Bovine Corneal Research Articles

In vitro culture of human fetal corneal endothelial cells

To explore and optimize a proper culture system for human fetal corneal endothelial cells (hFCECs), including the methods of primary culture, passage and cryopreservation. Fresh fetal corneas were explanted to propagate primary corneal endothelial cells. The cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) in the absence or presence of the extracts from bovine corneal endothelium cells (bCECs), and the cells were identified with immunocytochemical staining. The passage and cryopreservation of hFCECs were optimized according to previous reports on adult corneal endothelial cells. Using the explant culture method, hFCECs migrated successfully within 3 days and assumed polygonal-shaped corneal endothelial morphology. The optimizing methods were 0.125% trypsin + 0.02% EDTA for passage and 10% DMSO + 90% FBS for cryopreservation. Recovered hFCECs from cryopreservation remained typical morphology and immunological markers of corneal endothelial cells, including positive staining of NSE, Nestin, Ki67 and ZO-1, and negative staining of CK3/12, which demonstrated that they retained the characterizations of corneal endothelial cells and proliferative capacity. Moreover, the extracts from bCECs can promote the proliferative capacity of hFCECs significantly, while maintaining their typical endothelial morphology. The culture conditions of human fetal corneal endothelial cells were firstly optimized, including the primary culture, passage and cryopreservation. Meanwhile, we confirmed that the extracts from bovine corneal endothelium promoted the proliferative capacity while maintaining the morphology of hFCECs in vitro.

Hydration and rehydration in camel and bovine cornea

Abstract Purpose T0 investigate the hydration and rehydration of fresh camel and bovine cornea in sodium salt solutions during 20 minutes, 46 hours and 14 hours. Methods During the 20mins study hydration of fresh corneal stroma was investigated in sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium acetate (CH3COONa), sodium thio‐cyanate (NaSCN) and sodium fluoride (NaF) with 2 minutes time interval. During a 46hrs time period, the hydration study was carried out in NaCl (150, 300mM), NaF (150mM) at random intervals. The 14 hrs study was carried out to assess the rehydration of corneal stroma in NaCl (150, 300mM), NaF (150mM) after six hours of drying. Results During the 20 minute swelling studies in the first two minutes the rate of hydration in both camel and bovine corneas was high but gradually reduced in the 2 – 20 minute period. The rate of hydration and level of hydration of camel and bovine cornea was not significantly different from each other in all the strengths of solutions. During the 46 hours swelling studies the rate of initial hydration (0 to 2 hours) of camel and bovine stroma, in all solutions was significantly higher compared to hydration during later hours (2‐46hrs). Camel stromal hydration in150mM NaCl was significantly higher compared to bovine stromal hydration in the same solution during 10 – 24 hrs, and 24 to 46 hrs time period. Rehydration in camel stroma was significantly lower than bovine in 150mM NaF. Conclusion Above study showed that there was no selective affinity for particular ions in camel or bovine corneal stroma. The swelling in camel cornea is higher compared to bovine corneal stroma. This could be due to higher negatively charged KS‐PGs in the stroma. Less rehydration in camel cornea suggests the higher leaching of of PGs from stroma in NaF.

Long‐term effects of plasma membrane hyperpolarization on cell‐junction stability of corneal endothelial cells in organ and cell culture

Abstract Purpose One of the most important challenges in corneal preservation is to maintain the epithelial and endothelial integrity. In previous work (Cell Motil. Cytoskeleton 66:1087, 2009) we found that short term (1 hour) hyperpolarization of the plasma membrane potential (HPMP) produces a reorganization of the actin cytoskeleton and increases the stability of cell‐cell junctions of bovine corneal endothelial (BCE) cells in organ and cell cultures. The purpose of this work is to study the effect of long term (several days) HPMP on cell viability, actin organization and intercellular junction stability in cell and organ culture, with the general objective of designing appropriate solutions to use in the maintenance of corneas. Methods Cells and corneas were grown in culture media supplemented by hyperpolarizing agents (valinomycin, choline chloride, lithium chloride). We studied their effects on plasma membrane potential (oxonol V), cell viability (propidium iodide and Tunel), actin organization (FITC‐phalloidin) and cell junction stability (response to calcium deprivation). Results The results showed that cells maintained up to three days in media supplemented with 0.1 µg/mL valinomycin improved their viability in culture and increased the stability of their intercellular junctions, similarly to that obtained in short‐term HPMP treatments. Accordingly, the corneal epithelium and endothelium of treated corneas in culture were better preserved than control ones Conclusion These results suggest that HPMP solutions might be useful in the design of media for preservation of corneas that will be used in transplants.

