associated with rhegmatogenous retinal detachment

Purpose

To explore factors related to pathogenesis of rhegmatogenous retinal detachment (RRD) and development of proliferative vitreoretinopathy (PVR), vitreous levels of angiopoietin-1 and ?2 (Ang-1 and ?2), previously undefined in RRD, transforming growth factor-(TGF) β1, vascular endothelial growth factor (VEGF), erythropoietin (EPO) and proteolytic mediators of extracellular matrix remodelling (MMP-2 and ?9) were compared in eyes with RRD and eyes with idiopathic macular hole or pucker.

Methods

Vitreous samples were collected from 117 eyes with RRD (study group) and 40 eyes with macular hole or pucker (control group). Growth factors were measured by ELISA and matrix metalloproteinases (MMPs) by gelatin zymography.

Results

The mean vitreous concentrations of Ang-2, MMP-2, and MMP-9 were higher (all p?<?0.01), whereas concentration of VEGF was lower (p?=?0.01) in eyes with RRD relative to controls. Logistic regression analysis identified Ang-2 concentration as a novel marker of RRD (p?=?0.0001, OR 48.7). Ang-1, EPO, and total TGF-β1 levels were not significantly different between the groups. However, TGF-β1 and MMP-2 were increased in eyes with total RRD compared to those with local RRD (p?≤?0.05). In eyes with PVR, no differences were observed in any studied marker as compared with non-PVR eyes.

Conclusions

Current results reveal Ang-2 as a key factor upregulated in RRD. It may co-operate with fibrosis-associated factors and contribute to vascular complications such as breakdown of blood–eye barrier and PVR development.     

Combined silencing of TGF-β2 and Snail genes inhibit epithelial-mesenchymal transition of retinal pigment epithelial cells under hypoxia

Background

The formation of scar-like fibrous tissue in age-related macular degeneration (AMD) is associated with hypoxia. Under hypoxia, retinal pigment epithelial (RPE) cells can secret more transforming growth factor-β₂ (TGF-β₂), which is determined to induce epithelial-mesenchymal transition (EMT) at certain concentrations. Whether hypoxia can induce EMT by stimulating RPE cell line secrets TGF-β₂ or not remains unknown. To gain a better understanding of the signaling mechanisms of fibrosis in AMD under hypoxic conditions, we investigated EMT in retinal pigment epithelial (RPE) cells and the effect of TGF-β₂ and Snail in this process.

Methods

Human RPE cell line (ARPE-19) was incubated with 5 % O₂ for different periods of time. The expression of N-cadherin, α-smooth muscle actin (α-SMA), TGF-β₂ , and Snail were determined by Western blot and real-time PCR. Cell proliferation was assessed by CCK8 kit. RNA interference was used for multi-gene silencing of TGF-β₂ and Snail genes.

Results

N-cadherin was decreased and mesenchymal cell marker α-SMA was increased after the ARPE-19 cell line was incubated with 5 % O₂. Meanwhile, the proliferation capability of the cell line was increased. TGF-β₂ and Snail expression were increased in a time-dependent manner under hypoxia. After multi-silencing TGF-β₂ and Snail genes, N-cadherin was increased and α-SMA was reduced. Meanwhile, the proliferation of the cell line was suppressed.

Conclusions

Under hypoxic conditions, RPE cells undergo EMT. Endogenic TGF-β₂ and Snail are involved in this process. Furthermore, knockdown of both TGF-β₂ and Snail inhibited EMT to a greater extent than knockdown of either gene individually.

     

Vitamins Inhibit Oxidant-Induced Apoptosis of Corneal Endothelial Cells

Purpose

To determine the effects of vitamins A, C, and E supplementation on lipid peroxidation and apoptosis in corneal endothelial cells.

Methods

Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. The concentration of antioxidative vitamins (ascorbic acid, tocopherol, and retinoic acid) in the cells and supernatant was determined using reversed-phase high-performance liquid chromatography. Apoptosis was assessed by quantification of caspase-3-like activity, using annexin-V/propidium iodide stains for flow cytometry. Lipid peroxidation was assessed using the malondialdehyde method. Supplementation of antioxidative vitamins was tested in the setting of apoptosis.

Results

Increasing levels of free iron led to a rapid loss of antioxidative vitamins in the supernatant and corneal endothelial cells. This was correlated with rising levels of malondialdehyde and increased apoptosis. Supplementation with ascorbic acid or α-tocopherol alone was not sufficient to prevent lipid peroxidation in the cells, whereas a combination of vitamins C and E was able to do so. In contrast, supplementation with vitamin A alone significantly reduced oxidative stress and apoptosis.

