transforming-growth-factor-beta and Keratoconus

This study aims to investigate the genetic abnormalities in a two-generation Chinese family affected by keratoconus (KC). A two-generation Chinese family affected by KC was studied.. A total of 118 unrelated healthy individuals without KC were recruited as controls. The family history, clinical data, and peripheral blood leukocytes were collected from all subjects. Whole exome sequencing was performed using the genomic DNA of the proband (II.2) and the other two affected family members (I.1 and II.3). Afterwards, polymerase chain reaction was performed for the other enrolled subjects to verify the variants identified in family members with KC. The PolyPhen2, SIFT, PROVEIN and Mutation Taster software programs were applied to analyze the functional consequences of the variants.. A single nucleotide polymorphism (VARIANT) (c.1406 G > A [rs759370852]) in the transforming growth factor beta-induced (. A novel variant c.1406 G > A in

Topics: Asian People; China; Corneal Dystrophies, Hereditary; DNA Mutational Analysis; Extracellular Matrix Proteins; Humans; Keratoconus; Mutation; Pedigree; Polymorphism, Single Nucleotide; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study is to summarize the concurrent keratoconus (KC) and granular corneal dystrophy (GCD) phenotype and identify the underlying genetic cause in a 23-year-old male patient.. A detailed family history and clinical data from the patient and his parents were collected by ophthalmologic examination. The candidate genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing.. The proband was clinically diagnosed as a case of concurrent KC and GCD, which is a very rare presentation. His father and grandmother were diagnosed as GCD in both eyes. There was no character of KC in his father's and grandmother's eyes. A heterozygous TGFBI mutation in exon 4 (c.370G > A) was identified in the proband, which was predicted to generate a missense mutation (p.R124H). The mutation also existed in his father and grandmother. A heterozygous KRT12 mutation in exon 8 (c.1456-1457ins GTA) was identified in the proband, which was predicted to generate an insert mutation and created a premature termination codon. The mutation did not exist in his father and grandmother. The two mutations did not exist in his mother and 200 unrelated normal controls.. KC can co-exist with GCD. The missense mutation (c.370G > A) in the TGFBI gene and insert mutation (c.1456-1457ins GAT) in the KRT12 gene were identified in a 23-year-old male patient with concurrent KC and GCD.

Topics: China; Corneal Dystrophies, Hereditary; DNA; DNA Mutational Analysis; Female; Heterozygote; Humans; Keratin-12; Keratoconus; Male; Middle Aged; Mutation, Missense; Pedigree; Polymerase Chain Reaction; Transforming Growth Factor beta; Young Adult

To understand better the factors contributing to keratoconus (KTCN), we performed comprehensive transcriptome profiling of human KTCN corneas for the first time using an RNA-Seq approach. Twenty-five KTCN and 25 non-KTCN corneas were enrolled in this study. After RNA extraction, total RNA libraries were prepared and sequenced. The discovery RNA-Seq analysis (in eight KTCN and eight non-KTCN corneas) was conducted first, after which the replication RNA-Seq experiment was performed on a second set of samples (17 KTCN and 17 non-KTCN corneas). Over 82% of the genes and almost 75% of the transcripts detected as differentially expressed in KTCN and non-KTCN corneas were confirmed in the replication study using another set of samples. We used these differentially expressed genes to generate a network of KTCN-deregulated genes. We found an extensive disruption of collagen synthesis and maturation pathways, as well as downregulation of the core elements of the TGF-β, Hippo, and Wnt signaling pathways influencing corneal organization. This first comprehensive transcriptome profiling of human KTCN corneas points further to a complex etiology of KTCN.

Topics: Case-Control Studies; Collagen; Cornea; Down-Regulation; Hippo Signaling Pathway; Humans; Keratoconus; Protein Serine-Threonine Kinases; Transcriptome; Transforming Growth Factor beta; Wnt Signaling Pathway

