Tranilast inhibits TGF-β-induced collagen gel contraction mediated by human corneal fibroblasts

AIM： To determine if tranilast affects human corneal fibroblast（HCFs） contraction.METHODS： HCFs cultured in a three-dimensional type I collagen gel were treated with or without transforming growth factor beta（TGF-β） or tranilast. Gel diameter was measured as an indicator for collagen contraction. Immunoblot was performed to evaluate myosin light chain（MLC） and paxillin phosphorylation. Confocal microscopy was employed to examine the focal adhesions and actin stress fiber formation. Immunoblot analysis and gelatin zymography were performed to detect tissue inhibitors of metalloproteinases and matrix metalloproteinases（MMPs） in supernatant.RESULTS： The inhibitory effect of tranilast on HCFsmediated collagen gel contraction induced by TGF-β was dose-dependent. The significant effect of tranilast was started from 100 μmol/L and maximized at 300 μmol/L. The peak effect of 300 μmol/L tranilast also relied on the duration of treatment, which showed statistical significance from day 2. TGF-β-induced paxillin and MLC phosphorylation, stress fiber formation, focal adhesions, and MMP-1, MMP-2, and MMP-3 secretion in HCFs were also inhibited by tranilast.CONCLUSION： Tranilast suppresses the HCFs-cultured collagen gel contraction induced by TGF-β. It attenuates actin stress fibers formation, focal adhesions, and the secretion of MMPs, with these actions likely contributing to the inhibitory effect on HCF contractility. By attenuating the contractility of corneal fibroblasts, tranilast treatment may inhibit corneal scarring.

Alarmins from conjunctival fibroblasts up-regulate matrix metalloproteinases in corneal fibroblasts

AIM:To explore the effects of alarmins produced by necrotic human conjunctival fibroblasts on the release of matrix metalloproteinases(MMPs)by human corneal fibroblasts(HCFs).METHODS:A necrotic cell supernatant(NHCS)was prepared by subjecting human conjunctival fibroblasts to three cycles of freezing and thawing.The amounts of interleukin(IL)-1βand tumor necrosis factor(TNF)-αin NHCS were determined by enzyme-linked immunosorbent assays.HCFs exposed to NHCS or other agents in culture were assayed for the release of MMPs as well as for intracellular signaling by immunoblot analysis.The abundance of MMP m RNAs in HCFs was examined by reverse transcription and real-time polymerase chain reaction analysis.RESULTS:NHCS increased the release of MMP-1 and MMP-3 by HCFs as well as the amounts of the corresponding m RNAs in the cells.NHCS also induced activation of mitogen-activated protein kinase(MAPK)signaling pathways mediated by extracellular signal-regulated kinase(ERK),p38,and c-Jun NH2-terminal kinase(JNK)as well as elicited that of the nuclear factor(NF)-κB signaling pathway by promoting phosphorylation of the endogenous NF-κB inhibitor IκB-α.Inhibitors of MAPK and NF-κB signaling as well as IL-1 and TNF-αreceptor antagonists attenuated the NHCS-induced release of MMP-1 and MMP-3 by HCFs.Furthermore,IL-1βand TNF-αwere both detected in NHCS,and treatment of HCFs with these cytokines induced the release of MMP-1 and MMP-3 in a concentration-dependent manner.CONCLUSION:Alarmins,including IL-1βand TNF-α,produced by necrotic human conjunctival fibroblasts triggered MMP release in HCFs through activation of MAPK and NF-κB signaling.IL-1βand TNF-αare therefore potential therapeutic targets for the amelioration of corneal stromal degradation in severe ocular burns.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane（CAM） and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane （AM）. The lyophilized amniotic membrane（LAM） has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings（ Ahn＇s supporter） were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, foUowed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

AIM:To study the effect of zymosan,a ligand found on the surface of fungi,on gap junctional intercellular communication(GJIC)in cultured human corneal fibroblasts(HCFs).METHODS:Zymosan was added to the medium of cultured HCFs with or without the administration of mitogenactivated protein kinase(MAPK)inhibitors or the inhibitor kappa B kinase 2(IKK2)inhibitor IV.The protein and m RNA levels of connexin 43(Cx43)in HCFs were measured by Western blot,immunofluorescence,and quantitative reverse transcription-polymerase chain reaction(q RT-PCR)analyses.The GJIC activity was tested using a dye-coupling assay.RESULTS:The reduction of Cx43 protein and m RNA levels as well as a significant decrease in GJIC activity were observed in cultured HCFs when zymosan was added into the culture medium.Compared with controls(no zymosan),the protein level of Cx43 was reduced by 45%and 54%in the presence of zymosan at 200 and 600μg/m L,respectively(P<0.05);and it was reduced by 45%,48%,and 75%in the presence of zymosan(600μg/m L)for 24,36,and 48 h,respectively(P<0.05).The m RNA expression of Cx43 was reduced by 98%in the presence of zymosan(P<0.05).The effects of zymosan on Cx43 expression and GJIC activity were attenuated by the administration of PD98059[an extracellular signal-regulated kinase(ERK)signaling inhibitor](P<0.05),c-Jun NH2-terminal kinase(JNK)inhibitor II(P<0.05),and IKK2 inhibitor IV(P<0.05).CONCLUSION:Zymosan inhibits the activity of GJIC in cultured HCFs.This effect is likely regulated via the nuclear factor-κB(NF-κB),MAPK/ERK,and JNK signaling pathways.The inhibitory effects of zymosan on Cx43 expression and GJIC activity in HCFs may induce damage of corneal stroma during corneal fungal infection.