Swelling studies of camel and bovine corneal stroma.

In the present study we investigated the swelling characteristics of fresh camel and bovine cornea in sodium salt solutions. Swelling studies were carried out at 20 minutes, 14 hours, and 46 hours on five fresh camel and 5 five fresh bovine corneas. During the 20-minute hydration of fresh corneal stroma was investigated using sodium chloride (NaCl), sodium bicarbonate (NaHCO(3)), sodium acetate (CH(3)COONa), sodium thiocyanate (NaSCN), and sodium floride (NaF) at 2-minute time intervals. During a 46-hour time period, the hydration study was carried out using NaCl (150, 300 mM) and NaF (150 mM) at random intervals. The 14-hour study was carried out to assess the rehydration of corneal stroma after 6 hours of drying. During the 20-minute swelling studies in the first 2 minutes the rate of hydration in both camel and bovine corneas was high but gradually reduced in the 2-20-minute period. The rates and levels of hydration of camel and bovine cornea were not significantly different from each other in all the strengths of solutions. During the 46-hour swelling studies, the initial rate of hydration (0-2 hours) of camel and bovine stroma, in all solutions was significantly higher (Z = 0.056) compared to hydration during later hours (2-46 hours). Camel stromal hydration (high) in 150 mM NaCl was significantly higher compared to bovine stromal hydration in the same solution during the 10-24, and 24-46-hour time periods. Rehydration in camel stroma was significantly lower than bovine in 150 mM NaF. The 20-minute study showed that there was no selective affinity for particular ions in camel or bovine corneal stroma. Initial swelling in both corneal and bovine stroma is faster and more prominant compared to later swelling. The swelling in camel cornea is more prominant compared to bovine corneal stroma. This could be due to higher negatively charged keratin sulfate-proteoglycans in the stroma. Lower rehydration in camel cornea suggests stronger leaching of proteoglycans from stroma in NaF.

Intramolecular loop/tail interactions are essential for connexin 43‐hemichannel activity

Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 μM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.

An inverse finite element method for determining the anisotropic properties of the cornea

An inverse finite element method was developed to determine the anisotropic properties of bovine cornea from an in vitro inflation experiment. The experiment used digital image correlation (DIC) to measure the three-dimensional surface geometry and displacement field of the cornea at multiple pressures. A finite element model of a bovine cornea was developed using the DIC measured surface geometry of the undeformed specimen. The model was applied to determine five parameters of an anisotropic hyperelastic model that minimized the error between the measured and computed surface displacement field and to investigate the sensitivity of the measured bovine inflation response to variations in the anisotropic properties of the cornea. The results of the parameter optimization revealed that the collagen structure of bovine cornea exhibited a high degree of anisotropy in the limbus region, which agreed with recent histological findings, and a transversely isotropic central region. The parameter study showed that the bovine corneal response to the inflation experiment was sensitive to the shear modulus of the matrix at pressures below the intraocular pressure, the properties of the collagen lamella at higher pressures, and the degree of anisotropy in the limbus region. It was not sensitive to a weak collagen anisotropy in the central region.

