Bovine Corneal Endothelial Cells Research Articles

Effects of prostaglandin E 2 on growth, morphology, morphometry and keratin pattern of bovine corneal epithelial cells cultured in vitro

In this work the relationship between the proliferation of bovine corneal epithelial cells and PGE2 has been studied. Our data indicate that PGE2 plays an important role in the growth of corneal epithelial cells. Actually, epithelial cells cultured on a keratocyte feeder-layer and exposed to indomethacin, a cyclooxygenase inhibitor, have shown a decrease in growth rate at drug concentrations which otherwise did not induce a reduction in the viability of the keratocytes as well as in epithelial cells in separate cultures. This effect has been reversed by an exogenous PGE2 addition to the culture media. Moreover, significant increases have been found in the growth of epithelial cells cultured in the presence of keratocytes, with basal medium and with conditioning medium after adding exogenous PGE2 at concentrations equal to or lower than 10(-6) M. Significant decreases in the dimensions of the corneal epithelial cells have been found only when PGE2 has been added to basal and to conditioning medium, suggesting that the autacoid maintains cell dimension and morphology. The appearance of keratins with high molecular weight (54 and 57 kDa) coupled with the tendency to stratification of the cells cultivated with media supplemented with PGE2, indicates that the autacoid could favour cell differentiation. The action of PGE2 on the corneal epithelial cells does not seem to be influenced by the presence of the fibroblasts and their products, since PGE2 has induced increases in cell growth and morphological variations, independent of cultural conditions and therefore also only in the presence of basal medium.

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines.

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.

Enhancement of long‐term testosterone secretion and steroidogenic enzyme expression in human Leydig cells by co‐culture with human Sertoli cell‐enriched preparations

We have shown previously that long‐term testosterone secretion, which decreases when human Leydig cells are cultured alone, increases when purified human Leydig and Sertoli cells are cultured together. In this work, human Leydig cell functions were studied further during in vitro culture, either alone or with human Sertoli cells, on a basal membrane derived from bovine corneal endothelial cells. The secretion of testosterone increased during the first week of co‐culture and remained elevated up to day 12 of culture. In one prolonged co‐culture, testosterone secretion decreased progressively after day 12 and, after 1 month of culture, was at a level similar to that observed during the first 48 h. After culture for 48 h, testosterone secretion in the co‐culture was enhanced by 162 ± 5% ( p < 0.0001) compared with values observed when Leydig cells were cultured alone (42.6 ± 10.6 ng/106 Leydig cells/48 h; mean ± SEM). This change was associated with increase in mRNA levels for 3β‐hydroxysteroid dehydrogenase Δ5–Δ4‐isomerase (2.49 ± 0.58‐fold), cytochrome P450c17 (2.81 ± 0.99‐fold), cytochrome P450scc (5.20 ± 0.13‐fold) and cytochrome P450 aromatase (1.73 ± 0.21‐fold) when Leydig cells were co‐cultured with Sertoli cells ( p < 0.05 for each enzyme). IGF‐I mRNA levels were higher (2.77 ± 0.72‐fold for 7.6 kb and 1.41 ± 0.07‐fold for 1.1–1.3 kb transcripts) in the Leydig–Sertoli cell co‐cultures than the sum of the levels in Leydig and in Sertoli cells cultured alone. Both basal and hCG‐induced testosterone secretion were enhanced by treatment of the co‐culture with human recombinant FSH (50mIU/mL). For basal testosterone secretion, this increase amounted to 163 ± 5% compared with Leydig cells cultured alone ( p < 0.0001) and by 112 ± 4% compared with non‐FSH treated co‐cultures ( p < 0.01); for hCG‐stimulated testosterone secretion this increase was 220 ± 12% compared with Leydig cells cultured alone ( p < 0.0001) and 132 ± 8% compared with non‐FSH treated co‐cultures ( p < 0.01). This study confirms the enhancement of long‐term testosterone secretion by adult human Leydig cells by co‐culture with adult human Sertoli cells and shows that this effect is associated with an enhancement of the expression of several steroidogenic enzymes; it might be mediated, as in other species, through increased production of IGF‐I by co‐culture.

Enhancement of long-term testosterone secretion and steroidogenic enzyme expression in human Leydig cells by co-culture with human Sertoli cell-enriched preparations.