Conclusions

We present an in vitro model to test the direct influence of vitamin supplementation on corneal endothelial cells with regard to lipid peroxidation and apoptosis. We show that supplementation with antioxidative vitamins of corneal endothelial cells significantly prevents the generation of free-radical injury, lipid peroxidation, and consequent apoptosis. Jpn J Ophthalmol 2005;49:355–362 © Japanese Ophthalmological Society 2005     

A Novel H572R Mutation in the Transforming Growth Factor-β-Induced Gene in a Thai Family with Lattice Corneal Dystrophy Type I

Purpose

To describe a large Thai family with lattice corneal dystrophy (LCD) type I and to determine whether this LCD is associated with mutations within the transforming growth factor-β-induced (TGFBI) gene.

Methods

A six-generation family with LCD type I was identified and diagnosed on the basis of clinical and/or histopathologic evaluation. Visual acuity testing and slit-lamp biomicroscopic evaluation were carried out and corneal photography was documented. All 17 exons and flanking intron sequences of the TGFBI gene were sequenced.

Results

Thirty-three participants demonstrated LCD in both eyes, most of which was symmetrical. Age at onset of decreased vision was the mid- to late twenties. Visual acuity varied from 6/6 to no light perception. Two patients, 74 and 42 years of age, demonstrated a thick yellowish plaque covering the corneal surfaces. DNA sequencing revealed a heterozygous mutation in exon 13 (A1762G), changing histidine to arginine at codon 572 (H572R). Ten of 42 clinically unaffected family members, all under 25 years of age, exhibited the same mutation.

Conclusions

This is the first report of a molecular analysis of LCD type I in Thai patients. The novel mutation identified is associated with distinct phenotypes and later onset of the disease compared with the more common R124C mutation.?Jpn J Ophthalmol 2006;50:403–408 © Japanese Ophthalmological Society 2006     

In vitro extraction of intra-corneal iron using reverse iontophoresis and vitamin C

Purpose

The aim of this study was to optimize reverse iontophoretic (RI) extraction of ferric/ferrous ions from the cornea.

Methods

Group I consisted of the right eye corneas from 20 normal rabbits. Corneal blood staining was induced in 60 right eyes. The corneal depths from the endothelium to the epithelium layers were divided into three groups by slit-lamp examination: Group II, one-third corneal thickness; Group III, one-half corneal thickness; Group IV, full corneal thickness. RI was performed using vertical diffusion cells. The lower chamber was loaded with glutathione bicarbonate Ringer’s buffer (GBR; pH 7.0) or vitamin C (12.5 mg/mL) and GBR (pH 7.0), while the upper chamber was filled with 1 mL GBR. Progress of corneal blood staining removal was evaluated.

Results

Application of 1.5 mA to the cornea increased flux by 1.72- and 2.19-fold in Groups III and IV, respectively, but not in Groups I or II, compared to the control. When vitamin C was included, we observed significant flux increases in the controls (1.5-, 2.06-, 2.60-, and 4.59-fold) for Groups I, II, III, and IV, and under RI conditions for Groups III and IV. Following RI, the corneal endothelia appeared similar to corneas from untreated control rabbits, while Draize scores were zero.

Conclusions

These results suggested that extracellular ferric/ferrous ions could be extracted from the cornea in vitro by RI, and that vitamin C reduced Fe³⁺ to Fe²⁺ in the cornea and altered its permselectivity, thus increasing the RI contribution to iron extraction.     

Effects of all-trans retinoic acid nanoparticles on corneal epithelial wound healing

Background

We have developed inorganically-coated all-trans retinoic acid (atRA) nanoparticles, nano-sized egg-like particles of atRA (NANOEGG®-atRA). The purpose of this study was to determine the effects of NANOEGG®-atRA on corneal wound healing in vivo and in vitro.

Methods

A rabbit corneal epithelial wound healing model was exposed to different concentrations of NANOEGG®-atRA. Wound healing was serially quantified as the ratio of fluorescein-stained area at the selected times to that at baseline. After wound closure, the barrier function of the cornea was determined using low concentrations of tropicamide. At the completion of the experiments, the corneal epithelium was histologically examined. For the in vitro studies, linear scratch wounds were made on cultured SV40-immortalized human corneal epithelial cells (HCE-T). Then, the cells were exposed to different concentrations of NANOEGG®-atRA, and wound healing was determined by the degree of closure of the scratch wound. In addition, the effects of NANOEGG®-atRA on the proliferation of HCE-T cells were determined by WST-8 assays.

