transforming-growth-factor-beta and Corneal-Neovascularization

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

An Arg-Gly-Asp (RGD) motif in the fourth FAS1 domain of the human BIGH3 (transforming growth factor-β1-inducible gene-h3) protein has been reported to play an important role in mediating tumor angiogenesis. The aim of this study was to investigate the inhibitory effect of a modified C-terminal fragment BIGH3 protein with an RGDRGD motif on corneal neovascularization in vitro and in vivo. Recombinant C-terminal fragment BIGH3 protein with wild-type sequence and modified C-terminal fragment BIGH3 protein containing an RGDRGD motif were successfully expressed and purified. We demonstrated that both proteins significantly inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) adhesion, migration, and tube formation and induced cell apoptosis but failed to inhibit HUVEC proliferation. We determined that the mechanism underlying this activity was an interaction between BIGH3 and αvβ3 integrin, which blocked the phosphorylation of PI3K/Akt and ERK signaling pathways. The inhibitory effects of wild-type and modified C-terminal fragment BIGH3 proteins on angiogenesis were confirmed by a rabbit corneal neovascularization assay. More importantly, we provided evidence that the modified C-terminal fragment BIGH3 protein with an RGDRGD motif inhibited angiogenic activity far more effectively than did wild-type C-terminal fragment BIGH3. Collectively, our data show that a C-terminal fragment BIGH3 protein containing an RGDRGD motif might be promising as an effective drug in treating corneal neovascularization.

Topics: Amino Acid Motifs; Angiogenesis Inhibitors; Animals; Apoptosis; Blood Vessels; Blotting, Western; Caspases; Cell Adhesion; Cell Movement; Cell Proliferation; Corneal Neovascularization; Disease Models, Animal; Extracellular Matrix Proteins; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Integrin alphaVbeta3; Mutagenesis, Site-Directed; Oligopeptides; Peptide Fragments; Phosphatidylinositol 3-Kinases; Phosphorylation; Plasmids; Protein Folding; Proto-Oncogene Proteins c-akt; Rabbits; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Inflammatory processes within the cornea are known to be associated with corneal neovascularization (CN). We examined the effects of inflammatory mediators on the expression of angiogenic factors by corneal cells. TNF-alpha and IL-1 induced VEGF-A secretion by corneal fibroblasts (HCRF) and this was inhibited significantly by IFN-gamma. Constitutively secreted VEGF-A by corneal epithelial cells (HCE) was not affected by these cytokines. Moreover, sVEGF-R1(sFlt-1) secretion by HCRF was stimulated significantly by IFN-gamma. JAK-STAT pathway inhibitor reversed the effects of IFN-gamma on VEGF-A and sFlt-1 secretion by HCRF. RT-PCR analysis showed that IFN-gamma influences the expression of VEGF-A and sFlt-1 by affecting their mRNA level. IFN-gamma inhibited TGF-beta induced VEGF-A secretion but not sVEGF-R1 secretion. This is the first report demonstrating the inhibitory and stimulatory effects of IFN-gamma on VEGF-A and sFlt-1 secretion, respectively. Our results suggest that IFN-gamma acts as an anti-angiogenic cytokine in the human cornea.

Topics: Cell Line; Cornea; Corneal Neovascularization; Fibroblasts; Humans; Interferon-gamma; Interleukin-1; Janus Kinase 1; Receptor, Fibroblast Growth Factor, Type 1; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To examine the role of tumor necrosis factor alpha (TNFalpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro.. An in vitro model of angiogenesis was used to examine the roles of TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription-polymerase chain reaction.. HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice.. In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.

Topics: Animals; Coculture Techniques; Corneal Neovascularization; Corneal Stroma; Disease Models, Animal; Endothelium, Vascular; Extracellular Matrix Proteins; Fibroblasts; Fluorescent Antibody Technique, Indirect; Mice; Mice, Inbred C57BL; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Endothelial Growth Factor A

To analyze presence and distribution of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)alpha, and TGFbeta1 in human corneas with neovascularization due to different corneal diseases.. Indirect immunohistochemistry for VEGF, TGFalpha, and TGFbeta1, was performed on paraffin-embedded corneas obtained by keratoplasty. Corneas from each of the four main groups of histopathologic diagnoses associated with corneal neovascularization were analyzed (scarring after keratitis, graft rejection/insufficiency, acute necrotizing keratitis, scarring after mechanical/chemical injury). Subclassification of inflammatory infiltrates was done using immunohistochemistry for CD3 (T-lymphocytes) and CD68 (macrophages).. The analyzed angiogenic factors were detectable in corneas from all four histopathologic groups in a similar distribution; capillary endothelial cells, stromal and intravascular inflammatory cells (T-lymphocytes, macrophages), and basal corneal epithelial cells stained positive for the tested angiogenic factors.. The angiogenic factors VEGF, TGFalpha, and TGFbeta1 are detectable in human corneas with neovascularization. Their distribution is quite uniform in different corneal diseases, resulting in corneal angiogenesis. An antiangiogenic therapy inhibiting corneal neovascularization by antagonizing angiogenic factors would have to counteract several angiogenic factors.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; CD3 Complex; Cornea; Corneal Neovascularization; Corneal Transplantation; Endothelial Growth Factors; Humans; Immunoenzyme Techniques; Lymphokines; Macrophages; Retrospective Studies; T-Lymphocytes; Transforming Growth Factor alpha; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To examine the effect of donor-specific anterior chamber-associated immune deviation (ACAID) induction on the survival of orthotopic corneal allografts in neovascularized graft beds.. To induce donor-specific ACAID in recipients, peritoneal exudate cells (PEC) from C57BL/6 mice were incubated overnight with transforming growth factor (TGF)-beta. Cultured PEC were injected intravenously (i.v.) into BALB/c mice, and, 1 week later, these animals received orthotopic corneal allografts from C57BL/6 donors into neovascularized graft beds. Control mice received i.v. injection of syngeneic (BALB/c) PEC, cultured overnight with TGF-beta, and then received orthotopic corneal allografts from C57BL/6 donors.. All corneal allografts (15 out of 15) were rejected within 2 weeks after grafting in the neovascularized graft beds of control animals. However, only 6 out of 16 (37.5%) of corneal allografts were rejected in recipients in which donor-specific ACAID had been induced by injection of allogeneic PEC cultured with TGF-beta.. Previous studies revealed that rejection of orthotopic corneal allografts in neovascularized graft beds in mice correlated with acquisition of donor-specific delayed hypersensitivity (DH). The results of this study suggest that induction of donor-specific ACAID, which selectively impairs DH responses to donor antigens, effectively prolongs corneal allograft survival in "high-risk" eyes.

Topics: Animals; Anterior Chamber; Cornea; Corneal Neovascularization; Corneal Transplantation; Graft Survival; Hypersensitivity, Delayed; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Peritoneal Cavity; Tissue Donors; Transforming Growth Factor beta; Transplantation, Homologous