Keratoconus (KTCN) is a degenerative disorder of the eye that results in the conical shape and thinning of the cornea and is a leading cause for corneal transplantations. A number of studies suggest that genetic factors play a role in KTCN etiology. Some candidate gene variants have recently been shown to be associated with KTCN. The purpose of our study was to verify the role of VSX1, TGFBI, DOCK9, IPO5, and STK24 sequence variants in Polish KTCN patients.. Forty-two Polish patients with sporadic KTCN and 50 control individuals were enrolled into this study. Both affected and unaffected individuals underwent detailed ophthalmic examination. The mutations screening in the candidate genes was performed by the direct sequencing method.. Analysis of VSX1, TGFBI, DOCK9, IPO5, and STK24 genes identified numerous sequence variants. Variants c.-264_-255delGGGGTGGGGT, c.627 + 23G > A, c.809-6_809-5insT, and c.*200G > T in the VSX1 gene, and heterozygous c.1598G > A mutation (Arg533Gln) in exon 12 of TGFBI were detected for the first time in KTCN patients. Two known sequence variants of TGFBI c.1620T > C (Phe540Phe) and c.1678 + 23G > A were observed in KTCN patients and control individuals. The newly reported c.717 + 43A > G substitution in intron 7 of DOCK9 was identified in both KTCN patients and healthy individuals.. Our investigation showed that KTCN-related sequence variants of analyzed genes were found in a very small proportion of the studied patients indicating that genes other than VSX1, TGFBI, DOCK9, IPO5, and STK24 are involved in the development and progression of KTCN in Polish patients. Our results support the hypothesis about the genetic heterogeneity of KTCN.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; beta Karyopherins; Extracellular Matrix Proteins; Eye Proteins; Female; Genetic Predisposition to Disease; Genetic Testing; Genomic Structural Variation; Guanine Nucleotide Exchange Factors; Homeodomain Proteins; Humans; Keratoconus; Male; Middle Aged; Poland; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Transforming Growth Factor beta; Young Adult

Keratoconus (KC) is a complex corneal dystrophy with multifactorial etiology. Previous studies have shown evidence of mitochondrial abnormalities in KC; however, the exact cause of these abnormalities remains unknown. The aim of this study was to identify if transforming growth factor-β (TGF-β) isoforms play a role in the regulation of mitochondrial proteins in human KC cells (HKC).. Human corneal fibroblasts (HCF) and HKC were isolated and cultured for 4 weeks in three different conditions: (a). MEM + 10%FBS, (b) MEM + 10%FBS + TGF-β1 and (c) MEM + 10%FBS + TGF-β3. All samples were processed for mitochondrial damage analysis using real-time PCR.. We quantified and analyzed 84 mitochondrial and five housekeeping genes in HCFs and HKCs. Our data showed that when TGF-β1 and/or TGF-β3 were compared with control in HCFs, nine genes were significantly different; however, no genes were significantly regulated by the TGF-β isoforms in HKCs. Significant differences were also seen in seven genes when HFCs were compared with HKCs, in all three conditions.. Overall, our data support the growing consensus that mitochondrial dysfunction is a key player in KC disease. These in vitro data show clear links between mitochondrial function and TGF-β isoforms, with TGF-β1 severely disrupting KC-mitochondrial function, while TGF-β3 maintained it, thus suggesting that TGF-β may play a role in KC-disease treatment.

Topics: Cells, Cultured; Cornea; Fibroblasts; Humans; Keratoconus; Ligands; Mitochondria; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; RNA; Transforming Growth Factor beta

Keratoconus is a corneal disorder characterized by a thinning of stromal tissue, and the affected patients have induced astigmatism and visual impairment. It is associated with a loss of corneal stromal keratocytes (CSKs). Hence, reconstructing stromal tissue with autologous CSK replacement can be a viable alternative to corneal transplantation, which is restricted by the global donor material shortage and graft rejection. Human CSKs are normally quiescent and express unique markers, like aldehyde dehydrogenases and keratocan. In serum culture, they proliferate, but lose their characteristic phenotype and become stromal fibroblasts. Here we report a novel culture cocktail to ex vivo propagate and maintain CSKs. Primary human CSKs were obtained from adult donors and cultured with soluble human amnion stromal extract (ASE), rho-associated coiled-coil-forming protein serine/threonine kinase inhibitor Y-27632, and insulin-like growth factor-1 (collectively named as ERI). Protein profiling using mass spectrometry followed by MetaCore™ pathway analysis predicted that ASE proteins might participate in transforming growth factor-β (TGF-β) signaling and fibroblast development, cell adhesion, extracellular matrix remodeling, and immune response. In culture with 0.5% fetal bovine serum and ERI, the population of "activated keratocytes" was expanded. They had much lowered expression of both keratocyte and fibroblast markers, suppressed TGF-β-mediated Smad2/3 activation, and lacked fibroblast-mediated collagen contractibility. These "activated keratoctyes" could be propagated for six to eight passages ex vivo, and they regained CSK-specific dendritic morphology and gene marker expression, including aldehyde dehydrogenases, lumican, and keratocan biosynthesis, expression, and secretion when returned to serum-depleted ERI condition. This novel cocktail maintained human CSKs in both adherent and suspension cultures with proper keratocyte features and without the transformation to stromal fibroblasts. Thus, human CSKs can be ex vivo propagated as transient "activated keratocytes." This could provide sufficient number of genuine CSKs for corneal tissue engineering.