Effects of small interfering RNA approach to silence the connective tissue growth factor gene on the expression of the protein p27

To investigate the effects of the small interfering RNA (siRNA) approach to silence the expression of connective tissue growth factor (CTGF) gene expression on the expression of the protein p27. Experimental research. The specific siRNA of CTGF was designed and synthesized, then transfected into cultured bovine corneal endothelial cells by liposomes. The blank control group was only treated with the culture medium and not be transfected with siRNA. Reverse transfection-polymerase chain reaction (RT-PCR) was applied to show the effect of gene-silencing, and western blot was used to observe the protein p27 expression of bovine corneal endothelial cells, which were treated with 1.00 µg/L transforming growth factor β(2) (TGF-β(2)). The results were analyzed by one-factor analysis of variance. CTGF siRNA was successfully transfected into cultured bovine corneal endothelial cells and effectively silence the expression of CTGF mRNA. The CTGF mRNA expression decreased to 17.3%, 24.4% and 41.7% of control values at 24, 48 and 72 h after transfection, there was significant difference between these groups (F = 389.9, P < 0.05). Significant decrease of protein p27 expression was detected at 36 h after transfection (F = 299.3, P < 0.05). CTGF-specific siRNA was able to inhibit the expression of CTGF mRNA effectively and down-regulate the expression of p27.

Protection of Thymosin Beta-4 on Corneal Endothelial Cells from UVB-Induced Apoptosis

Cornea absorbs most of daily ultraviolet (UV) light. An excess of UV damages results in not only keratopathy and cataract but also maculopathy. It has been reported that thymosin beta-4 (Tbeta4) promotes wound healing, decreases inflammatory response and prevents apoptosis of corneal epithelial cells. However, it is not clear whether Tbeta4 protects UVB-induced corneal injury, particularly in corneal endothelial cells because of its non-proliferation in nature. The purpose of this study is to compare the protective effects of Tbeta4 on bovine corneal endothelial (BCE) cells from low- and high-dose UVB damage. In this study, 1 microg/ml of Tbeta4 was added to BCE cells 2 h before low (12.5 mj/cm2) or high dosage (100 mj/cm2) UVB exposure. Using a fluorogenic substrate cleavage assay, we found that Tbeta4 diminished the reactive oxygen species level in BCE cells elicited by UVB. However, the protection of viability by Tbeta4 could only be detected under low-dose UVB exposure. Moreover, both caspase-9 activity and annexin V/propidium iodine staining demonstrated that Tbeta4 only protected BCE cells from low-dose UVB-induced apoptosis but not high-dose UVB-induced necrosis. Together, Tbeta4 protected corneal endothelial cells from UVB-induced oxidative stress and apoptosis after low-dose UVB exposure. The results support further investigation towards topical use or anterior chamber injection of this small hydrophilic peptide in treating and preventing UVB-induced corneal endothelial damage.

Validation of porcine corneal opacity and permeability assay as an alternative to eye irritation tests

The Draize eye irritation test has been changed to alternative methods based on 3 Rs (Replacement, Reduction, Refinement) of animal experiments. The bovine corneal opacity and permeability (BCOP) assay is well established as one of the alternative methods to the Draize test but the supply of bovine eyes from a slaughterhouse is not sufficient in actuality. The porcine corneal opacity and permeability(PCOP) assay have been proposed as a reliable alternative to the BCOP assay and the Draize test because porcine corneas have numerous advantages including structural similarities with human corneas and the stable capacity for a corneal supply. In this study, three cosmetics and seven chemicals that are already known as irritants at Draize score and the bovine corneal opacity(BCO) assay were tested by the PCOP assay for the first time in Korea. The porcine corneal opacity was measured by quantifying the ability of light to pass through a cornea using an opacitometer. The permeability was measured at 10 and 30 min time points after chemical exposure. The PCOP assay showed the high correlation with the Draize score and BCO assay. These results showed that the PCOP assay can be used as an alternative method for the BCO assay and the Draize test although it needs further studies about insoluble chemicals and others to increase reliability.

