Missense mutations in COL8A2, the gene encoding the alpha2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy.
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Corneal clarity is maintained by its endothelium, which functions abnormally in the endothelial dystrophies, leading to corneal opacification. This group of conditions includes Fuchs' endothelial dystrophy of the cornea (FECD), one of the commonest indications for corneal transplantation performed in developed countries, posterior polymorphous dystrophy (PPCD) and the congenital hereditary endothelial dystrophies (CHED). A genome-wide search of a three-generation family with early-onset FECD demonstrated significant linkage with D1S2830 (Z(max) = 3.72, theta = 0.0). Refinement of the critical region defined a 6-7 cM interval of chromosome 1p34.3-p32 within which lies the COL8A2 gene. This encodes the 703 amino acid alpha2 chain of type VIII collagen, a short-chain collagen which is a component of endothelial basement membranes and which represented a strong candidate gene. Analysis of its coding sequence defined a missense mutation (gln455lys) within the triple helical domain of the protein in this family. Mutation analysis in patients with FECD and PPCD demonstrated further missense substitutions in familial and sporadic cases of FECD as well as in a single family with PPCD. This is the first description of the molecular basis of any of the corneal endothelial dystrophies or of mutations in type VIII collagen in association with human disease. This suggests that the underlying pathogenesis of FECD and PPCD may be related to disturbance of the role of type VIII collagen in influencing the terminal differentiation of the neural crest derived corneal endothelial cell. (+info)
Upregulation of collagen VIII following porcine coronary artery angioplasty is related to smooth muscle cell migration not angiogenesis.
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Type VIII collagen is upregulated after vessel injury, and this collagen has been implicated in both smooth muscle cell migration and angiogenesis. This study examines the temporal and spatial pattern of expression of type VIII collagen in porcine coronary vessels at specific time points after balloon angioplasty. In situ hybridization studies demonstrated that collagen VIII messenger ribonucleic acid (mRNA) was markedly elevated in the neoadventitia at 3 days post-angioplasty. By 14 days, elevated collagen VIII message was seen mainly in the neointima and this expression decreased to background levels by 90 days. The distribution of collagen VIII protein, detected using immunohistochemistry, was similar but the up-regulation lagged behind the mRNA increase by a few days. Pre-treatment of sections with pepsin highlighted variations in the organization and appearance of extracellular collagen VIII containing structures in both injured and normal vessels. New vessel formation was evident in the neoadventitia after 3 days, but there was no colocalization of type VIII collagen immunostaining with that of von Willebrand factor (a marker of endothelial cells) in the neoadventitia. These data show that up-regulation of collagen VIII in the neoadventitia is an important early marker of the coronary arterial response to injury, and is not associated with new vessel formation. (+info)
Human corneal epithelial cell viability and morphology after dilute alcohol exposure.