Results

Exposure to NANOEGG®-atRA decreased the injured area 24 hrs after the ablation. The maximum effect of NANOEGG®-atRA was observed at a concentration of 33 mM. Histologically, no abnormal or differentiated corneal epithelial cells were observed in the histological sections treated with NANOEGG®-atRA. The tropicamide-induced pupillary dilation was significantly slowed in the eyes treated with NANOEGG®-atRA. NANOEGG®-atRA at concentrations of 3.3 and 33 nM induced earlier wound closure in vitro, but did not induce proliferation of HCE-T cells.

Conclusion

NANOEGG®-atRA promotes wound healing and should be considered for the treatment of wounds of the corneal epithelium.

     

Vernetzung einer humanen künstlichen Hornhaut durch Induktion von Tissue-Transglutaminasen

Purpose

In recent years many three-dimensional cornea models have been developed. However, they show poor collagen stability in the stroma. Transglutaminases (Tgases) are calcium-dependent proteins which play an important role in cross-linking of the corneal stroma. The purpose of this study was to find out whether it is possible to induce in vitro cross-linking of the stroma in an artificial hemicornea model with the help of Tgases.

Materials and methods

For the construction of the hemicornea, human SV40 adenovector corneal epithelial cells (HCE) and human SV40 adenovector corneal keratocytes (HCK) were cultivated. Confluent HCK cells were treated for 24?h with transforming growth factor beta (TGFb) 1, 2 and 3 at different concentrations as well as with other growth factors and the treated cells were compared to untreated cultivated cells. The quantification of the expression of the Tgases by HCKs was examined with the use of real time PCR, Western blot imaging and immunochemistry.

Results

All concentrations of TGFbs used resulted in a significant increase of Tgase-mRNA, Tgase protein level and Tgase activity. The Tgases remained unaffected after treatment with other growth factors in comparison to untreated control cells. Treatment of the hemicornea with TGFb2 showed a very strong contraction and haze in comparison to the untreated hemicornea.

Conclusion

It has been shown for the first time that TGFb induces a strong expression of Tgases in HCK cells. This effect caused an undesired contraction and haze of the human hemicornea model. Further research is necessary in order to find out whether the induction of Tgases in the HCK cells can be regulated without losing stability of the constructed hemicornea.     

Exclusion of Transforming Growth Factor-b1 as a Candidate Gene for Myopia in the Japanese

Purpose

To determine whether single-nucleotide polymorphisms (SNPs) within the transforming growth factor-β1 (TGF-β1) gene are associated with high myopia in Japanese. Previous studies have indicated that the gene expression products, regulators of the TGF-β1 gene, are involved in high myopia.

Methods

Genomic DNA samples were obtained from 330 Japanese patients with high myopia and 330 Japanese controls without high myopia who were chosen at random. SNPs were genotyped by the TaqMan system, using primer extension and polymerase chain reaction amplification.

Results

Ten SNPs were identified in the high-myopia patients and controls, with four of the ten SNPs having nonsynonymous changes. However, no statistical differences in the SNPs were detected between the high-myopia cases and the controls.

Conclusions

Sequence variants of the TGF-β1 gene were not associated significantly with high myopia, and further studies are needed to identify which genes are responsible for high myopia.?Jpn J Ophthalmol 2007;51:96–99 © Japanese Ophthalmological Society 2007

     

Expression von Matrixmetalloproteinase-19 in der humanen Kornea

Background

At present there are no data in the literature on the expression of matrix metalloprotein-19 in the human cornea. The aim of this study was to analyze the expression of matrix metalloproteinase-19 in the human cornea and to investigate its potential role in corneal wound healing using a MMP-19 knock-out mouse model.

Methods

A method with Western blotting and immunohistological staining for MMP-19 was performed using paraffin embedded human corneas. Excimer laser keratectomy was performed in wild type (wt) and MMP-19 knock-out (ko) mice and the rate of re-epithelialization was analyzed after 8 h and 18 h.