Topics: Adult; Amides; Amnion; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Chromatography, High Pressure Liquid; Corneal Stroma; Female; Gene Expression Regulation; Humans; Insulin-Like Growth Factor I; Keratinocytes; Keratoconus; Male; Microscopy, Fluorescence; Middle Aged; Proteoglycans; Proteome; Pyridines; Real-Time Polymerase Chain Reaction; Smad2 Protein; Smad3 Protein; Tandem Mass Spectrometry; Tissue Engineering; Transforming Growth Factor beta

Keratoconus(KC) is an ecstatic corneal disease leading to corneal-thinning and the formation of a cone-like cornea. Elevated lactate levels, increased oxidative stress, and myofibroblast formation have all been previously reported. In the current study, we assess the role of Quercetin on collagen secretion and myofibroblast formation in KC in vitro. Human corneal fibroblasts(HCFs) and human keratoconus cells(HKCs) were treated with a stable Vitamin C derivative and cultured for 4 weeks, stimulating formation of a self-assembled extracellular matrix. All samples were analyzed using Western blots and targeted tandem mass spectrometry. Our data showed that Quercetin significantly down regulates myofibroblast differentiation and fibrotic markers, such as α-smooth muscle actin (α-SMA) and Collagen III (Col III), in both HCFs and HKCs. Collagen III secretion was reduced 80% in both HCFs and HKCs following Quercetin treatment. Furthermore, Quercetin reduced lactate production by HKCs to normal HCF levels. Quercetin down regulated TGF-βR2 and TGF-β2 expression in HKCs suggesting a significant link to the TGF-β pathway. These results assert that Quercetin is a key regulator of fibrotic markers and ECM assembly by modulating cellular metabolism and TGF-β signaling. Our study suggests that Quercetin is a potential therapeutic for treatment of corneal dystrophies, such as KC.

Topics: Cells, Cultured; Collagen; Extracellular Matrix; Fibrosis; Humans; Keratoconus; Lactic Acid; Metabolome; Metabolomics; Quercetin; Signal Transduction; Transforming Growth Factor beta

Keratoconus (KC) is a corneal thinning disease of unknown etiology whose pathophysiology is correlated with the presence of a thin corneal stroma and altered extracellular matrix (ECM). Transforming growth factor-β (TGF-β) signaling is a key regulator of ECM secretion and assembly in multiple tissues, including the anterior segment of the eye, and it has been linked to KC. We have previously shown that human keratoconus cells (HKCs) have a myofibroblast phenotype and altered ECM assembly compared to normal human corneal fibroblasts (HCFs). Moreover, TGF-β3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-β signaling that contribute to the observed fibrotic phenotype in HKCs.. HCFs and HKCs were stimulated with TGF-β1, TGF-β2, or TGF-β3 isoforms (0.1 ng/mL) in the presence of a stable vitamin C derivative (0.5 mM) for 4 weeks. All samples were examined using RT-PCR and western blotting to quantify changes in the expressions of key TGF-β signaling molecules between HCFs and HKCs.. We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05). Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7. HCFs also showed an upregulation in TGF-βRI, TGF-βRII, and TGF-βRIII following TGF-β3 treatment, whereas HKCs showed a significant two-fold downregulation.. Overall, our data shows the decreased expressions of the regulatory SMADs SMAD6 and SMAD7 by HKCs contribute to the pathological ECM structure observed in KC, and TGF-β3 may attenuate this mechanism by downregulating the expression of the key profibrotic receptor, TGF-βRII. Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.

Topics: Case-Control Studies; Cells, Cultured; Cornea; Down-Regulation; Extracellular Matrix; Fibroblasts; Humans; Keratoconus; Myofibroblasts; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Smad4 Protein; Smad6 Protein; Smad7 Protein; Transforming Growth Factor beta

We report a Korean case of concomitant keratoconus and granular corneal dystrophy type II.. Case report.. A 29-year-old man visited our clinic for a routine ocular check-up. Slit-lamp examination revealed a few well-circumscribed, greyish-white, discrete granular opacities in the central corneal stromae of both eyes. Direct sequencing of exon 4 of the BIGH3 gene revealed a heterozygous transversion from G to A in the second-nucleotide position of codon 124. In addition, a Fleischer ring and Vogt's striae were evident in the cornea. The corneal topography was suggestive of keratoconus.. Granular corneal dystrophy type II can co-exist with keratoconus and should be included in the differential diagnosis.