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PURPOSE: To determine the effect of dilute alcohol on human corneal epithelial cellular morphology and viability. Dilute alcohol is used for epithelial removal during photorefractive keratectomy (PRK) and laser subepithelial keratomileusis (LASEK). METHODS: Corneal epithelial sheets harvested from human eyes after alcohol application during PRK were examined by light and electron microscopy (specimens I-IV). In addition, tissue cultures of human epithelial sheets were monitored for epithelial migration and attachment (specimens V-VII). To determine the effect of dilute alcohol on epithelial cell viability, simian virus (SV)40-immortalized human corneal epithelial cells were exposed to dilute alcohol in distilled water (EtOH-H2O) or to keratinocyte serum-free medium (EtOH-KSFM) for incubation periods of 20 to 45 seconds and concentrations of 10% to 70%. Cell membrane permeability and intracellular esterase activity were analyzed by calcein-acetoxymethyl ester (AM)/ethidium homodimer assay. TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells at 0, 8,12, 24, and 72 hours. RESULTS: Electron microscopy showed varying degrees of basement membrane alterations after alcohol application, including disruptions, discontinuities, irregularities, and duplication (specimens I-IV). Cellular destruction and vacuolization of basal epithelial cells associated with absent basement membrane were also observed (specimen III). One of three cultured epithelial sheets showed attachment and outgrowth in the tissue culture until day 15 (specimen V). Twenty-second exposure of cultured immortalized human cells to various concentrations of EtOH-H2O showed significant reduction of viable cells when EtOH-H2O concentration exceeded 25% (P = 0.005). Increasing the duration of application of 20% EtOH-H2O beyond 30 seconds resulted in a significant reduction in viable cells (69.69% +/- 16.34% at 30 seconds compared with 2.14% +/- 2.29%, 10.45% +/- 7.11%, and 11.09% +/- 15.73% at 35, 40, and 45 seconds, respectively; P = 0.01). TUNEL assay of cultured human corneal epithelial cells exposed to 20% EtOH-H(2)O for 20 and 40 seconds showed maximal labeling at 24 hours (58.05% +/- 33.10%) and 8 hours (94.12% +/- 1.21%), respectively. Exposure to 20% EtOH-KSFM for 20 and 40 seconds resulted in substantially lower TUNEL positivity (3.51% +/- 0.20% at 24 hours and 7.11% +/- 0.49% at 8 hours). CONCLUSIONS: The viability and electron microscopic findings in the basement membrane zone showed significant variation after treatment of the epithelium in vivo with dilute alcohol. The application of dilute alcohol on the monolayer of cultured corneal epithelial cells resulted in increasing cell death in a dose- and time-dependent manner. (+info)
Estrogen receptor specificity for the effects of estrogen in ovariectomized mice.
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Estrogen exerts a variety of important physiological effects, which have been suggested to be mediated via the two known estrogen receptors (ERs), alpha and beta. Three-month-old ovariectomized mice, lacking one or both of the two estrogen receptors, were given estrogen subcutaneously (2.3 micro g/mouse per day) and the effects on different estrogen-responsive parameters, including skeletal effects, were studied. We found that estrogen increased the cortical bone dimensions in both wild-type (WT) and double ER knockout (DERKO) mice. DNA microarray analysis was performed to characterize this effect on cortical bone and it identified four genes that were regulated by estrogen in both WT and DERKO mice. The effect of estrogen on cortical bone in DERKO mice might either be due to remaining ERalpha activity or represent an ERalpha/ERbeta-independent effect. Other effects of estrogen, such as increased trabecular bone mineral density, thymic atrophy, fat reduction and increased uterine weight, were mainly ERalpha mediated. (+info)
Identification of genes induced in regenerating Xenopus tadpole tails by using the differential display method.
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To identify candidate gene(s) involved in the tail regeneration of Xenopus laevis tadpoles, we used the differential display method to isolate four genes (clones 1, 2, 13a, and 13b) whose expression is induced in regenerating tadpole tails. Among them, clones 13a and 13b were found to encode the Xenopus homologues of the alpha1 chain of type XVIII collagen and neuronal pentraxin I, respectively. Expression of clone 2 and neuronal pentraxin I genes increased dramatically in the blastema 3 days after amputation, whereas that for the clone 1 and type XVIII collagen genes was induced gradually after amputation. In situ hybridization revealed that the neuronal pentraxin I gene is expressed specifically in the regenerating tail epidermis but not in the normal tail epidermis or the most distal margin of the tail blastema, suggesting that it has a tissue-inductive role in tail regeneration. Expression of the four genes was induced in the limb and in the tail blastema, suggesting that they are involved in the regeneration of both organs. Finally, expression of clone 2 and neuronal pentraxin I genes was scarce during embryonic stages in comparison to the tail blastema, suggesting that their main functions are in organ regeneration. Our results demonstrate unique features of spatial and temporal gene expression patterns during Xenopus tadpole tail regeneration. (+info)
Molecular targets for tumour progression in gastrointestinal stromal tumours.