We have shown previously that long-term testosterone secretion, which decreases when human Leydig cells are cultured alone, increases when purified human Leydig and Sertoli cells are cultured together. In this work, human Leydig cell functions were studied further during in vitro culture, either alone or with human Sertoli cells, on a basal membrane derived from bovine corneal endothelial cells. The secretion of testosterone increased during the first week of co-culture and remained elevated up to day 12 of culture. In one prolonged co-culture, testosterone secretion decreased progressively after day 12 and, after 1 month of culture, was at a level similar to that observed during the first 48 h. After culture for 48 h, testosterone secretion in the co-culture was enhanced by 162 +/- 5% (p < 0.0001) compared with values observed when Leydig cells were cultured alone (42.6 +/- 10.6 ng/10(6) Leydig cells/48 h; mean +/- SEM). This change was associated with increase in mRNA levels for 3 beta-hydroxysteroid dehydrogenase delta 5-delta 4-isomerase (2.49 +/- 0.58-fold), cytochrome P450c17 (2.81 +/- 0.99-fold), cytochrome P450scc (5.20 +/- 0.13-fold) and cytochrome P450 aromatase (1.73 +/- 0.21-fold) when Leydig cells were co-cultured with Sertoli cells (p < 0.05 for each enzyme). IGF-I mRNA levels were higher (2.77 +/- 0.72-fold for 7.6 kb and 1.41 +/- 0.07-fold for 1.1-1.3 kb transcripts) in the Leydig-Sertoli cell co-cultures than the sum of the levels in Leydig and in Sertoli cells cultured alone. Both basal and hCG-induced testosterone secretion were enhanced by treatment of the co-culture with human recombinant FSH (50 mIU/mL). For basal testosterone secretion, this increase amounted to 163 +/- 5% compared with Leydig cells cultured alone (p < 0.0001) and by 112 +/- 4% compared with non-FSH treated co-cultures (p < 0.01); for hCG-stimulated testosterone secretion this increase was 220 +/- 12% compared with Leydig cells cultured alone (p < 0.0001) and 132 +/- 8% compared with non-FSH treated co-cultures (p < 0.01). This study confirms the enhancement of long-term testosterone secretion by adult human Leydig cells by co-culture with adult human Sertoli cells and shows that this effect is associated with an enhancement of the expression of several steroidogenic enzymes; it might be mediated, as in other species, through increased production of IGF-I by co-culture.

ETAReceptor Mediated Inhibition of Intracellular pH Regulation in Cultured Bovine Corneal Epithelial Cells

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHiregulation of exposure to 1 μmET-1 under control and acid loaded conditions. With the pH sensitive dye, 2′,7′ Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHiwas 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 μmdimethylamiloride (DMA) rapidly decreased pHiby 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 μmbafilomycin A1decreased pHiby 0.22 units. Following acid loading in NaCl Ringers with a 20 mmNH4Cl prepulse, pHirecovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 μmDMA or 20 μmbafilomycin A1. Based on decreases in H+efflux resulting from selective inhibition of NHE and V-type H+pump activity, NHE activity accounts for 76% of the pHirecovery following acid loading. Under control conditions, ET-1 (1 μm) had no effect on pHiwhereas ET-1 completely suppressed pHirecovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETAbecause in the presence of BQ-123 (10 μm), a selective ETAreceptor antagonist, pHirecovery was completely restored. Suppression of pHirecovery also occurred following stimulation of protein kinase C (PKC) with 10−7mphorbol myristate (PMA) whereas 10−7m4 α phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHirecovery after inhibition of PKC with 0.5 μmcalphostin C suggesting that the inhibition of pHirecovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 μm) reversed the suppression of pHirecovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 μmphenylarsine oxide, 10 nmokadaic acid, 10 μmcyclosporin A1or 20 μmBAPTA, also obviated the suppression of pHirecovery by ET-1. Therefore ETAreceptor mediated inhibition of pHiregulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+pump. This alteration may prevent them from becoming adequately stimulated to elicit pHirecovery in response to acid loading.

