transforming-growth-factor-beta and Eye-Burns

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-β (TGF-β) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-β was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

Topics: Alkalies; Animals; Burns, Chemical; Corneal Injuries; Disease Models, Animal; Eosine Yellowish-(YS); Eye Burns; Fibroblast Growth Factor 10; Hematoxylin; Humans; Inflammation; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Nerve Growth Factor; Rabbits; Transforming Growth Factor beta; Transforming Growth Factors; Wound Healing

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-β (TGF-β) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.

Topics: Alkalies; Aminosalicylic Acids; Animals; Benzenesulfonates; Burns, Chemical; Cell Line; Chemokine CXCL1; Chemotaxis; Epithelial Cells; Epithelium, Corneal; Eye Burns; Fibrosis; Gene Expression Regulation; Glycoproteins; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Neutrophils; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

The aim was to evaluate the ability of gelatin methacryloyl (GelMA) hydrogel eye pads loaded with amniotic extract to prevent symblepharon in rabbits.. Forty-eight rabbits were divided into 3 groups. After ocular alkali burn, Group A (n=16) was treated with amniotic extract-loaded hydrogel eye pads placed in the conjunctival sac, Group B (n=16) was treated with amniotic membrane transplantation, and Group C (n=16) received no treatment. At 1, 2, 3, and 4 weeks post-injury, 4 rabbits from each group were selected to evaluate for symblepharon, determine epithelial healing rate and corneal neovascularization, conduct histopathology, and to quantify the expression of TGF-β1.. At 1 week post-injury, the epithelial healing rate in Groups A and B was higher than Group C (p=0.002, 0.001, respectively). At 2 weeks, corneal neovascularization in Group B was less than Group C (p=0.004). At 3 and 4 weeks, no symblepharon has been found in Group A, but it was found in some eyes in Group B and C (p=0.009, 0.013). Further, the expression of TGF-β1 in Group A was lower than in Group B and C (p<0.001). H&E staining showed that the controls in Group C had more edema and inflammatory cell infiltration in the first 2 weeks, relative to Groups A and B. At 4 weeks, Masson's Trichrome staining showed that fibers were most regularly aligned in Group A and that immuno-histochemical staining found that proliferating cell nuclear antigen was highest expressed in Group C.. Treatment with GelMA hydrogel eye pads loaded with amniotic extract shortly after chemical injury prevented symblepharon in rabbits.

Topics: Amnion; Animals; Burns, Chemical; Caustics; Corneal Neovascularization; Drug Delivery Systems; Eye; Eye Burns; Gelatin; Hydrogels; Male; Rabbits; Sodium Hydroxide; Transforming Growth Factor beta

Epidemiological studies have indicated that smoking is a pivotal risk factor for the progression of several chronic diseases. Nicotine, the addictive component of cigarettes, has powerful pathophysiological properties in the body. Although the effects of cigarette smoking on corneal re-epithelialization have been studied, the effects of nicotine on corneal wound healing-related neovascularization and fibrosis have not been fully demonstrated. The aim of this study was to evaluate the effects of chronic administration of nicotine on corneal wound healing following acute insult induced by an alkali burn. BALB/C female mice randomly received either vehicle (2% saccharin) or nicotine (100 or 200 μg/ml in 2% saccharin) in drinking water ad libitum. After 1 week, animals were re-randomized and the experimental group was subjected to a corneal alkali burn, and then nicotine was administered until day 14 after the alkali burn. A corneal alkali burn model was generated by placing a piece of 2 mm-diameter filter paper soaked in 1N NaOH on the right eye. Histopathological analysis and the expression level of the pro-angiogenic genes vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) revealed that chronic nicotine administration enhanced alkali burn-induced corneal neovascularization. Furthermore, the mRNA expression of the pro-fibrogenic factors α-smooth muscle actin (αSMA), transforming growth factor-β (TGF-β), and collagen α1 (Col1) was enhanced in the high-concentration nicotine-treated group compared with the vehicle group after corneal injury. Immunohistochemical analysis also showed that the αSMA-positive area was increased in chronic nicotine-treated mice after corneal alkali burn. An in vitro assay found that expression of the α3, α7, and β1 nicotinic acetylcholine receptor (nAChR) subunits was significantly increased by chemical injury in human corneal fibroblast cells. Moreover, alkali-induced fibrogenic gene expression and proliferation of fibroblast cells were further increased by treatment with nicotine and cotinine. The proliferation of such cells induced by treatment of nicotine and cotinine was reduced by inhibition of the PI3K and PKC pathways using specific inhibitors. In conclusion, chronic administration of nicotine accelerated the angiogenic and fibrogenic healing processes in alkali-burned corneal tissue.

Topics: Actins; Animals; Burns, Chemical; Cell Line; Collagen Type I; Cornea; Corneal Injuries; Cotinine; Disease Models, Animal; Eye Burns; Female; Fibroblasts; Humans; Mice, Inbred BALB C; Nicotine; Protective Agents; Random Allocation; Receptors, Nicotinic; Sodium Hydroxide; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wound Healing

In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor β, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.

