Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas.

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.

Keratocan expression is increased in the stroma of keratoconus corneas.

BACKGROUND: Keratoconus is a noninflammatory disease characterized by thinning and scarring of the central portion of the cornea. The etiology is unclear. In this study, we sought to identify mRNAs that are differentially expressed in the stroma of keratoconus corneas in comparison to those of corneas from normal individuals and patients with other corneal diseases. MATERIALS AND METHODS: Total RNA was isolated from the stromal layer of normal human, keratoconus, and pseudophakic bullous keratopathy corneas. cDNA was synthesized and PCR-select subtractive hybridization experiments were performed. The differentially expressed genes noted were verified by dot blot analysis, cloned, and sequenced. Immunohistochemical staining, in situ hybridization, and/or reverse transcription polymerase chain reaction were used to assess expression of the identified genes at protein and/or mRNA levels in normal, keratoconus, and other diseased corneas. RESULTS: A number of genes were found to be up-regulated in keratoconus specimens. These included heat shock protein 90, decorin, fibronectin, ferritin heavy chain, and keratocan. Among them, keratocan mRNA transcript and protein were demonstrated to be expressed at a higher level specifically in the keratoconus stroma. CONCLUSIONS: Keratocan expression in the stoma was increased in keratoconus corneas. This up-regulation appears to be keratoconus specific. Keratocan is one of the three keratan sulfate proteoglycans in the cornea speculated to be important for structure of the stromal matrix and maintenance of corneal transparency. The overexpressed keratocan may conceivably alter the fibrillogenesis in the stroma, leading to structural defects and contributing to the development of keratoconus.

Cell density regulated expression of transcription factor Sp1 in corneal stromal cultures.

Sp1, a ubiquitously expressed transcription factor, has been implicated to have a role in cell differentiation and cell proliferation. In keratoconus, a corneal disease characterized by thinning and scarring of the central cornea, Sp1 is found up-regulated. In the present study, we examined the expression of Sp1 in stromal cells cultured from normal human and keratoconus-afflicted corneas and evaluated the influence of varying cell densities. Immunohistochemical staining, Western blotting and electrophoretic mobility shift assays indicated that in both normal human and keratoconus cultures, Sp1 protein levels and binding activities increased with the density of cells. The basal level of Sp1 in keratoconus cultures was higher than that in normals. These results demonstrate a marked density mediated up-regulation of Sp1 in corneal stromal cells, suggesting that the Sp1 expression may be regulated by differentiation states of the cells in the cornea. In addition, cells from keratoconus corneas in vitro appear to carry and retain the Sp1 abnormality as in vivo. The Sp1 defect may be an inborn error in keratoconus.

Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues.

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.

Signal transduction mediated by adhesion of human trabecular meshwork cells to extracellular matrix.

In this study we investigated the signaling event induced by adhesion of human trabecular meshwork (TM) cells to extracellular matrix (ECM) elements such as fibronectin. The role of tyrosine phosphorylation in adhesion was evaluated. A number of intracellular entities involved in the adhesion-mediated pathways were identified. For the experiments, human TM cells were seeded onto fibronectin- or polylysine (negative control)-coated plates. Fifteen, 30, 90 and 240 min after the seeding, cell lysates were collected. Immunoblotting analysis revealed that tyrosine phosphorylation occurred within 15 min of adhesion of TM cells to fibronectin and the level increased with time. The phosphotyrosyl proteins had molecular masses 25-220 kDa. A much lower level of tyrosine phosphorylation was observed when cells were plated on polylysine. Immunoprecipitation experiments indicated that the phosphotyrosine-containing proteins included focal adhesion kinase, paxillin, phosphatidylinositol 3-kinase and mitogen activated protein kinase. Within 30 min of adherence to fibronectin, human TM cells immunostained for paxillin and phosphotyrosine and exhibited prominent focal contacts. When treated with tyrosine kinase inhibitors genistein and herbimycin A and a protein kinase C (PKC) pseudosubstrate peptide inhibitor, cell adhesion to fibronectin was compromised and focal contact formation was limited. These results demonstrated that in human TM cells, tyrosine kinase was activated upon their adherence to fibronectin. PKC also appeared to play a role in modulation of the cell-matrix adhesion process. The current study provides insight into the signaling pathways that are linked to the ECM-induced events in TM cells. Elucidation of the hierarchy of signal responses may help develop strategies manipulating the cell-matrix interactions in the TM system.

Expression of integrin receptors in the human trabecular meshwork.

PURPOSE: To examine the expression of integrin receptors in the human trabecular meshwork. METHODS: The expression of integrins in human tissues was visualized by immunohistochemical staining using integrin-specific antibodies. Immunoprecipitation was performed after biotin labeling of cell surface proteins. Reverse transcriptase-polymerase chain reaction was used to detect the presence of mRNA species for integrin subunits. RESULTS: Human trabecular meshwork tissues obtained from donors 2 to 65 years old stained positively for integrins alpha1, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 and beta5. The staining was observed mostly around the edges of trabecular beams in association with trabecular meshwork cells. The staining intensity did not appear to vary with donor age. In addition, immunoprecipitation of tissue extracts revealed the presence of integrin alpha2 and confirmed the absence of beta2. Results from polymerase chain reactions were consistent with these findings. CONCLUSIONS: A spectrum of integrin receptors that may have important roles in the cell-matrix interactions are demonstrated in the human trabecular meshwork. The repertoire identified in tissues is similar to that found in cultured cells except that the beta4 expression in tissues is lost in cultures.

Risk factors for low birthweight in Singapore: strategies for prevention.

An analysis of 9399 consecutive singleton births delivered at this unit is presented. The incidence of low birthweight (less than 2500 g) was 7.4%. Further analysis of the 698 low birth weight babies indicated that a variety of socio-demographic risk factors are operational in the etiology of low birthweight. Many of these are preventable before pregnancy so that the implementation of preventive Public Health measures utilising appropriate technology at least in developing countries may be socially and economically preferable to continued financial investment in intensive perinatal services.