transforming-growth-factor-beta and Corneal-Diseases

In the cornea, the epithelial basement membrane (EBM) and corneal endothelial Descemet's basement membrane (DBM) critically regulate the localization, availability and, therefore, the functions of transforming growth factor (TGF)β1, TGFβ2, and platelet-derived growth factors (PDGF) that modulate myofibroblast development. Defective regeneration of the EBM, and notably diminished perlecan incorporation, occurs via several mechanisms and results in excessive and prolonged penetration of pro-fibrotic growth factors into the stroma. These growth factors drive mature myofibroblast development from both corneal fibroblasts and bone marrow-derived fibrocytes, and then the persistence of these myofibroblasts and the disordered collagens and other matrix materials they produce to generate stromal scarring fibrosis. Corneal stromal fibrosis often resolves completely if the inciting factor is removed and the BM regenerates. Similar defects in BM regeneration are likely associated with the development of fibrosis in other organs where perlecan has a critical role in the modulation of signaling by TGFβ1 and TGFβ2. Other BM components, such as collagen type IV and collagen type XIII, are also critical regulators of TGF beta (and other growth factors) in the cornea and other organs. After injury, BM components are dynamically secreted and assembled through the cooperation of neighboring cells-for example, the epithelial cells and keratocytes for the corneal EBM and corneal endothelial cells and keratocytes for the corneal DBM. One of the most critical functions of these reassembled BMs in all organs is to modulate the pro-fibrotic effects of TGFβs, PDGFs and other growth factors between tissues that comprise the organ.

Topics: Animals; Basement Membrane; Corneal Diseases; Fibrosis; Heparan Sulfate Proteoglycans; Humans; Transforming Growth Factor beta

Maintenance of the transparency and regular shape of the cornea are essential to the normal vision, whereas opacification of the tissue impairs vision. Fibrogenic reaction leading to scarring in an injured cornea is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of fibrous extracellular matrix. Inflammatory/fibrogenic growth factors/cytokines produced by inflammatory cells play a pivotal role in fibrogenic response. Signaling systems involved in myofibroblast formation and fibrogenesis are activated by various growth factors, i.e., transforming growth factor beta or others. Modulation of transforming growth factor beta signal transduction molecules, e.g., Smad and mitogen-activated protein kinases, by gene transfer and other technology provides a new concept of prevention/treatment of unfavorable fibrogenesis in the cornea.

Topics: Cell Transdifferentiation; Cornea; Corneal Diseases; Endothelium, Corneal; Fibrosis; Gene Transfer Techniques; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Myofibroblasts; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wound Healing

Authors present one of the ocular diabetic complications--diabetic keratoepitheliopathy. The aim of this article is to summarize the knowledge about diabetic keratoepitheliopathy, its causes, manifestations and treatment options. Diabetes mellitus is associated with structural and functional disturbances in eyelids, conjunctiva and cornea. There are also changes in tear film present. Diabetic neuropathy resulting from the biochemical poor control of diabetes is the probable basic cause of the pathology. Mechanisms responsible for these changes are still not well understood. The corneal and conjunctival complications may occur spontaneously. But more often they arise from undue stress of intraocular surgery procedures. The incidence of diabetic keratoepitheliopathy in diabetic patients is high. However, it is rarely diagnosed. Effectiveness of symptomatic treatment with use of common medications is not satisfactory and it needs further investigation.

Topics: Adult; Aged; Causality; Comorbidity; Conjunctival Diseases; Corneal Diseases; Diabetes Complications; Dry Eye Syndromes; Endothelium, Corneal; Extracellular Matrix Proteins; Eyelid Diseases; Humans; Prevalence; Risk Factors; Tears; Transforming Growth Factor beta

In 1997, a group of hereditary corneal dystrophies was related to mutations in the TGFBI (BIGH3) gene. Within this group, some corneal dystrophies present particular biochemical features in that they are characterized by corneal amyloid deposition. Contrary to clinical and genetic knowledge, the biochemical characteristics of the encoded protein (Big-h3) and the mechanisms of its amyloid conversion remain unclear. We review the current knowledge on the Big-h3 protein and focus on the behavior of the codon 124 region. We discuss this protein's mechanisms of amyloid conversion from our results and previous reports as well as from other types of amyloidosis. These data provide a better understanding of the putative processes leading to the phenotypic variations linked with their respective codon 124 mutation.

Topics: Amino Acid Sequence; Amyloidosis; Base Sequence; Codon; Corneal Diseases; Extracellular Matrix Proteins; Eye Proteins; Humans; Molecular Sequence Data; Mutation; Transforming Growth Factor beta

The shortage of human donor corneas has raised important concerns about engineering of corneal endothelial cells (CECs) for clinical use. However, due to the limited proliferative capacity of human CECs, driving them into proliferation and regeneration may be difficult. Unlike human CECs, rabbit CECs have a marked proliferative capacity. To clarify the potential reason for this difference, we analysed the proteomes of four human corneal endothelium samples and four rabbit corneal endothelium samples with quantitative label-free proteomics and downstream analysis. We discovered that vitamin and selenocompound metabolism and some signaling pathways such as NF-kappa B signaling pathway differed between the samples. Moreover, TGFβ, PITX2 and keratocan were distinctively expressed in rabbit samples, which might be associated with active proliferation in rabbit CECs. This study illustrates the proteomic differences between human and rabbit CECs and might promote CEC engineering strategies.

Topics: Aged; Animals; Cell Differentiation; Cells, Cultured; Corneal Diseases; Corneal Transplantation; Disease Models, Animal; Endothelium, Corneal; Homeobox Protein PITX2; Homeodomain Proteins; Humans; Male; Middle Aged; Proteome; Proteomics; Rabbits; Tissue Preservation; Transcription Factors; Transforming Growth Factor beta

Recessive variants in LTBP2 are associated with eye-restricted phenotypes including (a) primary congenital glaucoma and (b) microspherophakia/megalocornea and ectopia lentis with/without secondary glaucoma. Nosology of LTBP2 pathology in humans is apparently in contrast with the consolidated evidence of a wide expression of this gene in the developing embryo. Accordingly, in previously published patients with LTBP2-related eye disease, additional extraocular findings have been occasionally reported and include, among others, high-arched palate, tall stature, and variable cardiac involvement. Anyway, no emphasis was put on such systemic manifestations. Here, we report two unrelated Roma/Gypsy patients first ascertained for a multisystem disorder mainly characterized by primary congenital glaucoma, complex congenital heart defect, tall stature, long fingers, skin striae and dystrophic scarring, and resembling Marfan syndrome. Heart involvement was severe with polyvalvular heart dysplasia in one, and transposition of great arteries, thoracic arterial tortuosity, polyvalvular heart dysplasia, and neo-aortic root dilatation in the other. Both patients were homozygous for the recurrent c.895C>T[p.(R299X)] variant, typically found in individuals of Roma/Gypsy descent with an eye-restricted phenotype. Our findings point out LTBP2 as responsible of a systemic phenotype coherent with the community of syndromes related to anomalies in genes involved in the TGFβ-pathway. Among these disorders, LTBP2-related systemic disease emerges as a distinct condition with expanding prognostic implications and autosomal recessive inheritance.