Connective Tissue Growth Factor Expression in Rabbit Corneal Fibroblast and Its Effect on Fibroblast Proliferation In Vitro

The connective tissue growth factor (CTGF) expression in cultured corneal fibroblasts and its effect on corneal fibroblasts proliferation in vitro were examined. Total RNA was extracted from early passaged rabbit corneal fibroblasts by guanidine isothiocyanate one-step method and mRNA was reversely transcripted into complementary DNA (cDNA). Specific CTGF primers were used in the PCR reaction and the products were analyzed by electrophoresis to determine the expression of mRNA for CTGF against DNA marker. House-keeping gene GAPDH was used as control. Different concentrations of CTGF (0.5, 5, 50, 500, 5 000, 50 000 ng/ml) were added into the third passaged corneal fibroblast culture system, and its effect on corneal fibroblast proliferation was measured by MTT method. The results showed that compared with a GAPDH 450 bp band, CTGF RT-PCR product showed a specific 120 bp band as expected. CTGF produced a dose-dependent increase in the proliferation of corneal fibroblasts but it inhibited fibroblast proliferation at higher concentrations (5 000 and 50 000 ng/ml). It was concluded that proliferating corneal fibroblasts produce CTGF and CTGF helps to promote corneal fibroblast proliferation. The results indicated that CTGF might be involved in the corneal wound healing after photorefractive keratectomy in which corneal fibroblasts are activated to proliferate and secrete growth factors that in turn promote corneal fibroblast proliferation.

Inhibitory effects of luteolin on TLR3-mediated inflammation caused by TAK/NF-κB signaling in human corneal flbroblasts

AIM: To study the role of luteolin(LUT) in the expression of toll-like receptors 3(TLR3) ligand poly I:C stimulated inflammatory factors in human corneal fibroblasts(HCFs).METHODS: HCFs cells were cultivated with or without LUT or poly I:C.The expression levels of interleukin(IL)-6, IL-8, monocyte chemotactic protein-1(MCP-1), vascular cell adhesion molecule(VCAM)-1, as well as intercellular adhesion molecule(ICAM)-1 were measured using enzymelinked immunosorbent assay(ELISA), immunoblotting or reverse transcription-quantitative polymerase chain reaction(PCR) analyses.Immunoblotting was used to assess toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β(TRIF), TLR3, transforming growth factor-bactivated kinase 1(TAK1), tumor necrosis factor receptor-associated factor 6(TRAF6), the transcription factor AP-1, as well as transcription factor nuclear factor(NF-κB)–inhibitory protein IκB-α degradation and phosphorylation.Immunofluorescence assays were used to localize the cellular location of the p65 subunit of NF-κB.RESULTS: Corneal fibroblasts exposed to poly I:C demonstrated decreased VCAM-1, ICAM-1, MCP-1, IL-6, and IL-8 expression levels upon exposure to LUT in a time-dependent and concentration-dependent manner.LUT was observed to suppress poly I:C-triggered expression of TLR3, the translocation of NF-κB p65 into cell nuclei, as well as the phosphorylation of TAK, c-Jun, and IκB-α, while no impact on the expression levels of TRIF and TRAF6 were observed.CONCLUSION: LUT suppress the expression of proinflammatory adhesion molecules, chemokines, and cytokines in poly I:C exposed HCFs.These effects are likely mediated through TAK/NF-κB signal attenuation.Therefore, LUT is a candidate molecule that can prevent the TLR3-mediated inflammation response associated with corneal viral infection.

Anti-inflammatory effects of a synthetic peptide derived from pigment epithelium-derived factor on H2O2-induced corneal injury in vitro

Background The common pathological characteristics of corneal injury include inflammatory factors activation, vascular endothelial cells or inflammatory cells infiltration into lesions, corneal edema, corneal neovascularization （CNV）, and scar formation. PEDF-34 is the functional fragment of pigment epithelium-derived factor （PEDF） that has anti-angiogenic and anti-inflammatory properties and contains an N-terminal 34-amino acid peptide. This study was to investigate the anti- inflammatory effects of PEDF-34 on H202-induced corneal injury in vitro. Methods After cultured in H202 （0.1 mmol/L） for 2 hours, human corneal fibroblasts （HCFs） and human umbilical vein endothelial cells （HUVECs） were treated with PEDF-34-nanoparticles （NPs） at different concentrations （0.1, 0.5, 1.0, 2.0 μg/ml） or 2.0 μg/ml controI-NPs for 24 hours. The viable cells were quantified using the MTT assay. Western blotting or ELISA analysis was performed for measuring the human vascular endothelial growth factor （VEGF） and intercellular adhesion molecule-1 （ICAM-1） expression of both HCFs and HUVECs. VEGF and nuclear factor KB （NF-KB） mRNA levels of HCFs were semi-quantified by RT-PCR. Results The survival rates of HCFs or HUVECs stimulated by H202 did not decrease significantly （P 〉0.05） compared to those in the normal conditions. As compared to controI-NP group, PEDF-34-NPs had dose-dependent inhibitive effect on HUVECs with the MTT assay, but not HCFs. Western blotting analysis showed that the VEGF and ICAM-1 levels in the HCFs and HUVECs stimulated by H202 were significantly higher than those in the normal conditions, which were decreased dramatically in those treated with PEDF-34-NPs. RT-PCR analysis revealed that the VEGF mRNA and NF-KB mRNA levels increased in H202-stimulated HCFs, while both of them decreased in PEDF-34-NP groups dose dependently. Conclusions PEDF-34-NPs may play an important role in regulating the NF-kB pathway, inhibiting inflammatory activity. PEDF-34-NPs may be a potential new drug for treating corneal injury in the future.