Topics: Adult; Corneal Dystrophies, Hereditary; Corneal Topography; Extracellular Matrix Proteins; Humans; Keratoconus; Male; Polymorphism, Single Nucleotide; Transforming Growth Factor beta

Keratoconus (KC) is a complex thinning disease of the cornea that often requires transplantation. The underlying pathogenic molecular changes in this disease are poorly understood. Earlier studies reported oxidative stress, metabolic dysfunctions and accelerated death of stromal keratocytes in keratoconus (KC) patients. Utilizing mass spectrometry we found reduced stromal extracellular matrix (ECM) proteins in KC, suggesting ECM-regulatory changes that may be due to altered TGFβ signals. Here we investigated properties of stromal cells from donor (DN) and KC corneas grown as fibroblasts in serum containing DMEM: F12 or in serum-free medium containing insulin, transferrin, selenium (ITS). Phosphorylation of SMAD2/3 of the canonical TGFβ pathway, was high in serum-starved DN and KC fibroblast protein extracts, but pSMAD1/5/8 low at base line, was induced within 30 minutes of TGFβ1 stimulation, more so in KC than DN, suggesting a novel TGFβ1-SMAD1/5/8 axis in the cornea, that may be altered in KC. The serine/threonine kinases AKT, known to regulate proliferation, survival and biosynthetic activities of cells, were poorly activated in KC fibroblasts in high glucose media. Concordantly, alcohol dehydrogenase 1 (ADH1), an indicator of increased glucose uptake and metabolism, was reduced in KC compared to DN fibroblasts. By contrast, in low glucose (5.5 mM, normoglycemic) serum-free DMEM and ITS, cell survival and pAKT levels were comparable in KC and DN cells. Therefore, high glucose combined with serum-deprivation presents some cellular stress difficult to overcome by the KC stromal cells. Our study provides molecular insights into AKT and TGFβ signal changes in KC, and a mechanism for functional studies of stromal cells from KC corneas.

Topics: Cell Culture Techniques; Cell Proliferation; Cell Survival; Cells, Cultured; Corneal Stroma; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Humans; Insulin; Keratoconus; Mass Spectrometry; Selenium; Signal Transduction; Tissue Donors; Transferrin; Transforming Growth Factor beta

To understand the region point mutations and single nucleotide polymorphisms characteristic of keratoconus candidate gene in Chinese population, the TGFBI.. Polymerase chain reaction-single strand conformation polymorphism and DNA direct sequencing were performed on blood samples from 30 cases of keratoconus patients and 30 normal controls. 17 exons from the coding region of TGFBI gene were examined for point mutations and single nucleotide polymorphisms.. Two types of base mutation were found in exon 12, which were both heterozygous. In 1 patient the site 535 showed GGA→TGA substitution, which was the change from glycine to stop codon (G535X). This was not found in all control cases. In 2 patients and 1 control case the site 540 showed TTT→TTC substitutions without changing of the coding for phenylalanine (F540F), suggesting for the polymorphism.. The candidate keratoconus gene TGFB1 showed genetic variation and mutation in keratoconus population. The gene might play a role in the development of keratoconus in Chinese population.

Topics: Adolescent; Adult; Asian People; Base Sequence; Child; Exons; Extracellular Matrix Proteins; Female; Humans; Keratoconus; Male; Middle Aged; Molecular Sequence Data; Point Mutation; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Sequence Analysis, DNA; Transforming Growth Factor beta; Young Adult

