Michelle Kurpakus-Wheater

Thymosin β4 promotes human conjunctival epithelial cell migration

Purpose. In this study the effects of thymosin ß4 (Tß4) on migration and production of laminin-5 in the human conjunctival cell line HC0597 was analyzed. Methods. Boyden chamber assays assessed the ability of Tß4 to stimulate in vitro cell migration. Control or Tß4-treated cells were processed for immunofluorescence microscopy using antibodies to vinculin or laminin-5. Cell lysates were processed for Western blot and densitometric analysis using antibodies to laminin-5 a3 or ?2 chains. Results. Tß4 stimulated migration in a dose-dependent manner. Focal adhesions present in Tß4-treated cells were smaller and more rounded compared to the “streaks” characteristic of controls. Western blot analysis and densitometry revealed that Tß4-treated cells expressed more laminin-5 a3 and ?2 chain protein. Conclusions. Tß4 stimulates in vitro conjunctival epithelial cell migration, and results in altered focal adhesion formation and increased extracellular laminin-5 deposition. The increased migration may be correlated with increased production of laminin-5.

Maintaining Corneal Integrity How the “Window” Stays Clear

The anterior surface of the eye is composed of the cornea, conjunctiva, and the zone between the two called the limbus. The cornea must maintain optical clarity to retain good vision. However, the ocular surface is vulnerable to trauma, microbial infection, and exposure to environmental toxins. This places the cornea, especially, at risk for disruptions of the epithelial barrier and subsequent immunopathological events. Cell-cell and cell-matrix attachment junctions incorporating adhesion molecules ensure that the epithelial barrier remains intact. Protein components of the basement membrane, including laminins, are vital to the adhesion of corneal epithelial cells to the underlying stroma and function to enhance the strength of the bond between epithelium and connective tissue. Epithelial cells also play an early and crucial role in the initiation of ocular surface responses should a potentially antigenic molecule enter into deeper corneal tissues. For example, epithelial cells may produce and release cytokines such as interleukin-1 (IL-1). The delicate balance between the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are central to mechanisms regulating dissolution of the extracellular matrix that may be a consequence of infection or wound healing. Adhesion molecules, cytokines and chemokines, and MMPs and TIMPs thus participate in the corneal response to immunologic challenge or wounding. They may also be involved in corneal pathologies associated with genetic diseases, diabetes, and vitamin A deficiency. In addition these molecules are components of cellular pathways underlying the clinical complications often observed with contact lens wear and refractive surgeries used to improve visual acuity.

Laminin-5 is a component of preserved amniotic membrane

Purpose. To determine if laminin-5 is retained in the matrix of cryopreserved human amniotic membrane tissue prepared for ocular surgeries. Methods. Amniotic membrane was solubilized in urea/SDS buffer. Constituent proteins were resolved by SDS-PAGE and laminin-5 content was determined by Western blot analysis using a panel of antibodies directed against the a3, ß3 or ?2 chains of the molecule. Human corneal epithelial cells were seeded on amniotic membrane and cultured in the presence or absence of EGF. The cell-membrane construct was examined for laminin-5 content using Western blot analysis and immunofluorescence microscopy. Results. In preserved amniotic membrane the laminin-5 a3 chain is present in both the unprocessed (190-kDa) and processed (160-kDa) forms. The ß3 chain is found in the 145-kDa form. The ?2 chain appears to be predominantly in the processed (105-kDa) form. Very little of the unprocessed form of the ?2 chain (155-kDa) could be detected using immunoblot analysis. A similar distribution of laminin-5 was also present in extracts of corneal epithelial cells cultured on amniotic membrane. Immunofluorescence analysis of cells cultured on the membrane demonstrated polarization of laminin-5 at the cell-membrane interface. Conclusions. The presence of both the unprocessed and processed forms of laminin-5 a3 and ?2 chains in preserved human amniotic membrane suggests that when used as a substrate in ocular surgeries, this membrane may be capable of promoting corneal epithelial cell motility and adhesion. Regulation of the motile or adhesive function may lie with factors secreted by the corneal epithelium that populates the membrane following surgery.

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.

Caspase-9 activation in hypoxic human corneal epithelial cells

The purpose of this study was to determine the effect of hypoxia on caspase-8 and -9 gene and protein expression and activity in corneal epithelium. Non-transformed human corneal epithelial cells (HCEC) were cultured in 2% oxygen. A cDNA expression array coupled with densitometric analysis was used to compare relative mRNA expression levels of 96 apoptosis-related genes in hypoxic and normoxic HCEC. Caspase-8, caspase-9, FLIP, Fas, FasL, and TNFα protein expression was assessed further using Western blot analysis and ELISA. Caspase-8 and -9 activities were measured using a fluorometric activity assay. Hypoxia did not affect caspase-8 or -9 gene or protein expression in HCEC, however caspase-9 activity was significantly increased. Hypoxia significantly suppressed the activity of caspase-8. FLIP and Fas gene and protein expression were not significantly altered in hypoxic cells compared to normoxic controls. mRNA and protein levels of TNFα and TNFR-1 were significantly decreased, while FasL mRNA and proteins levels were significantly increased in hypoxic HCEC. In corneal epithelium stressed by hypoxia caspase-9 activity is upregulated, suggesting that apoptosis proceeds via the mitochondrial pathway. Caspase-8 activity may be suppressed because the loss of TNFα and TNFR-1 gene and protein expression inhibits the initial formation of a death signaling complex.