Topical retinoic acid induces corneal strengthening by upregulating transglutaminase 2 in murine cornea.

Transglutaminase 2 (TG2) is the most abundant crosslinking enzyme in murine and human cornea, while retinoids are well-known inducers of TG2 expression. This study aims to determine if the retinoic acid supplementation can increase corneal stiffness by crosslinking through upregulating the corneal TG2 expression. The right eyes of C57BL/6 mice were treated with 2 × 10-2M retinol palmitate (VApal) eyedrops or control eyedrops and hold for 30 min, once a day for 28 consecutive days. The WB and qPCR results showed increased expression of TG2 in murine cornea with the prolongation of VApal eyedrop application. After 28 days of VApal eyedrop treatment, the increased TG2 were found catalytically active and distributed in corneal epithelium and stroma as detected by 5-(biotinamido) pentylamine (5-BP) incorporation method and immunofluorescence staining. The transmission electron microscope image revealed that VApal treated cornea manifested with increased collagen density in anterior and middle layer of stroma. The higher elastic module was found among VApal treated cornea by nano-indentation test. In cultured corneal epithelial cells and keratocytes, all-trans retinoid acid (ATRA) treatment increased the content of TG2 in cell lysis and in culture medium. These results indicate that retinoic acid induce the reinforcement of the cornea by TG2 mediated crosslinking via increasing the TG2 expression in corneal epithelium and keratocyte. As TG2 was found to be less in the cornea of keratoconus patients in several RNA-sequencing studies, retinoic acid could serve as a non-invasive prevention method for keratoconus progression.

Keratocytes create stromal spaces to promote corneal neovascularization via MMP13 expression.

PURPOSE: To investigate the exact mechanism by which keratocytes promote corneal neovascularization. METHODS: The expression of matrix metalloproteinase 13 (MMP13), cluster of differentiation 146 (CD146), VEGFa, VEGFc, VEGF receptor (r)2, and VEGFr3 by normal and alkali-burned rat corneas was determined via quantitative (q)RT-PCR and/or Western blot analysis or in situ hybridization. Corneal neovascularization was observed under a slit lamp microscope and evaluated via immunohistochemistry. The cells that expressed MMP13 in the corneas were determined via sequential immunohistochemistry and in situ hybridization. The degradation of type I collagen was evaluated via the detection of hydroxyproline content and Western blot analysis. The effects of VEGFa and VEGFc on MMP13 expression were determined via luciferase reporter assay for the MMP13 promoter and primary keratocyte culture. RESULTS: Matrix metalloproteinase 13 was predominantly expressed by epithelial cells in normal rat corneas, but it was expressed by cells in corneal stromas after alkali burns. The formation of new blood vessels was consistent with MMP13 expression and attenuated by a selective MMP13 inhibitor in alkali-burned corneas. Keratocytes were the major cells expressing MMP13 in corneal stromas after alkali burns. Through MMP13 expression, keratocytes directly degraded collagen type I to create stromal spaces, which were convenient for newly formed blood vessels to grow into. Expression of MMP13 and collagen type I degradation via keratocytes were induced by VEGFc through VEGFr3 and inhibited by antibodies for VEGFc and VEGFr3. CONCLUSIONS: Keratocytes could directly degrade type I collagen and create stromal spaces, promoting corneal neovascularization through VEGFc/VEGFr3-induced MMP13 expression.

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

OBJECTIVE: 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. PROCEDURE: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFß1 to induce myofibroblast formation. The cultures were treated with either 5 µm or 10 µm SAHA for 72 h in the presence of TGFß1. 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. RESULTS: Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFß1-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. CONCLUSIONS: Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFß-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.

Inhibition of interleukin-1ß-induced matrix metalloproteinase expression in human corneal fibroblasts by tranilast.

PURPOSE: Matrix metalloproteinases (MMPs) mediate the degradation of extracellular matrix proteins and are implicated in the pathogenesis of corneal ulceration. Tranilast, a clinically approved antiallergy drug, has been found to exert various anti-inflammatory effects. We examined the effects of this agent on MMP expression in cultured corneal fibroblasts. METHODS: Human corneal fibroblasts were cultured in the absence or presence of interleukin-1ß (IL-1ß) or tranilast. The release of MMPs into culture supernatants was assessed by immunoblot analysis and gelatin zymography, and the cellular abundance of MMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-κB (NF-κB) inhibitor IκB-α was examined by immunoblot analysis. RESULTS: The IL-1ß-induced expression of MMP-1, -2, and -3 in corneal fibroblasts was inhibited by tranilast in a concentration- and time-dependent manner. It was also attenuated by synthetic inhibitors of MAPK or NF-κB signaling pathways. Tranilast inhibited the IL-1ß-induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK) as well as the phosphorylation and degradation of IκB-α. Tranilast did not exhibit cytotoxicity for corneal fibroblasts. CONCLUSIONS: Tranilast inhibits the IL-1ß-induced production of MMP-1, -2, and -3 by human corneal fibroblasts, with this action likely being mediated through suppression of MAPK and NF-κB signaling pathways. Tranilast thus warrants further investigation as a potential treatment for corneal ulceration on the basis of its inhibition of MMP expression in corneal fibroblasts.