Development of a new opacitometer for the bovine corneal opacity and permeability (BCOP) assay

The bovine corneal opacity and permeability (BCOP) assay is an in vitro eye irritation test that relies on the measurement of induced opacity and permeability evaluated in isolated bovine corneas. Routinely, opacity is measured by an OP-KIT opacitometer which provides a centre-weighted reading of light transmission through the corneal tissue. However, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of isolated cornea. Consequently, these findings question the reliability of the obtained data when opacity is manifested in a non-homogeneous manner. In addition, the BCOP test is only recommended to identify corrosive and severe eye irritants, and has not proven as sensitive in distinguishing among mild and moderate eye irritants. Within this context, a new opacitometer was developed which uses an adjustable laser beam in combination with a calibrated photometer. This work focused on a technical optimisation and describes the optical characteristics of the new device. The laser-based opacitometer allowed the analysis of the complete corneal surface, showed improved sensitivity to detect subtle changes in corneal transparency and was able to detect more efficiently opaque spots located along the sides of the excised corneas.

Probing Collagen/Enzyme Mechanochemistry in Native Tissue with Dynamic, Enzyme-Induced Creep

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal and enzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance, and disease in vertebrate animals. However, extracting a quantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissue due to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay and diffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and the cutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing of enzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initial strain. The results, obtained on uniaxially loaded strips of bovine corneal tissue (0.1, 0.25, or 0.5 N), demonstrate that small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that a change in tissue elongation of only 1.5% (at approximately 5% strain) reduces the maximum cutting rate of the enzyme by more than half. Estimation of the average load per monomer in the tissue strips indicates that this protective "cutoff" occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime. The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep response indicates that the decrease in the cleavage rate of the BC is nonlinear (initially steep between 4.5 and 6.5% and then flattens out from 6.5 to 9.5%). The high sensitivity to strain at low strain implies that even lightly loaded collagenous tissue may exhibit significant strain protection. The dynamic, enzyme-induced creep assay described herein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in many different tissue types.

A Hierarchy of Endothelial Colony–Forming Cell Activity Displayed by Bovine Corneal Endothelial Cells

To test the hypothesis that the robust expansion of bovine corneal endothelial cells (BCECs) in vitro is due to the presence of individual endothelial cells with various levels of proliferative potential. BCECs and bovine vascular endothelial cells (ECs) derived from aorta, coronary artery, and pulmonary artery were cultivated in optimized medium. These cell populations were confirmed by morphologic features, functional assays, and gene expression profiles. Moreover, ECs were plated in a single-cell clonogenic assay to evaluate colony-forming ability. Both corneal and vascular ECs were confirmed to be pure populations of endothelium uncontaminated with hematopoietic cells. A complete hierarchy of endothelial colony-forming cells (ECFCs) was identified in BCECs by a single-cell clonogenic assay. The distribution of the various types of ECFCs was similar to the control ECs removed from the systemic vessels. Cultured BCECs display clonal proliferative properties similar to those of vascular ECs.

Lovastatin Inhibits the Thrombin-Induced Loss of Barrier Integrity in Bovine Corneal Endothelium

Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the efficacy of statins, which inhibit activation of RhoA, in opposing the thrombin-induced loss of barrier integrity of monolayers of cultured bovine corneal endothelium. Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confirmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly. Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin- and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin- and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution. Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mM ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin beta(4), and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.

Validating and troubleshooting ocular invitro toxicology tests

In vitro organotypic models for testing ocular irritants have warranted sufficient interest as methods to replace in vivo ocular testing. The in vitro organotypic models claim to maintain short-term normal physiological and biochemical functions of the mammalian cornea in an isolated system. In these test methods, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability using opacitometry and spectrophotometry, respectively. Both measurements are used quantitatively for irritancy classification for prediction of the in vivo ocular irritation potential of a test substance. Examples of organotypic models that incorporate these criteria include: the bovine corneal opacity and permeability (BCOP) assay, the isolated chicken eye (ICE) test method and the isolated rabbit eye (IRE) assay. A fourth method, the hen's egg test–chorioallantoic membrane (HET-CAM) assay, differs in the evaluation criteria but is also normally included among this class of in vitro protocols. Each of these protocols is discussed in detail as representative candidate in vitro methods for assessing ocular irritation and corrosion. The methodologies, protocol details, applications, and their validation status are discussed. A brief historical perspective of the development of original in vitro ocular testing models is also mentioned. More importantly, improving and troubleshooting the current techniques, in order to present the models as stand-alone in vitro tools for ocular toxicity assessment, is emphasized.