Topics: Adolescent; Child; Corneal Diseases; Ectopia Lentis; Eye Diseases, Hereditary; Female; Genetic Diseases, X-Linked; Glaucoma; Heart; Heart Defects, Congenital; Homozygote; Humans; Iris; Latent TGF-beta Binding Proteins; Male; Marfan Syndrome; Phenotype; Roma; Transforming Growth Factor beta

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor β-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming

Topics: Aged, 80 and over; Amyloid; Chromatography, High Pressure Liquid; Cornea; Corneal Diseases; Extracellular Matrix Proteins; High-Temperature Requirement A Serine Peptidase 1; Humans; Mutagenesis, Site-Directed; Peptides; Protein Domains; Protein Folding; Recombinant Proteins; Tandem Mass Spectrometry; Transforming Growth Factor beta

Loeys-Dietz syndrome (LDS), an autosomal-dominant connective tissue disorder, is characterised by systemic manifestations including arterial aneurysm and craniofacial dysmorphologies. Although ocular involvement in LDS has been reported, detailed information on those manifestations is lacking.. Retrospective chart review of patients with diagnosed LDS and comparison with age-matched control patients.. Mean age was 37.8±14.6 years (patients with LDS) and 38.4±13.5 years (controls). Patients with LDS less frequently had iris transillumination, cataract and glaucoma compared with controls. Scleral and retinal vascular abnormalities were not found in any of the LDS eyes. Ectopia lentis was found in one patient with LDS. The eyes of patients with LDS tended to be more myopic (spherical equivalent, -2.47±2.70 dioptres (dpt) vs -1.30±2.96dpt (controls); P=0.08) and longer (24.6±1.7mm vs 24.1±1.5mm (controls); P=0.10). Central corneal thickness was significantly reduced in LDS eyes (521±48µm vs 542±37µm (controls); P=0.02). Corneal curvature (43.06±1.90dpt (LDS) versus 43.00±1.37dpt (controls); P=0.72) and interpupillary distance (65.0±6.0mm (LDS) vs 64.3±4.8mm (controls); P=0.66) did not differ significantly between both groups. Visual acuity was similar between both groups (0.03±0.09logarithm of the minimum angle of resolution (logMAR) for LDS eyes and 0.05±0.17logMAR for control eyes, P=0.47).. Ocular features of LDS include decreased central corneal thickness and mild myopia. Ectopia lentis may be slightly more common than in controls but appears less common than in Marfan syndrome. Hypertelorism, scleral and retinal vascular abnormalities were not features of LDS.

Topics: Adolescent; Adult; Aged; Biometry; Cataract; Corneal Diseases; Ectopia Lentis; Female; Glaucoma; Humans; Iris Diseases; Loeys-Dietz Syndrome; Male; Middle Aged; Myopia; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Retrospective Studies; Smad3 Protein; Transforming Growth Factor beta; Visual Acuity; Young Adult

To protect corneal transparency, we tried to develop a new therapeutic strategy for corneal neovascularization, corneal scar, and TGFBI-related corneal dystrophy using nucleic acid drug. 1. The expression of angiopietin-like protein 2 (Angptl2) markedly increased in the neovascularized corneas compared to the normal cornea, and Angtpl2 was(a potent inducer of inflammatory corneal neovascularization. We have produced a single-stranded proline-modified short hairpin anti-Angptl2 ribonucleric acid interference (RNAi) molecule that is carried in a lipid nanoparticle for topical application. We have found this agent can penetrate all layers of the cornea. Angptl2 mRNA expression and corneal neovascularization were inhibited in a mouse alkari injury model by topical application of this agent. Thus, this modified RNAi agent is a new topical formulation for use against corneal neovascularization and scar. 2. Human umbilical vein endothelial cells (HUVECs) were cultured with human corneal keratocytes under serum-free conditions. We performed microarray gene-expression analysis in the coculture system and selected angiopoietin-like protein 7 (Angptl7). In vivo, intrastromal injections of an anti-Angptl7 RNAi agent into the avascular corneal stroma of mice resulted in the growth of blood vessels. Further, we examined the effects of Angptl7 on corneal nerves using culture rat trigeminal cells and this molecule had neurotrophic property on the cornea. Thus, Angpt17 is a unique molecule, which contain its bilateral character (anti-angiogenic and neurotrophic) in the cornea; an agonistic nucleic acid drug for Angptl7 may be a new therapeutic tool for protecting corneal transparency. 3. We examined local gene editing for TGFBI-related corneal dystrophy using CRISPR-Cas9 mediated homology directed repair (HDR). Cultured corneal keratocytes were obtained from a patient of R124H granular dystrophy. The R124H gene arrangement was corrected by a tranfection of guide RNA and HDR repair template single strand DNA in vitro. Thus, CRISPR-Cas9 medi-ated HDR could be a future radical treatment for TGFBI-related corneal dystrophy.

Topics: Angiopoietins; Animals; Corneal Diseases; Disease Models, Animal; Extracellular Matrix Proteins; Genetic Therapy; Humans; Neovascularization, Pathologic; Transforming Growth Factor beta

To investigate the role of palladin in the cornea, we examined expression of this actin assembly-related protein in normal, diseased, or injured corneal tissue as well as in cultured corneal fibroblasts.. Expression of palladin and α-smooth muscle actin (α-SMA) in the rat cornea with an incision wound, in the normal and diseased human cornea, and in cultured human corneal fibroblasts was examined by immunofluorescence or immunoblot analysis.. The expression of both palladin and α-SMA was detected at the lesion site during wound healing in the rat cornea. Whereas neither palladin nor α-SMA was detected in the normal human cornea, the colocalization of both proteins was detected in diseased human corneas with underlying conditions characterized by the presence of fibrosis. The expression of both palladin and α-SMA in cultured human corneal fibroblasts was increased by transforming growth factor-β (TGF-β) in a manner sensitive to inhibition by blockers of Smad or mitogen-activated protein kinase (MAPK) signaling. Finally, RNA interference-mediated depletion of palladin attenuated the TGF-β-induced upregulation of α-SMA expression in human corneal fibroblasts as well as TGF-β-induced collagen gel contraction mediated by these cells.. Palladin is expressed in the rat and human cornea in association with scar formation. Expression of palladin in human corneal fibroblasts is increased by TGF-β in a manner dependent on Smad and MAPK signaling and is required for the TGF-β-induced upregulation of α-SMA.

Topics: Actins; Animals; Cells, Cultured; Cornea; Corneal Diseases; Corneal Injuries; Cytoskeletal Proteins; Fibroblasts; Humans; MAP Kinase Signaling System; Phosphoproteins; Rats; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wound Healing

Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

Topics: Adolescent; Adult; Amino Acid Motifs; Caveolae; Child; Corneal Diseases; Endocytosis; Endoplasmic Reticulum; Extracellular Matrix Proteins; Female; Fibroblasts; Golgi Apparatus; Humans; Integrin alphaVbeta3; Lysosomes; Male; Proteasome Endopeptidase Complex; Proteolysis; Transforming Growth Factor beta; Ubiquitin; Young Adult

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

Endothelial-to-mesenchymal transition (EnMT) is a cell transformation process involved in both morphogenesis and pathogenesis. EnMT of corneal endothelial cells happens after endothelial injury and during ex vivo culture. Previous studies have shown that the transforming growth factor-β signaling pathway is involved in this transition. In this study, we found that rat corneal endothelial cells could spontaneously undergo EnMT during ex vivo culture. This change in rat corneal endothelial cells was associated with Notch signaling pathway activation after the first passage, which was blocked by the Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). This inhibitor also prevented transforming growth factor β1-, β2-, and β3-induced EnMT and reversed transformed rat corneal endothelial cells to a normal phenotype. Furthermore, DAPT treatment blocked retrocorneal membrane formation in a rat corneal endothelium damage model. Our study indicates that the Notch signaling pathway is involved in the corneal EnMT process, which may be a novel therapeutic target for treating corneal endothelial fibrogenic disorders.