A novel mechanism of UV-A and riboflavin-mediated corneal cross-linking through induction of tissue transglutaminases.

PURPOSE: Collagen cross-linking using UV-A irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. The purposes of this study were to examine whether primary human corneal keratocytes (HCKs) are capable of expressing and secreting fibronectin and tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix protein, and to examine whether fibronectin and tTgase are increased after the treatment of HCK cells with UV-A irradiation combined with riboflavin (RFUV-A), thus providing another possible physiological mechanism of the cross-linking pathway. METHODS: Cell cultures established from HCKs were treated with 0.025% riboflavin solution and UV-A (370 nm) irradiance 3 mW/cm2 for 30 minutes. Induction of fibronectin and tTgase was investigated by immunohistochemistry, real-time polymerase chain reaction, and Western blot analysis. Cell viability was quantified by a microscopic live-dead assay. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. RESULTS: Treatment of cultured HCK cells with RFUV-A increased the fibronectin and tTgase messenger RNA and protein levels. This effect was not observed in cells treated with riboflavin or UV-A radiation alone. Incorporation of biotinylated cadaverine was significantly increased when HCK cells were treated with RFUV-A. CONCLUSIONS: The enzymes tTgase and fibronectin are expressed by RFUV-A treatment in cultured HCK cells. This mechanism provides more information about the physiology of corneal cross-linking.

Effect of connective tissue growth factor on protein kinase expression and activity in human corneal fibroblasts.

PURPOSE: To investigate signal transduction pathways for connective tissue growth factor (CTGF) in human corneal fibroblasts (HCF). METHODS: Expression of 75 kinases in cultures of serum-starved (HCF) were investigated using protein kinase screens, and changes in levels of phosphorylation of 31 different phosphoproteins were determined at 0, 5, and 15 minutes after treatment with CTGF. Levels of phosphorylation of three signal transducing phosphoproteins (extracellular regulated kinase 1 [ERK1], extracellular regulated kinase 2 [ERK2] [MAPKs], and signal transducer and activator of transcription 3 [STAT3]) were measured at nine time points after exposure to CTGF using Western immunoblots. Inhibition of Ras, MEK1/2 (MAPKK), and ERK1/2, on CTGF-stimulated fibroblast proliferation and collagen gel contraction was assessed using selective inhibitors farnesylthiosalicylic acid, PD-98059, and SB203580, respectively. RESULTS: Thirty two of the 75 kinases (43%) evaluated by the kinase screen were detected in extracts of quiescent HCF, suggesting these kinases are available to respond acutely to CTGF exposure. Addition of CTGF increased levels of phosphorylation of five phosphoproteins (ERK1 and 2, MEK1/2 [MAPKK], STAT3, and SAPK/JNK), and decreased levels of phosphorylation of 14 phosphoproteins (including protein kinases B and C) after 5 and 15 minutes. Further analysis of ERK1 and 2 and STAT3 phosphorylation showed rapid increases within 1 minute of CTGF exposure that peaked between 5 and 10 minutes then returned to pretreatment levels by 30 minutes. Treatment of HCF with selective inhibitors of Ras, MEK 1/2, and ERK1/2 individually blocked both CTGF induced cell proliferation, and collagen gel contraction. CONCLUSIONS: Results from protein kinase screens and selective kinase inhibitors demonstrate Ras/MEK/ERK/STAT3 pathway is required for CTGF signaling in HCF.

Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per µg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/µg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/µg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

[Cross-linking in an artificial human cornea via induction of tissue transglutaminases]. / 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.

In vitro comparison of the cytotoxic effects of clinically available ophthalmic solutions of fluoroquinolones on human keratocytes.

OBJECTIVE: To compare the cytotoxic effects of preserved versus unpreserved commercially available ophthalmic preparations of fluoroquinolones on human keratocytes in vitro. DESIGN: Experimental study. METHODS: Human keratocytes in vitro were incubated for 15 or 60 minutes with commercially available preparations containing different types of fluoroquinolones, with or without benzalkonium chloride. We examined the morphologic aspects of the cultures by an inverted-phase contrast microscope and the release of cytoplasmic enzyme lactate dehydrogenase into the medium immediately or 24 hours after exposure to drugs. RESULTS: Whereas preparations of ofloxacin, norfloxacin, and gatifloxacin, all containing benzalkonium chloride, and moxifloxacin, which is preservative-free, displayed various degrees of cytotoxicity in our model, the unpreserved monodose preparation of norfloxacin was virtually devoid of harmful effects under our experimental conditions. CONCLUSIONS: Our in vitro results indicated the cytotoxic role of preservatives in commercial preparations of fluoroquinolones and the relative nontoxicity of monodose unpreserved norfloxacin, even when keratocytes were incubated with this formulation for 6 hours.