Topics: Actins; Alkalies; Animals; Cornea; Corneal Opacity; Eye Burns; Fibrosis; Gene Expression Regulation; Gene Knockout Techniques; Inflammation; Interleukin-6; Mice; Transforming Growth Factor beta; TRPV Cation Channels; Vascular Endothelial Growth Factor A

The aim of this study was to investigate the effects of systemically administered bone-marrow-derived mesenchymal stromal cells (MSCs) on the early acute phase of inflammation in the alkali-burned eye. Mice with damaged eyes were either untreated or treated 24 h after the injury with an intravenous administration of fluorescent-dye-labeled MSCs that were unstimulated or pretreated with interleukin-1α (IL-1α), transforming growth factor-β (TGF-β), or interferon-γ (IFN-γ). Analysis of cell suspensions prepared from the eyes of treated mice on day 3 after the alkali burn revealed that MSCs specifically migrated to the damaged eye and that the number of labeled MSCs was more than 30-times higher in damaged eyes compared with control eyes. The study of the composition of the leukocyte populations within the damaged eyes showed that all types of tested MSCs slightly decreased the number of infiltrating lymphoid and myeloid cells, but only MSCs pretreated with IFN-γ significantly decreased the percentage of eye-infiltrating cells with a more profound effect on myeloid cells. Determining cytokine and NO production in the damaged eyes confirmed that the most effective immunomodulation was achieved with MSCs pretreated with IFN-γ, which significantly decreased the levels of the proinflammatory molecules IL-1α, IL-6, and NO. Taken together, the results show that systemically administered MSCs specifically migrate to the damaged eye and that IFN-γ-pretreated MSCs are superior in inhibiting the acute phase of inflammation, decreasing leukocyte infiltration, and attenuating the early inflammatory environment.

Topics: Alkalies; Allografts; Animals; Antiviral Agents; Burns, Chemical; Eye Burns; Female; Inflammation; Interferon-gamma; Interleukin-1alpha; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred BALB C; Stem Cell Niche; Transforming Growth Factor beta

We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing ion channel, or TRPA1 antagonist treatment affects the severity inflammation and scarring during tissue wound healing in a mouse cornea injury model. In addition, the effects of the absence of TRPA1 on transforming growth factor β1 (TGF-β1)-signaling activation were studied in cell culture. The lack of TRPA1 in cultured ocular fibroblasts attenuated expression of TGF-β1, interleukin-6, and α-smooth muscle actin, a myofibroblast the marker, but suppressed the activation of Smad3, p38 MAPK, ERK, and JNK. Stroma of the healing corneas of TRPA1(-/-) knockout (KO) mice appeared more transparent compared with those of wild-type mice post-alkali burn. Eye globe diameters were measured from photographs. An examination of the corneal surface and eye globes suggested the loss of TRPA1 suppressed post-alkali burn inflammation and fibrosis/scarring, which was confirmed by histology, immunohistochemistry, and gene expression analysis. Reciprocal bone marrow transplantation between mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO mouse wound healing with reduced inflammation and fibrosis. Systemic TRPA1 antagonists reproduced the KO phenotype of healing. In conclusion, a loss or blocking of TRPA1 in mice reduces inflammation and fibrosis/scarring in the corneal stroma during wound healing following an alkali burn. The responsible mechanism may include the inhibition of TGF-β1-signaling cascades in fibroblasts by attenuated TRPA1 signaling. Inflammatory cells are considered to have a minimum involvement in the exhibition of the KO phenotype after injury.

Topics: Animals; Corneal Diseases; Eye Burns; Fibrosis; Inflammation; Mice; Mice, Knockout; Real-Time Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Transient Receptor Potential Channels; TRPA1 Cation Channel; Wound Healing

We investigated the wound healing effect of retinyl palmitate eyedrops following a corneal alkali burn in rats.. A total of 160 Sprague-Dawley male rats were divided into two groups and central corneas were injured by contacting eyes with filter paper saturated with 0.01 m NaOH for 45 seconds. Vitamin A group was treated with retinyl palmitate and antibiotic (Cravit(®) : 0.5% levofloxacin) eye drops four times daily for 3 days and the control group with vehicle and antibiotic eye drops. Corneal wound healing by fluorescein staining and impression cytologic analysis were conducted at 0, 24, 48 and 72 hr after injury. Vascular endothelial growth factor A (VEGF-A), thrombospondin 2, matrix metalloproteinase 9 (MMP 9) and transforming growth factor-β (TGF-β) were measured in corneas by ELISA, immunofluorescent staining and real-time PCR.. Corneal wound healing was better in the vitamin A group than in the control group. Early sprouting of new vessel was observed in the control group at 72 hr, but not in the vitamin A group. Corneal thrombospondin 2 proteins in ELISA were higher in the vitamin A group, but VEGF-A, MMP 9 and TGF-β proteins were higher in the control group (p < 0.05). Similarly, thrombospondin 2 immunofluorescent staining was stronger, whereas VEGF-A, MMP 9 and TGF-β staining were weaker in the vitamin A group (p < 0.05). In addition, thrombospondin 2 mRNA levels were higher, whereas VEGF-A, MMP 9 and TGF-β mRNA levels were lower in the vitamin A group (p < 0.05).. Retinyl palmitate eye drops can inhibit VEGF-A and activate thrombospondin 2 and improve conjunctival impression cytologic findings. Furthermore, retinyl palmitate eye drops were found to promote corneal healing after an alkali burn in rats.

Topics: Animals; Burns, Chemical; Corneal Ulcer; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Burns; Fluorescent Antibody Technique, Indirect; Male; Matrix Metalloproteinase 9; Ophthalmic Solutions; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sodium Hydroxide; Thrombospondins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vitamin A; Vitamins; Wound Healing