Topics: Animals; Cell Line, Transformed; Cell Movement; Cell Proliferation; Cells, Cultured; Corneal Diseases; Dipeptides; Endothelial Cells; Endothelium, Corneal; Gene Expression Regulation; Humans; Mesoderm; Phenotype; Rats; Rats, Sprague-Dawley; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor β-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.

Topics: Amyloid; Benzalkonium Compounds; Corneal Diseases; Detergents; Extracellular Matrix Proteins; Humans; Ophthalmic Solutions; Peptides; Sodium Dodecyl Sulfate; Transforming Growth Factor beta

Platelet-rich plasma (PRP) harbors high concentrations of growth factors related to the promotion of wound healing. We evaluated the efficacy of PRP eyedrops in the treatment of persistent epithelial defects (PEDs).. Autologous PRP and autologous serum (AS) were prepared from whole blood. The concentrations of transforming growth factor (TGF)-β1, TGF-β2, epidermal growth factor (EGF), vitamin A and fibronectin in the PRP and AS were analyzed and compared. The corneal epithelial healing efficacy of PRP was compared with that of AS in patients with PED induced by post-infectious inflammation.. The concentrations of TGF-β1, TGF-β2, EGF, vitamin A and fibronectin in the PRP and AS were not statistically different. However, the concentrations of EGF in the PRP were significantly greater than in the AS. AS was used in 17 and PRP in 11 eyes of 28 patients. The healing rates of the corneal epithelia of the PRP-treated eyes were significantly higher than those treated with AS.. The PRP was effective in the treatment of PEDs. This may be attributable to its high concentration of platelet-contained growth factors, most notably EGF. PRP could be an effective, novel treatment option for chronic ocular surface disease.

Topics: Adult; Aged; Aged, 80 and over; Cell Proliferation; Chromatography, High Pressure Liquid; Corneal Diseases; Corneal Ulcer; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelium, Corneal; Eye Infections, Bacterial; Female; Fibronectins; Humans; Male; Middle Aged; Ophthalmic Solutions; Platelet Count; Platelet-Rich Plasma; Retrospective Studies; Serum; Transforming Growth Factor beta; Vitamin A; Wound Healing

The localized surface plasmon resonance (LSPR) optical property has recently been well employed as an effective platform for the quantitative detection of protein-protein interactions on the nanoscale. However, its advantage has not been fully explored yet in the DNA diagnosis field, especially in detecting point mutations of DNA. Point mutations of the BIGH3 gene are associated with the most common corneal dystrophies (CDs), such as Avellino corneal dystrophy, Reis-Bucklers corneal dystrophy, and lattice corneal dystrophy. Since the detection of these corneal dystrophies is urgently needed before laser-assisted in situ keratomileusis operation to prevent blindness, genetic analysis of the BIGH3 gene is critical in most ophthalmological clinics. In this study, we report LSPR-based detection of the BIGH3 gene mutations by using a multispot gold-capped nanoparticle array (MG-NPA) chip. The analytical range and sensitivity of the MG-NPA chip were determined by measuring different concentrations of each CD target DNA in the range of 1 fM to 1 microM. Under the optimal conditions, the detection of DNA hybridization with each CD target DNA was performed with a detection limit of 1 pM target DNA. The selective discrimination against a single-base mismatch DNA sequence was also achieved by using both homozygous and heterozygous CD samples. It demonstrates that the label-free LSPR-based optical biosensor system employing the MG-NPA chip provides a new diagnostic platform allowing the selective and sensitive detection of various DNA point mutations, leading to possible diagnosis of mutation-related diseases including corneal dystrophies reported here.

Topics: Base Sequence; Corneal Diseases; DNA; DNA Probes; Extracellular Matrix Proteins; Gold; Homozygote; Humans; Metal Nanoparticles; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Point Mutation; Surface Plasmon Resonance; Time Factors; Transforming Growth Factor beta

The aim of this study was to investigate transcriptional activities of genes encoding transforming growth factor (TGF)-beta isoforms in bullous keratopathy corneas.. The study group consisted of 45 patients with bullous keratopathy (22 females and 23 males). The control group included 45 corneal donors (21 females and 24 males). Quantification of TGF-beta1, TGF-beta2, and TGF-beta3 mRNAs was performed by real-time quantitative reverse transcription PCR (QRT-PCR).. TGF-beta1, TGF-beta2, and TGF-beta3 mRNAs were detected in both normal and pseudophakic bullous keratopathy (PBK) corneas. We found significantly lower transcriptional activity of TGF-beta3 mRNA in bullous keratopathy corneas compared to normal tissues. TGF-beta1 and TGF-beta2 expressions were at the same level in both PBK and healthy corneas.. Downregulation of TGF-beta3 gene expression may play a significant role in molecular changes observed in bullous keratopathy.

Topics: Aged; Cornea; Corneal Diseases; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Male; Middle Aged; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

A single layer of corneal endothelial cells covers the posterior surface of the Descemet membrane. Normally, corneal endothelial cells do not proliferate, suggesting that factors inhibit their undergoing mitosis. Fibroblastic transformation of corneal endothelial cells due to syphilitic interstitial keratitis or alkali burn may result in stromal opacification. Various growth factors such as fibroblast growth factor 2 and transforming growth factor β are involved in "endothelial-mesenchymal transition (EnMT)." Endothelial wound-healing assay experiments revealed that cell migration toward artificial wound defect was mediated by p38 and c-Jun N-terminal kinase. To understand whether Smad signal might have an important role in EnMT regulation, gene transfer of Smad7 was employed to block transforming growth factor β/Smad signal in rat corneal endothelium 3 days before alkali burning. EnMT during healing interval after alkali burn was markedly suppressed by Smad7 overexpression associated with upregulation of cell proliferation. Therefore, blocking Smad signal effectively suppresses injury-induced EnMT and fibrogenic reaction by corneal endothelium without impairing repair of wound defect. Strategies that block Smad may be useful for prevention and treatment of fibrogenic disorders in the corneal endothelium.

Topics: Cell Movement; Corneal Diseases; Endothelium, Corneal; Fibroblast Growth Factor 2; Humans; Mesenchymal Stem Cells; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

To examine the distribution of the alpha11 integrin chain in the human cornea during fetal development and in normal and diseased adult human corneas.. Six fetal corneas, 10 to 20 weeks of gestation (wg), and 18 adult corneas including 3 normal, 7 with keratoconus, 5 with pseudophakic bullous keratopathy (PBK), 2 with Fuchs' corneal dystrophy, and 1 with a scar after deep lamellar keratoplasty (DLKP) were processed for immunohistochemistry with specific antibodies against the alpha11 integrin chain; collagen I, IV, and V; and alpha-smooth muscle actin (alpha-SMA). The cellular source of alpha11 integrin chain was further investigated in cell cultures.. At 10 to 17 wg, the alpha11 integrin chain was predominantly present in the anterior corneal stroma. At 20 wg, in normal adult corneas and in Fuchs' dystrophy corneas there was weak staining in the stroma. The PBK corneas showed variable and weak staining, generally accentuated in the posterior stroma near Descemet's membrane. In contrast, the anterior portion of the stroma in the keratoconus corneas was strongly stained in an irregular streaky pattern. Human corneal fibroblasts/myofibroblasts produced alpha11 integrin chain in culture. Cultures treated with TGF-beta showed higher content of both alpha-SMA and the alpha11 integrin chain.. The presence of the alpha11 integrin chain during early corneal development and the enhanced expression in scarred keratoconus corneas indicates that this integrin chain is likely to play an important role in collagen deposition during corneal development and in keratoconus with a scarring component and compromised basement membrane integrity.