To assess the presence of transforming growth factor-β (TGFβ) pathway markers in the epithelium of keratoconus patient corneas.. Retrospective, comparative case series of laboratory specimens.. Immunohistochemistry results for TGFβ2, total TGFβ, mothers against decacentaplegic homolog (Smad) 2, and phosphorylated Smad2 was performed on formalin-fixed, paraffin-embedded sections of keratoconus patient corneas and normal corneas from human autopsy eyes. Keratoconus patient corneas were divided in two groups, depending on their severity based on keratometer readings and pachymetry. Autopsy controls were age-matched with the keratoconus cases. Immunohistochemistry signal quantification was performed using automated software. Real-time reverse-transcriptase polymerase chain reaction was performed on total ribonucleic acid of epithelium of keratoconus patient corneas and autopsy control corneas.. Immunohistochemistry quantification showed a significant increase in mean signal in the group of severe keratoconus cases compared with normal corneas for TGFβ2 and phosphorylated Smad2 (P < .05). Immunohistochemistry analysis using antibodies against total TGFβ and Smad2 did not show any significant increase in the keratoconus cases versus the autopsy controls. Reverse-transcriptase polymerase chain reaction exhibited elevated messenger ribonucleic acid levels of Smad2 and TGFβ2 in severe keratoconus corneal epithelium.. This work shows increased TGFβ pathway markers in severe keratoconus cases and provides the rationale for investigating TGFβ signaling further in the pathophysiology of keratoconus.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Epithelium, Corneal; Female; Humans; Immunoenzyme Techniques; Keratoconus; Male; Middle Aged; Phosphorylation; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta2

To investigate the point mutations and polymorphisms of transforming growth factor beta-induced gene (TGFBI) in Chinese patients with keratoconus and discuss the relationship between the feature of gene mutations and single nucleotide polymorphisms of TGFBI gene and keratoconus.. Polymerase chain reaction single strand conformation polymorphism and DNA direct sequencing were performed in 30 keratoconus cases and 30 healthy controls. All 17 exons of the TGFBI gene were analyzed for point mutations and single nucleotide polymorphisms.. Totally two heterozygous nucleotide changes were identified in exon 12 of the TGFBI gene. The codon 535 is changed from GGA to TGA in 1 patient, leading to a substitution of glycine to a stop codon at the protein level (G535X). The codon 540 is changed from TTT to TTC in 2 patients and 1 control individual, resulting in a nonsense mutation (F54F), and is a single nucleotide polymorphism of the gene.. Mutation and polymorphisms of the TGFBI gene were detected in Chinese patients with keratoconus in this study. The results suggest that TGFBI gene might play an important role in the pathogenesis of keratoconus.

Topics: Adolescent; Adult; Case-Control Studies; Child; China; Extracellular Matrix Proteins; Female; Glycine; Humans; Keratoconus; Male; Middle Aged; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Sequence Analysis, DNA; Transforming Growth Factor beta; Young Adult

To report the identification and characterization of stromal amyloid deposits in patients with keratoconus.. The excised corneal buttons from 2 patients diagnosed clinically with keratoconus underwent histochemical analysis with Masson trichrome, Congo red, Alcian blue, and periodic acid-Schiff stains, and immunohistochemical analysis for the transforming growth factor beta-induced gene (TGFBI) protein product (TGFBIp), prealbumin, lysozyme, and kappa and lambda light chain expression. After the collection of DNA from both patients, exons 4, 11, 12, 13 and 14 of TGFBI were amplified and sequenced to search for mutations previously associated with dystrophic corneal stromal amyloid deposition.. Light microscopic examination of the corneal buttons revealed stromal thinning, epithelial basement membrane abnormalities, and focal disruption of Bowman layer. Multiple stromal deposits were identified that stained red with Masson trichrome, pink with periodic acid-Schiff, and red with Congo red; the Congo red-stained deposits demonstrated birefringence and dichroism with crossed polarizing lenses. Immunohistochemical staining demonstrated reactivity of the stromal deposits with antibodies to TGFBIp but no reactivity with antibodies against prealbumin, lysozyme, or kappa and lambda light chains. Screening of TGFBI exons 4, 11, 12, 13, and 14 revealed 2 previously identified single nucleotide polymorphisms present in the heterozygous state in both individuals but no other coding region variants.. Two cases of keratoconus with clinically unsuspected, presumed secondary stromal amyloid deposition are described. Although TGFBIp is identified in the stromal deposits, no previously reported amyloidogenic mutations are identified in TGFBI in either affected individual, indicating a previously undescribed mechanism of stromal amyloid deposition.