Preserved and Unpreserved 12 Anti-allergic Ophthalmic Solutions and Ocular Surface Toxicity: In Vitro Assessment in Four Cultured Corneal and Conjunctival Epithelial Cell Lines

In the present study, we evaluated the cytotoxicity of anti-allergic ophthalmic solutions in cultured corneal and conjunctival cells, namely SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). The viability of cell cultures was determined following the exposure of cells to 12 commercially available anti-allergic ophthalmic solutions for varying exposure times and at various dilutions using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of different drugs. Based on CVS data, the order of cell viability after exposure to the drugs was Zepelin ≥ Tramelas PF ≥ Cumorol PF ≥ Ketotifen PF ≥ Eyevinal = Fumarton ≥ Cumorol > Intal ≥ Rizaben ≥ Tramelas ≥ Patanol Livostin. In conclusion, cell viability was mostly affected by the concentration of benzalkonium chloride rather than the active component and/or the anti-allergic action of the drug. The CVS was useful in comparing the toxicity of different drugs.

Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants

In the present work, we assessed the relationship between alkyl carbon chain length and ocular irritation potentials using the hen’s egg test–chorioallantoic membrane (HET-CAM) and bovine corneal opacity and permeability (BCOP) assays using 5 commercial alkyl polyglucoside surfactants with different compositions of alkyl chain lengths (C6–C16). With HET-CAM, there was a good correlation between the proportion of C10 alkyl polyglucoside and the eye irritation potential Q score (r2 = 0.912, p = .011). There were no significant differences between the proportion of C10 alkyl polyglucoside and corneal opacity in BCOP assays; however, there was a relatively high positive correlation between the proportion of C10 alkyl carbon chain lengths and corneal permeability (r2 = 0.736, p = .063).

Formation and Disassembly of Adherens and Tight Junctions in the Corneal Endothelium: Regulation by Actomyosin Contraction

Purpose. To determine the role of actin cytoskeleton in the disassembly and reformation of adherens junctions (AJs) and tight junctions (TJs) in bovine corneal endothelial monolayers. Methods. Disassembly and reformation of AJs and TJs were induced by extracellular Ca(2+) depletion and subsequent add-back of Ca(2+), respectively. Resultant changes in the transendothelial electrical resistance (TER), an indicator of integrity of TJs, were measured based on electrical cell-substrate impedance. Phosphorylated myosin light chain (ppMLC), a biochemical measure of actomyosin contraction, and activation of its upstream regulatory molecule RhoA-GTP were assessed by Western blot analysis. Results. Extracellular Ca(2+) depletion led to activation of RhoA, increase in ppMLC, decrease in TER, contraction of the perijunctional actomyosin ring (PAMR), and redistribution of zonula occludens-1 (ZO-1) and cadherins. These effects were reversed on Ca(2+) add-back. Pretreatment with Y-27632 and blebbistatin (as inhibitors of actomyosin contraction) reduced the rate of decline in TER, opposed the contraction of the PAMR, and blocked the redistribution of ZO-1 and cadherins. Both drugs reduced the recovery in TER and opposed the normal redistribution of ZO-1 and cadherins on Ca(2+) add-back. Cytochalasin D, which led to dissolution of the PAMR, also reduced the recovery of TER on Ca(2+) add-back. Conclusions. The (Ca(2+) depletion)-induced disassembly of AJs accelerates the breakdown of TJs through a concomitant increase in the actomyosin contraction of the PAMR. However, these data on reassembly show that a contractile tone of the PAMR is essential for assembly of the apical junctional complex.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and/or increasing insulin on the stratification and secretion of aligned matrix by fourth- to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. High-resolution differential interference contrast microscopy, quick-freeze/deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

Barrier Dysfunction of the Corneal Endothelium in Response to TNF-α: Role of p38 MAP Kinase

TNF-alpha is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF-alpha-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-alpha was visualized by immunofluorescence. The expression of TNF-alpha receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-alpha was determined by Western blot analysis. Exposure to TNF-alpha induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-alpha also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. TNF-alpha breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-alpha-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.