Topics: Actins; Adult; Aged; Aged, 80 and over; Aging; Cells, Cultured; Collagen Type I; Collagen Type IV; Collagen Type V; Cornea; Corneal Diseases; Corneal Transplantation; Embryonic Development; Female; Fibroblasts; Fluorescent Antibody Technique, Indirect; Gestational Age; Humans; Integrin alpha Chains; Male; Microscopy, Fluorescence; Middle Aged; Muscles; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Young Adult

Several inherited corneal disorders in humans result from mutations in the transforming growth factor beta induced gene (TGFBI), which encodes for the extracellular transforming growth factor beta induced protein (TGFBIp) that is one of the most abundant proteins in the cornea. We previously reported a significant amount of TGFBIp in plasma by immunoblotting using the only TGFBIp antiserum (anti-p68(beta ig-h3)) available at that time (anti-p68(beta ig-h3) was generated against residues Val210-His683 of TGFBIp). This observation raised the possibility that a fraction of corneal TGFBIp may originate from the plasma. However, recent experiments in our laboratory indicated that the anti-p68(beta ig-h3) antiserum cross-reacts with an environmental protein contaminant. Therefore, we investigated the specificity of the originally utilized anti-p68(beta ig-h3) antiserum and re-evaluated the amount of TGFBIp in human plasma by immunoblotting using a new specific antiserum.. The observed cross-reactivity of the previously utilized anti-p68(beta ig-h3) antiserum was tested by immunoblotting and the antigen identity was determined by mass spectrometry. A part of human TGFBI encoding an NH2-terminal 11.4 kDa fragment of TGFBIp (residues Gly134-Ile236) was amplified by polymerase chain reaction (PCR) and cloned in E. coli. The TGFBIp fragment was expressed in E. coli, purified by Ni2+-affinity chromatography, and used to immunize rabbits to produce a specific antiserum (anti-TGFBIp(134-236)). To enhance the detection of possible TGFBIp in plasma by allowing a higher sample load, albumin and immunoglobulin G (IgG) were specifically depleted from normal human plasma by affinity chromatography. The presence of TGFBIp in plasma was investigated by immunoblotting using the anti-TGFBIp(134-236) antiserum. Purified TGFBIp from porcine corneas was used for estimation of the TGFBIp detection limit.. The previously utilized TGFBIp antiserum, anti-p68(beta ig-h3), cross-reacted with human keratin-1, a common environmental protein contaminant. Thus, the anti-p68(beta ig-h3) antiserum recognizes both TGFBIp and keratin-1. In contrast, the anti-TGFBIp(134-236) antiserum reacted with TGFBIp but showed no indication of reactivity with other proteins in plasma. Using this antiserum, TGFBIp was not detected in crude or albumin/IgG-depleted human plasma and the detection limit of TGFBIp using immunoblotting was estimated to be 10 ng.. Our failure to detect TGFBIp in human plasma using a highly specific antiserum suggests that TGFBIp is not present in a physiologically relevant concentration in human plasma. The previous impression that normal human plasma contains a significant amount of TGFBIp by immunoblotting was due to the utilization of a less specific antiserum that recognizes both TGFBIp and human keratin-1. Together with other results, our observation makes it unlikely that TGFBIp is imported into the cornea from the circulation as reported for other abundant extracellular corneal proteins and suggests corneal origin of TGFBIp deposits in individuals with inherited corneal diseases caused by mutations in the TGFBI gene.

Topics: Animals; Artifacts; Cornea; Corneal Diseases; Epitopes; Extracellular Matrix Proteins; Humans; Immune Sera; Immunoblotting; Keratin-1; Mutation; Swine; Transforming Growth Factor beta

Beta igh3 is a transforming growth factor-beta-inducible cell adhesion molecule and its mutations are responsible for human autosomal dominant corneal dystrophies. Previously, we have studied the molecular properties of beta igh3 in vitro and reported that beta igh3 polymerizes to form a fibrillar structure and interacts with several extracellular matrix proteins including type I collagen. This study aimed to understand the role of elevated circulating levels of normal beta igh3 in eye development and corneal diseases.. We generated Alb-hss igh3 transgenic mice that have liver-specific expression of human beta igh3 (hss igh3) under the control of the albumin (Alb) enhancer/promoter and investigated the influence of beta igh3 overexpression in mouse eye. Polymerase chain reaction (PCR) genotyping, western blotting, and ELISA were performed to generate Alb-hss igh3 transgenic mouse lines. To identify the ocular pathology, electron microscopy and histological staining were employed in Alb-hss igh3 transgenic mice and wild-type mice.. Normal hss igh3 was ectopically overexpressed in the liver, secreted into blood stream, and reached the cornea of Alb-hss igh3 transgenic mice. Among transgenic mice, some mice had anterior segment defects including corneal opacity, disorganization of the collagen layers in the corneal stroma, and corneolenticular adhesion.. These results suggest that beta igh3 may be involved in anterior segment morphogenesis and eye development in mice. In addition, this indicates that the level of normal beta igh3 expression must be properly maintained during ocular development. The phenotype observed in Alb-hss igh3 transgenic mice is similar to human eye disorders such as anterior segment dysgenesis and Peters' anomaly. Thus, this model provides a very useful tool to study human eye diseases and the control of proliferation and differentiation of neural crest-originated cells.

Topics: Animals; Anterior Eye Segment; Collagen; Cornea; Corneal Diseases; Corneal Opacity; Corneal Stroma; Extracellular Matrix Proteins; Eye; Humans; Lens Diseases; Liver; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron; Phenotype; Tissue Distribution; Transforming Growth Factor beta

Amyloid is found in several corneal dystrophies, including distinct lattice corneal dystrophies (LCD) and Avellino corneal dystrophy. Recently, point mutations in the transforming growth factor-beta-induced gene (TGFBI) encoding for keratoepithelin (KE) have been demonstrated in these corneal disease entities. We intended to investigate if KE was also a component of the rarely seen secondary corneal amyloid deposits.. Immunohistochemical staining with a polyclonal antibody against KE was performed on formalin-fixed paraffin-embedded tissue of five corneal buttons with secondary amyloid obtained after keratoplasty. Secondary amyloidosis was due to Fuchs endothelial dystrophy (FED) with bullous keratopathy and/or recurrent erosions in all cases. The diagnosis had been established by light microscopy using Congo red staining. Two cases of LCD type I served as positive controls and three corneas with FED and one with keratoconus without amyloid served as negative controls.. All corneas with secondary amyloidosis as well as LCD type I revealed positive staining in the respective amyloid deposits. KE was localized in the subepithelial pannus and in the anterior stroma in the corneas with secondary amyloidosis. In the specimens with LCD type I it was distributed in the amyloid deposits located in the anterior and mid-stroma. Staining for KE showed a granular appearance in all cases. The intensity of staining was variable among the specimens.. KE is found not only in primary amyloid deposits of hereditary corneal dystrophies, but also in secondary amyloidosis of the cornea of diverse ethiologies.