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BACKGROUND AND AIMS: The distinction between benign and malignant gastrointestinal stromal tumours (GISTs) is often unclear at the clinical and histopathology levels. GISTs are believed to arise from the stem cells of Cajal. In order to define genetic biomarkers and identify target genes related to GIST progression, we analysed and compared benign and malignant GISTs with verified follow up data using cDNA expression arrays. METHODS: Eight genes were frequently overexpressed in malignant GISTs and their overexpression was confirmed using quantitative real time reverse transcription-polymerase chain reaction. These genes included ezrin (villin 2 (VIL2)), collagen 8 alpha 1 subunit (COL8A1), G2/mitotic specific cyclin B1 (CCNB1), high mobility group protein (HMG2), TSG101 tumour susceptibility protein, CENP-F kinetochore protein, protein tyrosine kinase 2 (FAK), and protein kinase DYRK2. To test these genes in a clinical setting, we obtained diagnostic samples of 16 additional GISTs that were classified at diagnosis as benign, malignant, and uncertain malignant potential (UMP). RESULTS: There was remarkable gene overexpression in all malignant GISTs. Statistical analyses revealed significant correlations between overexpression of several gene pairs in malignant GISTs. We found the strongest correlations (rho>0.70) among the significant correlations (p<0.01) between CCNB1-CENP-F (rho = 0.87) and CCNB1-FAK (rho = 0.73). Gene expression of the UMP GISTs suggested two different groups. Three UMP GISTs had gene expression consistent with malignant tumours and their follow up data revealed that indeed these patients had recurrences later on. On the other hand, UMP GISTs that had low gene expression levels continued free of disease for several years. CONCLUSIONS: These results provide insight into the oncogenesis of GISTs and suggest that testing the expression profile of a number of genes may segregate GISTs into groups of different tumour behaviour. (+info)
Expression and supramolecular assembly of recombinant alpha1(viii) and alpha2(viii) collagen homotrimers.
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Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, alpha1(VIII) and alpha2(VIII), that can form homotrimers in vitro and in vivo. Here, recombinant collagen VIII was expressed to study its supramolecular assembly following secretion. Cells transfected with alpha1(VIII) or alpha2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of approximately 180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated homotrimers with stable pepsin-resistant triple-helical domains. Size fractionation of native recombinant collagen VIII molecules expressed with or without prolyl 4-hydroxylase identified urea-sensitive high molecular mass assemblies eluting in the void volume of a Superose 6HR 10/30 column and urea-resistant assemblies of approximately 700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant alpha1(VIII)(3), alpha2(VIII)(3), and co-expressed alpha1(VIII)(3)/alpha2(VIII)(3). Microscopy analysis of recombinant collagen VIII identified rod-like molecules of 134 nm in length that assembled into angular arrays with branching angles of approximately 114 degrees and extensive networks. Based on these data, we propose a model of collagen VIII assembly in which four homotrimers form a tetrahedron stabilized by central interacting C-terminal NC1 trimers. Tetrahedrons may then act as building blocks of three-dimensional hexagonal lattices generated by secondary interactions involving terminal and helical sequences. (+info)
Endostatin promotes the anabolic program of rabbit chondrocyte.
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Endostatin is a natural occurred angiogenesis inhibitor derived from collagenXVIII. So far its function during the angiogenesis process of bone formation and arthropathy has not been well studied yet. The present study addresses the function of endostatin in rabbit articular chondrocytes (RAC). We found that endostatin can promote RAC adhesion and spreading as well as its proliferation. In monolayer cultured RAC, CollagenII, TIMP1 and collagenXVIII transcription were up regulated by endostatin while collagenI and MMP9 were down regulated. Moreover collagenXVIII and endostatin antigens are present at synovial fluid. These findings indicate new function of endostatin as a homeostatic factor in cartilage metabolism. (+info)