Topics: Adult; Amyloid; Cornea; Corneal Stroma; Extracellular Matrix Proteins; Genetic Testing; Heterozygote; Humans; Immunohistochemistry; Keratoconus; Male; Middle Aged; Polymorphism, Single Nucleotide; Staining and Labeling; Transforming Growth Factor beta

To investigate whether corneal epithelial cells of individuals with lattice corneal dystrophy (LCD) possess an intrinsic defect.. Retrospective case-control study.. The medical charts of nine individuals with LCD and those of 14 patients with keratoconus and 11 patients with corneal leukoma (controls), all of whom underwent penetrating keratoplasty (PKP) in one eye at Yamaguchi University Hospital between February 1998 and November 2001, were examined for the time for epithelial resurfacing after surgery.. The time required for resurfacing of the corneal epithelium after PKP was significantly greater in LCD patients (8.56 +/- 4.95 days, mean +/- SD) than in patients with either keratoconus (1.71 +/- 0.91 days, P = .006) or corneal leukoma (3.00 +/- 1.95 days, P = .03).. Corneal epithelial wound healing was delayed in LCD patients after PKP, suggesting that the keratoepithelin gene mutations responsible for this condition affect corneal epithelial cells.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Corneal Dystrophies, Hereditary; Epithelium, Corneal; Extracellular Matrix Proteins; Female; Humans; Keratoconus; Keratoplasty, Penetrating; Male; Middle Aged; Mutation; Retrospective Studies; Time Factors; Transforming Growth Factor beta; Wound Healing

Keratoconus is a noninflammatory, corneal thinning disorder leading to mixed myopic and irregular astigmatism and implicated as a major reason for cornea transplantations in the Western world. Genetic factors have been suggested as a cause of keratoconus. The levels of transforming growth factor beta-induced (TGFBI) protein have been reported to be altered in keratoconus tissues. Mutations in this gene are responsible for causing various corneal dystrophies. Given this strong evidence of the involvement of this gene in corneal dystrophies, we investigated possible mutations within this gene in 15 probands of families with keratoconus.. All patients and control individuals had complete ophthalmological examination by a corneal specialist to determine their affectation status. The entire transcript of the TGFBI gene was analyzed by direct sequencing from patient DNA.. We found 8 sequence variations within the gene, none of which was protein-altering changes. These changes were also observed in control individuals, and 4 are previously known polymorphisms.. We concluded that the TGFBI gene is not responsible for causing keratoconus in these patients.

Topics: Amino Acid Substitution; Case-Control Studies; Cohort Studies; DNA; Exons; Extracellular Matrix Proteins; Humans; Introns; Keratoconus; Mutation; Polymorphism, Genetic; Transforming Growth Factor beta

To observe the expression of betaig-h3 in normal cornea and keratoconus and to elucidate the role of extracellular matrix in keratoconus.. In situ hybridization was used to detect the expression of betaig-h3 in the cornea. The cDNA library was screened with human betaig-h3 cDNA probe to locate betaig-h3 mRNA in cells.. Expression of betaig-h3 was found mainly in the stroma of the normal cornea and keratoconus, but decrease depending on the degree of keratopathy. In some serious cases, no expression signal was detected. The strongest expression was seen at the border of the normal region and keratoconus.. betaig-h3, the structural component of the extracellular matrix, can affect cell adhensiveness in the development of corneal fibrous interstitial organization. During the development of keratoconus, decreasing levels of betaig-h3 cause the diminution of corneal steadiness, which is related to formation of keratoconus.

Topics: Cornea; Extracellular Matrix Proteins; Humans; Keratoconus; Neoplasm Proteins; RNA, Messenger; Transforming Growth Factor beta; Wound Healing

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

Keratoconus is a progressive ectatic disease of the cornea. Despite extensive clinical and laboratory investigations, its pathogenesis remains unclear. In this study, we examined the localization of betaig-h3, a recently described extracellular matrix protein in keratoconus corneas both in the absence and presence of subepithelial scarring.. Two normal corneas and central corneal buttons of 10 patients with keratoconus were excised during perforating keratoplasty and examined, including one case with acute corneal hydrops. In one case, keratoconus was associated with Down syndrome. Immunodetection was done with an antipeptide antibody reacting with the N-terminal part of betaig-h3.. We found decreased betaig-h3 levels in the basal epithelial layer and keratocytes of keratoconus corneas. In the scarred corneas, however, betaig-h3 levels were increased in the basal epithelial layers and in activated keratocytes at the places of scarring. In the cornea of the patient with Down syndrome, we found an additional betaig-h3-positive zone in the anterior stroma.. The decreased levels of betaig-h3 corneas seem to be specific for keratoconus. Considering the putative role of betaig-h3 as a cellular-attachment protein, paucity of betaig-h3 in the corneal stroma may lead to decreased mechanical stability and contribute to the development of keratoconus.

Topics: Adolescent; Adult; Cornea; Extracellular Matrix Proteins; Humans; Immunoenzyme Techniques; Keratoconus; Keratoplasty, Penetrating; Middle Aged; Neoplasm Proteins; Transforming Growth Factor beta