Topics: Adult; Aged; Aged, 80 and over; Amyloid; Amyloidosis; Corneal Diseases; Extracellular Matrix Proteins; Female; Humans; Immunohistochemistry; Keratoplasty, Penetrating; Male; Middle Aged; Transforming Growth Factor beta

To determine whether primary, polymorphic, corneal amyloid deposition is associated with a mutation of the TGFBI gene.. Interventional case series of 8 patients. Slit lamp examination of all patients and photodocumentation of 5 patients were performed. Genomic DNA was isolated from buccal mucosal swabs obtained from all patients and all 17 exons of the TGFBI gene were amplified and sequenced.. Multiple polymorphic, refractile deposits were noted throughout the central corneal stroma in all patients. The deposits appeared gray-white on direct illumination and translucent on retroillumination, characteristic of amyloid. In 2 patients, linear, branching opacities, reminiscent of lattice corneal dystrophy, were identified. Histopathologic examination confirmed the presence of stromal amyloid in the cornea of 1 patient who required corneal transplantation for pseudophakic corneal edema. Screening of the entire coding region of the TGFBI gene revealed 4 previously described synonymous substitutions, Leu217Leu, Val327Val, Leu472Leu, and Phe540Phe. A previously unreported missense change, Asp299Asn, was identified in one affected patient but not in her affected sister. No pathogenic mutations, including the Ala546Asp missense mutation previously associated with polymorphic corneal amyloidosis, were identified in any of the patients.. TGFBI gene mutations were not identified in a series of patients with polymorphic corneal amyloid deposition. As bilateral, discrete stromal amyloid deposits may be dystrophic or degenerative, differentiation between these phenotypically similar conditions is facilitated with the use of molecular genetic analysis.

Topics: Aged; Aged, 80 and over; Amyloid; Amyloidosis, Familial; Corneal Diseases; Corneal Stroma; DNA Mutational Analysis; Extracellular Matrix Proteins; Female; Humans; Middle Aged; Mutation; Polymerase Chain Reaction; Transforming Growth Factor beta

Amniotic membrane (AM) transplantation is an accepted procedure in ocular surgery. However, little is known of the interdonor and intradonor variability within the membrane. In addition, the effects of the methods of processing, storage, and preoperative preparation on the membrane are not fully elucidated. The purpose of this study was to use TGF-beta as an example to investigate interdonor and intradonor variability and to determine the effect of "handling " on TGF-beta1 within fresh, processed and stored, and transplantation-ready AM (TRAM).. Seventeen human AMs, both fresh and handled, were analyzed for TGF-beta1 by real-time polymerase chain reaction, immunohistochemistry, SDS-PAGE, and Western blotting.. TGF-beta1 was the highest normalized expressed isoform of TGF-beta in all samples, but it varied between membranes of different donors and at different sites within the same membrane. The highest concentration was noted in the spongy layer. Removal of the spongy layer successfully removed the bulk of TGF-beta1 from TRAM. Latency-associated protein (LAP) and a latent TGF-beta-binding protein (LTBP) were also detected.. TGF-beta1 is present in various regulatory forms in the AM. A degree of intermembrane and intramembrane variation is modified by handling. Unless a standardized protocol is adopted that delivers a membrane with consistent constituents, clinical outcomes may vary and comparisons may be invalid.

Topics: Amnion; Antigens, Viral; Blotting, Western; Corneal Diseases; Electrophoresis, Polyacrylamide Gel; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; Specimen Handling; Tissue Donors; Transforming Growth Factor beta; Transforming Growth Factor beta1

To report a case of stellate and branching linear corneal stromal amyloid deposits secondary to trichiasis and the use of molecular genetic analysis to exclude lattice corneal dystrophy.. Case report and review of the literature. A 30-year-old man with a history of chronic ocular irritation was found to have distichiasis, epiblepharon, and unilateral corneal amyloidosis indistinguishable from lattice corneal dystrophy. Screening of the TGFBI gene was performed to rule out a previously reported mutation associated with lattice corneal dystrophy.. A corneal biopsy performed before presentation to the authors confirmed the presence of corneal amyloidosis. Screening of exons 4, 11, 12, and 14 in the TGFBI gene identified 2 previously reported polymorphisms, Leu472Leu and Phe540Phe, but no other coding region changes.. Corneal stromal amyloidosis clinically resembling lattice corneal dystrophy may be associated with trichiasis. The exclusion of a TGFBI-associated corneal dystrophy in this case, leaving trichiasis as the most likely cause of the corneal amyloid deposition, demonstrates the utility of molecular genetic analysis in confirming or refuting a presumptive clinical diagnosis.

Topics: Adult; Amyloidosis; Corneal Diseases; Corneal Stroma; Exons; Extracellular Matrix Proteins; Eyelashes; Eyelid Diseases; Hair Diseases; Humans; Male; Polymorphism, Single Nucleotide; Transforming Growth Factor beta

An alkali burn in the cornea is a common serious clinical problem often leading to permanent visual impairment. Since transforming growth factor-beta (TGF-beta) is involved in the response to corneal injury, we evaluated the therapeutic effects of adenoviral gene transfer of mouse bone morphogenic protein-7 (BMP-7), which has antagonistic effects on TGF-beta in tissue fibrosis. Burned cornea did not express endogenous BMP-7 mRNA and protein. Resurfacing of the burned cornea by invading conjunctival epithelium was accelerated by adenoviral introduction of BMP-7. Exogenous BMP-7 expression also suppressed myofibroblast generation, appearance of monocytes/macrophages and expression of MCP-1, TGF-betas, and collagen I alpha2 chain in the affected stroma. Ectopic BMP-7 did not suppress stromal neovascularization throughout the interval studied and also did not reduce VEGF mRNA expression at Day 10. Ectopic BMP-7 in burned corneal tissue resulted in activation of Smad1/5/8 signaling and partial suppression of the phospho-Smad2 signal. These data suggest that overexpression of BMP-7 is an effective strategy for treatment of ocular alkali burns.

Topics: Adenoviridae; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Division; Corneal Diseases; Disease Models, Animal; DNA-Binding Proteins; Eye Injuries; Gene Transfer Techniques; Immunohistochemistry; Mice; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad Proteins; Smad1 Protein; Trans-Activators; Transforming Growth Factor beta

The present study was undertaken to create a conditional knockout of AP-2alpha in the corneal epithelium.. A line of mice expressing Cre-recombinase specifically in the early lens placode was crossed with mice in which the AP-2alpha allele is flanked by two loxP sites. The resultant Le-AP-2alpha mutants exhibited a targeted deletion of AP-2alpha in lens placode derivatives, including the differentiating corneal epithelium.. The Le-AP-2alpha mutant mice were viable and had a normal lifespan. The adult corneal epithelium exhibited a variation in the number of stratified epithelial layers, ranging from 2 to 10 cell layers. A substantial decrease in expression of the cell-cell adhesion molecule, E-cadherin, was observed in all layers of the Le-AP-2alpha mutant corneal epithelium. The basement membrane, or Bowman's layer, was thinner in the mutant cornea and in many regions was discontinuous. These defects corresponded with altered distribution of laminin and entactin, and to a lesser degree, type IV collagen. The Le-AP-2alpha mutant cornea also exhibited stromal defects, including disrupted organization of the collagen lamellae and accumulation of fibroblasts beneath the epithelium that showed increased immunoreactivity for proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (alpha-SMA), p-Smad2, and TGF-beta2.. In the absence of AP-2alpha, the corneal epithelium exhibits altered cell adhesion and integrity and defects in its underlying basement membrane. These defects likely caused the alterations in the corneal stroma.