Results

MMP-19 was strongly expressed in the human corneal epithelium mainly in the basal cell layer. MMP-19 was not expressed in the corneal stroma. In the mouse model the size of the corneal lesion after 8 h was 83% (wt) and 89.9% (ko) of the initial area (p=0.09). After 18 h the lesion was 17% (wt) and 13.3% (ko) of the initial area (p=0.01). Laminin-5 was expressed in the migrating epithelial cells with no differences between wild type and knock-out mouse.

Conclusion

MMP-19 showed a strong expression in the basal cells of the human corneal epithelium. Corneal re-epithelialization was slightly faster in the MMP-19 knock-out mouse. No differences in the expression of laminin-5 could be detected.     

The efficacy of autologous serum eye drops for severe dry eye syndrome: a randomized double-blind crossover study

Background

To evaluate the efficacy of autologous serum (AS) eye drops for the symptomatic relief of severe dry eye syndrome (DES), as compared to conventional preservative-free artificial tears (PFAT).

Methods

This prospective double-blind randomized crossover study used the Ocular Surface Disease Index (OSDI), tear film break-up time (TBUT), Schirmer’s Test, and OXFORD Scale at baseline and after each of two 1-month treatment periods to measure the effect of 20 % diluted AS eye drops vs. PFAT in 20 consecutive severe DES patients that were refractory to conventional treatment.

Results

The study included 20 (18 female and two male) severe DES patients (40 eyes). Significantly higher TBUT (P?<?0.001, Wilcoxon signed-rank test) and a greater decrease in OSDI score (55.18 % decrease in the AS treatment group vs. 19.50 % decrease in the PFAT treatment group) (P?<?0.001, Student’s paired samples t-test) were observed in the AS treatment group after 1 month of treatment. There wasn’t a significant difference in Schirmer’s test and OXFORD conjunctival and corneal vital dying grading scores between the two treatment groups after 1 month of treatment (P?>?0.05 [Mann–Whitney U test]).

Conclusions

AS eye drops were more effective than conventional eye drops for improving tear film stability and subjective comfort in patients with severe DES.     

Osmoprotective effects of supplemental epidermal growth factor in an ex vivo multilayered human conjunctival model under hyperosmotic stress

Background

To analyze the effects of supplemental epidermal growth factor (EGF) and the roles of inflammatory cytokines (interleukin [IL]-6) in an ex vivo dry-eye model under hyperosmotic stress using a multilayered culture of human conjunctival epithelial cells (HCECs).

Methods

Multilayered cultures of HCECs were exposed to hyperosmotic stress (400 mOsm/L) for 24 h in addition to 0.5 ng/mL EGF (low-EGF group) or 25 ng/mL EGF (high-EGF group). Apoptosis was analyzed using the TUNEL assay. Cell proliferation was measured using the [3H]-thymidine incorporation assay. The expression of IL-6, EGF, EGF receptor (EGFR), and phosphorylated extracellular signal-regulated kinase (p-ERK) was measured by western blot analysis. The secretion of IL-6 was measured using ELISA. Western blot analysis was also performed using antibodies against cleaved caspase-3.

Results

The percentage of apoptotic cells was lower in the high-EGF group (6.7 %) than in the low-EGF group (10.3 %). The high-EGF group demonstrated increased proliferation (323.7 counts/min in the low-EGF group vs 649.1 counts/min in the high-EGF group). EGF induced higher phosphor-EGFR expression and upregulated p-ERK in HCECs. In addition, EGF significantly decreased the secretion of IL-6 and cleaved caspase-3 in HCECs.

Conclusions

The level of IL-6 was increased in the ex vivo HCEC dry-eye model that was under hyperosmotic stress. Supplemental EGF reduces the level of IL-6, decreases apoptosis, and increases proliferation. These findings indicate that EGF has potential as a therapeutic agent for the treatment of dry eyes.     

Topische antiangiogene Therapie an der Hornhaut

Background

The efficacy and safety of novel topical inhibitors of corneal neovascularisation will be discussed.

Methods

A literature review after a PUBMED search and own clinical and experimental results are presented.

Results

The off-label use of Avastin® eye drops and GS101 eye drops against insulin receptor substrate (IRS)-1, which have been tested in phase II trial, both seem to be relatively efficient and safe ways to inhibit progressive corneal neovascularisation. Other VEGF antagonists, such as ranibizumab and pegaptanib eye drops also inhibit corneal neovascularisation.

Conclusions

Avastin® and GS101 eye drops are the first specific angiogenesis inhibitors for topical inhibition of corneal angiogenesis available for clinical use.