Topics: Actins; Animals; Basement Membrane; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Collagen Type IV; Corneal Diseases; Corneal Stroma; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Gene Deletion; Laminin; Membrane Glycoproteins; Mice; Mice, Knockout; Proliferating Cell Nuclear Antigen; Transforming Growth Factor beta; Transforming Growth Factor beta2

To characterize the clinicopathologic phenotype as well as the molecular genetic basis of an autosomal dominant form of corneal amyloidosis.. Clinicopathologic and molecular genetic study of a family with a form of corneal amyloidosis.. Forty-nine individuals from one family were studied.. The medical records of affected family members were reviewed, and corneal tissue from those who had undergone penetrating keratoplasty (PK) was examined. Several family members were examined clinically, and corneas were photographed. Deoxyribonucleic acid from blood or buccal swabs was extracted from each consenting family member to determine the status of their transforming growth factor beta-induced (TGFBI) gene. The coding region of the TGFBI gene was analyzed for mutations in the proband's DNA, and compared with the nucleotide sequences of normal individuals. This was performed by amplifying and sequencing all exons of the TGFBI gene. In all other family members, only exons 4, 8, 11, and 12 of the gene were amplified, sequenced, and analyzed for mutations.. Clinicopathologic manifestations in relation to mutational status of the TGFBI gene.. Slit-lamp biomicroscopy revealed bilateral multiple polymorphic, polygonal, refractile, chipped ice-appearing gray and white opacities. There were also occasional deep filamentous lines that did not form a distinct lattice pattern. Corneal tissue of affected individuals who underwent PK contained widespread deposits of amyloid within the corneal stroma, particularly in the deep central stroma. Twelve members of the family were found to have a heterozygous single mutation in the TGFBI gene leading to a predicted amino acid substitution of aspartic acid for alanine (A546D). Nine of these individuals had ophthalmologist-documented corneal disease. The remaining 3, who were 11, 14, and 15 years old, were asymptomatic. In addition, 4 inconsequential polymorphisms with the nucleotide changes 387 G/A (R129R), 981 G/A (V327V), 1416 T/C (L472L), and 1620 C/T (F540F) were found.. A distinct, progressive form of corneal amyloidosis with an autosomal dominant mode of inheritance is characterized clinically by the presence of refractile polymorphic corneal opacities. We have designated this entity, which is caused by an A546D mutation in the TGFBI gene, polymorphic corneal amyloidosis.

Topics: Adolescent; Adult; Aged; Amyloidosis; Child; Corneal Diseases; Corneal Opacity; DNA Mutational Analysis; Extracellular Matrix Proteins; Female; Genes, Dominant; Humans; Keratoplasty, Penetrating; Male; Middle Aged; Mutation, Missense; Pedigree; Polymerase Chain Reaction; Reoperation; Transforming Growth Factor beta

To describe histopathological and immunohistochemical findings in human corneas after myopic laser in situ keratomileusis (LASIK) followed by iatrogenic keratectasia and after hyperopic LASIK.. Department of Ophthalmology, University of Innsbruck, Innsbruck, Austria.. Clinical, histological, and immunohistochemical investigations were performed of 1 human cornea with iatrogenic keratectasia following myopic LASIK and 1 human cornea with irregular astigmatism and central scar formation after hyperopic LASIK. Corneal buttons were obtained during penetrating keratoplasty in both patients.. Histopathological examination showed thinning of the central stroma with a posterior residual thickness of 190 microm in the patient with iatrogenic keratectasia after myopic LASIK and significant midperipheral thinning in the patient who had hyperopic LASIK. However, this characteristic ablation profile of the stroma after hyperopic LASIK was partially mitigated and compensated by the epithelium, which was significantly thinned in the center and markedly thickened in the midperiphery. Traces of wound healing with minimal scar tissue were present at the flap margin after myopic and hyperopic LASIK. In a few sections of the cornea with keratectasia after myopia LASIK, only a few collagen lamellae were visible crossing between the posterior residual stroma and the superficial flap. Immunohistochemical examination revealed minimally increased staining of dermatan sulfate proteoglycan within the stroma adjacent to the interface of the microkeratome incision. Increased staining of hepatocyte growth factor was found on keratocytes/fibroblasts at the flap margin in both corneas.. The wound-healing response is generally poor after LASIK, which may result in significant weakening of the tensile strength of the cornea after myopic LASIK, probably due to biomechanically ineffective superficial lamella. After LASIK in patients with high hyperopia, compensatory epithelial thickening in the annular midperipheral ablation zone might be partly responsible for regression.

Topics: Adult; Chondroitin Sulfate Proteoglycans; Collagen; Cornea; Corneal Diseases; Dermatan Sulfate; Dilatation, Pathologic; Female; Hepatocyte Growth Factor; Humans; Hyperopia; Iatrogenic Disease; Immunoenzyme Techniques; Keratan Sulfate; Keratomileusis, Laser In Situ; Keratoplasty, Penetrating; Male; Middle Aged; Myopia; Platelet-Derived Growth Factor; Transforming Growth Factor beta

The closely related mycobacteria responsible for tuberculosis produce an unusually high number of secreted proteins, many of which are clearly implicated in pathogenesis and protective immunity. Falling within this category are the closely related proteins MPB70 and MPB83. The structure of MPB70 reveals a complex and novel bacterial fold, which has clear structural homology to the two C-terminal FAS1 domains of the cell adhesion protein fasciclin I, whose structures were reported very recently. Assessment of the surface features of MPB70, the sequence divergence between MPB70 and MPB83, the conservation of residues across a group of FAS1 domains, and the locations of disease-inducing mutations in betaig-h3 strongly suggests that MPB70 and MPB83 contain two functional surfaces on opposite faces, which are probably involved in binding to host cell proteins. This analysis also suggests that these functional surfaces are retained in the FAS1 proteins associated with mediating interactions between cells and the extracellular matrix (fasciclin I, periostin, and betaig-h3) and furthermore that some of the human corneal disease-inducing substitutions identified in betaig-h3 will perturb interactions at these sites.

Topics: Amino Acid Sequence; Antigens, Bacterial; Bacterial Proteins; Cell Adhesion Molecules; Cell Adhesion Molecules, Neuronal; Corneal Diseases; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Mutation; Mycobacterium tuberculosis; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Transforming Growth Factor beta; Tuberculosis

Pseudophakic bullous keratopathy (PBK) is a major indication for corneal transplantation. Previous studies showed that PBK corneas had increased levels of insulin-like growth factor-I (IGF-I), bone morphogenetic protein-4 (BMP-4), transforming growth factor-beta (TGF-beta), interleukin-1alpha (IL-1alpha) and IL-8. The PBK corneas also had accumulations of tenascin-C (TN-C), fibrillin-1 (Fib-1), matrix metalloproteinase-2 (MMP-2), inflammatory cells but not myofibroblasts. Our goal is to determine if the growth factors/cytokines that are elevated in PBK corneas alter the expression of extracellular matrix (ECM) and/or degradative enzymes in vitro.. Stromal cell cultures from normal and PBK human corneas were established and treated for 6 days with IGF-I, BMP-4, IL-1alpha, IL-8 or TGF-beta1/beta2. Immunostaining, Western blot and dot blot analyses for TN-C, Fib-1, alpha-smooth muscle actin (alpha-SMA, a marker for myofibroblasts) or tissue inhibitor of metalloproteinase-1 (TIMP-1) were performed. RNAs were collected and analyzed with Northern blots for TN-C, Fib-1 and beta(2)-microglobulin. Culture media were analyzed using gelatin zymography with or without ethylenediaminetetraacetic acid (EDTA). Some samples were activated with p-aminophenylmercuric acetate (APMA) and reduction/alkylation, and the degradative activities were measured by the MMP-gelatinase activity assay kit.. The IGF-I and TGF-beta1/TGF-beta2 increased (a) TN-C protein deposition, and (b) Fib-1 protein and RNA levels, but (c) had no significant affect on TIMP-1, matrix metalloproteinase-2 (MMP-2) or gelatinase activities. TGF-beta1/TGF-beta2 induced alpha-SMA protein (myofibroblasts) while IGF-I did not. BMP-4, IL-1alpha and IL-8 had little affect on the cells.. Based upon our data, the fibrotic markers, TN-C and Fib-1, found in PBK corneas may be accounted for by IGF-I and TGF-beta. These growth factors promote fibrosis and ECM deposition without promoting proteolysis. While the other growth factors/cytokines are elevated in PBK corneas, their role(s) in PBK pathogenesis are not clear. In addition, exogenous IGF-I most closely elicited a response that was most similar to the characteristics of the PBK/ABK corneas, i.e. accumulation of TN-C and Fib-1 proteins in the absence of myofibroblasts.

Topics: Blotting, Northern; Blotting, Western; Cells, Cultured; Corneal Diseases; Extracellular Matrix; Fibrillin-1; Fibrillins; Humans; Insulin-Like Growth Factor I; Matrix Metalloproteinases; Microfilament Proteins; Pseudophakia; Stromal Cells; Tenascin; Transforming Growth Factor beta

Excellent long-term prognosis of penetrating corneal grafts has been explained by the immunological privilege of the cornea and the anterior chamber. In animal models the secretion of transforming growth factor beta(2) (TGF-beta(2)) into the anterior chamber and the expression of the Fas ligand on corneal endothelial cells were identified as important for the integrity of the immunological privilege.. To determine the TGF-beta(2) and soluble Fas ligand (sFasL) levels in the aqueous humor of patients after penetrating keratoplasty (PK) who have and who do not have immune reactions.. Anterior chamber puncture was performed in 13 patients who had a cataract without PK (group 1), in 31 patients after PK who did not have immune reactions (group 2), and in 12 patients after PK newly diagnosed as having endothelial immune reactions (group 3). Total TGF-beta(2) and sFasL were determined via enzyme-linked immunosorbent assay.. Transforming growth factor beta(2) was detected in all patients, irrespective of the underlying condition; there was no difference in TGF-beta(2) levels between the different groups (P =.89, analysis of variance). None of the patients in group 1, 11 of 31 patients in group 2, and 8 of 12 patients in group 3 had detectable sFasL concentrations (P =.002, chi(2) test). Soluble Fas ligand averaged (mean [SD]) 20.8 (31.1) pg/mL in group 2, and 38.1 (33.2) pg/mL (P<.01, analysis of variance) in group 3.. It appears that total TGF-beta(2) is maintained at high steady-state levels, while the level of sFasL is up-regulated in patients who underwent PK, particularily in the advent of graft rejection.

Topics: Aged; Aqueous Humor; Cell Count; Corneal Diseases; Endothelium, Corneal; Fas Ligand Protein; Humans; Keratoplasty, Penetrating; Membrane Glycoproteins; Middle Aged; Solubility; Transforming Growth Factor beta; Transforming Growth Factor beta2

The purpose of this study was to identify the growth factors and cytokines present in normal and diseased corneas. Total RNA was isolated from normal and diseased corneas. cDNA was synthesized from individual corneas and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with primers to IL-1alpha, 1IL-8, PDGF-B, BMP-2, BMP-4, IGF-I, TGF-beta2, FGF-2, and VEGF. After normalization to beta2-microglobulin, several factors were identified that were significantly different from normal. Antibodies to IGF-I, BMP-2, VEGF and TGF-beta2 were used for immunohistochemistry. A total of 93 corneas were used for this study including 31 normal, 20 keratoconus, 19 bullous keratopathy (pseudophakic and aphakic, PBK/ABK), and 23 diabetic corneas. The VEGF RNA levels were significantly decreased in the keratoconus and PBK/ABK corneas but increased in the diabetic corneas. BMP-2 gene expression was lower than normal in the PBK/ABK and diabetic corneas. IGF-I and BMP-4 RNA levels were increased in PBK/ABK. In the immunohistochemical studies, the protein patterns paralleled those found at the mRNA level. The only exception was IGF-I in diabetic corneas that showed increased staining in the epithelium and its basement membrane without a significant increase in mRNA levels. TGF-beta2 mRNA and protein levels were similar to normal in all diseased corneas. Thus, no alterations in the tested growth factors/cytokines were unique to keratoconus corneas. In contrast, PBK/ABK corneas had specific significant elevations of BMP-4 and IGF-I. Diabetic corneas were unique in their increased VEGF mRNA levels. These data suggest that while some growth factor/cytokine alterations are non-specific and can be found in multiple corneal diseases, there are others that are unique to that disease.

Topics: Adult; Aged; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Case-Control Studies; Corneal Diseases; Cytokines; Diabetes Complications; Diabetes Mellitus; DNA, Complementary; Endothelial Growth Factors; Fibroblast Growth Factors; Gene Expression; Growth Substances; Humans; Insulin-Like Growth Factor I; Interleukin-1; Interleukin-8; Keratoconus; Platelet-Derived Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transforming Growth Factor beta

To investigate the relation between corneal haze formation and transforming growth factor-beta (TGF-beta) after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK).. Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.. White rabbits were divided into 4 groups, with each group receiving 1 of the following surgeries: manual epithelial abrasion, PRK, lamellar keratotomy, or LASIK. The degree of corneal haze was quantitatively analyzed by measuring the light scattering intensity of corneas before and 4 and 12 weeks after surgery. The expression of type IV collagen and TGF-beta1 in the corneas at baseline and at 4 weeks was examined immunohistochemically.. The light scattering intensity was significantly greater 4 and 10 weeks after PRK. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not influence the light scattering intensity of the corneas. Type IV collagen was detected in the basal lamina of the corneal epithelium and in Descement's membrane in the normal cornea. After epithelial abrasion, there was no change in the distribution of type IV collagen. Four weeks after PRK, the expression of type IV collagen was detected in the subepithelial layer of the laser-ablated area. Four weeks after lamellar keratotomy, type IV collagen was linearly and fragmentarily detected in the corneal stroma. Four weeks after LASIK, type IV collagen was linearly and continuously detected in the corneal stroma and was detected slightly in the subepithelial region of the laser-ablated area. In the normal corneas, the expression of TGF-beta1 was not detected in the keratocytes. Four weeks after PRK, the expression of TGF-beta1 increased in the keratocytes that proliferated in the subepithelial fibrous layer. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not change the expression pattern of TGF-beta1 in the keratocytes.. The multiplier effect of epithelial abrasion and excimer laser ablation in PRK may increase the expression of TGF-beta1 in keratocytes and induce corneal haze.

Topics: Animals; Collagen Type IV; Cornea; Corneal Diseases; Corneal Stroma; Fibrosis; Immunoenzyme Techniques; Keratomileusis, Laser In Situ; Lasers, Excimer; Light; Male; Photorefractive Keratectomy; Rabbits; Scattering, Radiation; Transforming Growth Factor beta; Transforming Growth Factor beta1

Due to the immune privilege of the anterior eye chamber, the success rate of corneal transplantation can reach 90%. The aim of this study was to determine cytokine profile in aqueous humor of patients undergoing corneal transplantation, and to establish whether cytokine profile at the time of surgery influenced corneal graft outcome.. Proinflammatory (TNF-beta and IFN-gamma) and immunosuppressive (TGF-beta2) cytokine levels were measured in aqueous humor and serum of 44 patients. Non-inflammatory corneal diseases included keratoconus (n=8), bullous keratopathy (n=7), and stromal dystrophy (n=3). Inflammatory diseases included corneal scars (n=10), graft rejection (n=5), pending perforation (n=4), chemical burns (n=4), rejection/ uveitis (n=1), infectious keratitis (n=1), and perforated ulcer (n=1). Control aqueous humor and sera were obtained from cadavers without corneal pathology.. The concentration of TGFbeta2 in the aqueous humor in non-inflammatory corneal diseases was similar to that of controls (2,605+/-204 pg/mL vs 2,200+/-100 pg/mL). In inflammatory corneal diseases, the concentration of TGFbeta2 in aqueous humor was significantly lower (1,400+/-375 pg/mL, p<0.001). TNF-beta was detected in the aqueous humor of 16 out of 26 patients with inflammatory corneal diseases and in all patients with stromal dystrophies, but was undetectable in cases of keratoconus and bullous keratopathy. Low levels of IFN-gamma were present in all aqueous humor samples. Patients' sera contained significantly less cytokine (up to 252 pg/mL) then their aqueous humor (p<0.001). We have set an arbitrary cut-off point for TGF-beta2 level in aqueous humor at 1,500 pg/mL and divided all investigated samples (from 44 patients and 10 controls) into two groups, one with high and the other with low TGF-beta2 concentration. The coefficient of contingency showed that patients with high TGF-beta2 concentration in their aqueous humor had significantly greater chance for graft acceptance than those with low TGF-beta2 concentration (p<0.001).. High TGFbeta2 concentrations in the eyes without intraocular inflammation suggest its immunosuppressive role in human eyes. High concentration of TGFbeta2 (>1,500 pg/mL) was associated with graft acceptance. Also, absence of proinflamatory TNFbeta increased the graft acceptance, but independently from TGFbeta2.

Topics: Adult; Aged; Aged, 80 and over; Aqueous Humor; Biomarkers; Cornea; Corneal Diseases; Corneal Transplantation; Enzyme-Linked Immunosorbent Assay; Female; Graft Rejection; Humans; Interferon-gamma; Lymphotoxin-alpha; Male; Middle Aged; Statistics, Nonparametric; Transforming Growth Factor beta; Treatment Outcome

BetaIG-H3 is a recently described extracellular matrix protein that is present in various organs. In rabbit corneas, increased betaIG (the rabbit form of betaIG-H3) mRNA levels were shown during corneal development and wound healing. In this study, we investigated the localization of betaIG-H3 protein in scarring human corneas.. Corneal buttons obtained during keratoplasty were examined. Immunohistological detection using a polyclonal antipeptide antibody against the betaIG-H3 protein was performed on 24 pathological corneas (9 ulcerations, 8 alkali burns, 2 perforating injuries, 5 bullous keratopathy) and 2 normal corneas.. In normal corneas, strong staining was present in the basal layer of the epithelium and in the endothelium; the stromal fibers showed faint, uniform immunoreactivity. In all scarring corneas, the epithelium was usually thickened and all of its layers were reactive with the betaIG-H3 antibody. The cytoplasm of the stromal fibroblasts, as well as the stromal fibers around them also showed staining with the antibody. These changes were present in all scarring corneas, irrespective of the pathological process leading to scar formation.. These results prove, at the protein level, the presence of increased amounts of betaIG-H3 at the sites of scarring in human corneas.

Topics: Alkalies; Cicatrix; Cornea; Corneal Diseases; Corneal Injuries; Epithelium, Corneal; Extracellular Matrix Proteins; Fibroblasts; Humans; Immunohistochemistry; Neoplasm Proteins; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Wound Healing

To evaluate the efficacy of autologous serum application for the treatment of persistent epithelial defect.. Prospective, clinical, noncomparative case series.. A total of 16 eyes were studied.. Autologous serum was prepared from the patients and diluted to 20% by saline. The patients were instructed to use the autologous serum six to ten times a day. The concentration of vitamin A, epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta) was measured at 1 week and 1 month stored in the refrigerator and 1 month and 3 months in the freezer.. Time to closure of epithelial defect.. Vitamin A, EGF, and TGF-beta were stable during the 1 month in the refrigerator and 3 months in the freezer. Among 16 persistent epithelial defects, 7 (43.8%) healed within 2 weeks, 3 (18.8%) healed within 1 month, and the remaining 6 (37.5%) did not respond within 1 month. No apparent side effect of autologous serum application was observed.. Autologous serum application healed 43.8% of persistent defect within 2 weeks and 62.5% within 1 month.

Topics: Adult; Aged; Blood; Cell Movement; Corneal Diseases; Dry Eye Syndromes; Epidermal Growth Factor; Epithelium, Corneal; Female; Humans; Male; Middle Aged; Prospective Studies; Transforming Growth Factor beta; Vitamin A; Wound Healing

Using a multiprobe RNase protection assay, we examined cytokine and chemokine mRNAs that were expressed after corneal infection with Pseudomonas aeruginosa in mice. Cytokines that were upregulated included interleukin-1alpha (IL-1alpha) and -1beta, IL-1 receptor antagonist, IL-6, IL-11, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, lymphotoxin beta, transforming growth factor beta1, and tumor necrosis factor alpha. Chemokine transcripts that were upregulated included Eotaxin; gamma-interferon-inducible protein 10; monocyte chemoattractant protein 1; macrophage inflammatory proteins 1alpha, 1beta, and 2; and RANTES. Peak expression of these cytokines and chemokines was observed between 1 and 3 days after infection. These responses returned to or approached baseline preinfection levels by 7 days after ocular challenge. Identification of the various cytokines and chemokines upregulated during corneal infection provides important information relevant to unraveling the pathogenesis induced by this bacterium and provides hope that specific molecules can be targeted for therapy.

Topics: Animals; Chemokine CCL11; Chemokine CCL2; Chemokine CCL4; Chemokine CCL5; Chemokine CXCL2; Chemokines; Chemokines, CC; Corneal Diseases; Cytokines; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-11; Interleukin-6; Lymphotoxin-alpha; Lymphotoxin-beta; Macrophage Colony-Stimulating Factor; Macrophage Inflammatory Proteins; Membrane Proteins; Mice; Mice, Inbred ICR; Monokines; Pseudomonas Infections; RNA, Messenger; Sialoglycoproteins; Stem Cell Factor; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Chronic exposure of the gray, short-tailed opossum, Monodelphis domestica, to ultraviolet radiation (UVR) induces highly vascularized mesenchymal tumors of the cornea. Cell lines derived from these UVR-induced corneal tumors and the corneal tumors themselves were examined for the presence of mRNA coding for basic and acidic fibroblast growth factors (FGF), transforming growth factors-beta and -alpha (TGF-beta and TGF-alpha), epidermal growth factor (EGF), and tumor necrosis factor-alpha (TNF-alpha). Basic FGF was expressed in the cell lines derived from corneal tumors and in the corneal tumors. Expression of basic FGF was high in one corneal tumor. Transcripts for acidic FGF were detected only in the corneal tumor cell lines, not in primary tumors. TGF-beta expression was detected in the corneal tumors and tumor-derived cell lines. TGF-alpha, EGF, and TNF-alpha transcripts were not detectable in any opossum material; however, homologous gene sequences for TGF-alpha and EGF were detected on Southern blots of opossum genomic DNA. Southern blot analysis revealed no evidence of amplification or rearrangement of the genes for basic FGF or acidic FGF in the UVR-induced corneal tumor that expressed high levels of basic FGF. Opossum basic FGF, which stimulated the proliferation of fetal bovine heart endothelial cells, was purified by heparin affinity chromatography from a UVR-induced corneal tumor and a corneal tumor cell line. Immunoblotting of opossum basic FGF from a corneal tumor cell line using antiserum to bovine basic FGF showed two prominent immunoreactive bands of 17.5 and 18.5 kDa. Expression of basic FGF and acidic FGF may play a role in the development and progression of UVR-induced corneal tumors in M. domestica.

Topics: Animals; Cell Line; Cornea; Corneal Diseases; DNA, Neoplasm; Eye Neoplasms; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression; Gene Rearrangement; Neoplasms, Radiation-Induced; Opossums; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Ultraviolet Rays