Oxidation Decreases Low Density Lipoprotein Association with the Subendothelium Extracellular Matrix

Atherosclerosis is initiated by accumulation of low density lipoprotein (LDL) in the subendothelium extracellular matrix (ECM) followed by oxidation and scavenger receptor mediated uptake by the vessel wall recruited macrophages. The effect of oxidation on LDL association with the ECM is not yet clear. In the present study we examined the hypothesis that excessive oxidation of LDL results in decreased LDL association with ECM, thereby increasing its accessibility to the scavenger receptor on macrophages. We have studied the relative association of Cu+2ion oxidized LDL to native LDL with the native or oxidized bovine corneal endothelial cells ECM. Oxidation of LDL decreased its binding to the ECM. The kinetic of this process was characterized by approximately 1 h lag phase followed by a significant decreased binding of 80% after 6.5 h oxidation. This kinetic more closely resembled the pattern of the oxidation product dienals accumulation rather than thiobarbituric acid reactive substance formation. Oxidation of ECM-bound LDL resulted in an increased LDL release from the ECM as a function of Cu+2ion concentration with a maximal increase of 2-fold at 3 μM. ECM oxidation prior to exposure to LDL resulted in a 30% decrease in LDL binding to the ECM. In conclusion, these results suggest that oxidation processes in the vessel wall result in increased dissociation of ECM-bound LDL, which in turn makes this oxidized LDL more accessible for binding and uptake by macrophages leading to foam cell formation.

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

PURPOSE. To characterize Ca 2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS. Changes in intracellular Ca 2+ ([Ca 2+ ] i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca 2+ -sensitive dye Fura-PE3. Relative rates of Ca 2+ influx were measured employing Mn 2+ as a surrogate for Ca 2+. RESULTS. Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca 2+ ] i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 µM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn 2+ influx following [Ca 2+ ] i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca 2+ -ATPase. Under Ca 2+ -free conditions, peak responses were similar to those in the presence of external Ca 2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 ± 18.8% but UTP responses by only 10.6 ± 9.5%. Repeated exposures to UTP or msATP reduced [Ca 2+ ] i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 mM) with temporal characteristics of [Ca 2+ ] i mobilization similar to that of cultured cells. CONCLUSION. BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+] i response, subsequently caused capacitative Ca 2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.

Tissue culture of human neurocytomas induces the expression of glial fibrilary acidic protein.

Cell cultures were established from three human neurocytoma specimens (primary and recurrent). The phenotypic evolution was analyzed by immunocytology in different culture conditions in the presence and absence of serum including the addition of epidermal growth factor, rat caudate extract, retinoic acid, and N-acetyl cystein. The cells were grown on glass cover slides or an extracellular matrix (ECM) from bovine corneal endothelial cells. Immunostainings were performed after overnight incubation and were repeated after 5 and 10 days of culture. The cultures were compared to an oligoastrocytoma also arising at the foramen of Monro and an ependymoma of the frontal lateral ventricle, two tumors supposedly originating from the same tissue matrix as the neurocytoma. After overnight incubation, 90% of the neurocytoma cells were positive for A2B5 and synaptophysin. GFAP reactivity appeared in the periphery of cell processes in less than 1% of the cells. The staining patterns and morphology were nearly identical under the different culture conditions. After 5 days, almost all cells were strongly positive for GFAP, while the number of cells remaining positive for synaptophysin and A2B5 was unchanged from the earlier time point. Again, there were no fundamental differences between the incubation conditions. At this point, cultures maintained on ECM were compared to their counterparts on untreated glass cover slides with identical staining results, although many fewer cells had attached. An identical immuno-reactive pattern was found on day 10. In contrast to the neurocytoma cultures, there was an immediate strong GFAP signal in both the mixed glioma and the ependymoma. A2B5 was also positive, but synaptophysin was absent. Because the neurocytoma specimens were synaptophysin positive but GFAP negative by immunohistochemistry, it is concluded that neurocytomas may represent a human neuronoglial precursor tumor that switches its phenotype in culture to astroglial differentiation despite very diverse culture conditions.

A Novel Alternatively Spliced Form of Murine Vascular Endothelial Growth Factor, VEGF 115

Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115. It differs from VEGF 120 by 37 amino acids at the carboxyl terminus. VEGF 115-specific sequence reacted to a single transcript in mouse tissues. Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes. The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter. The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody. A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells. Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones. We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115. The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

Mechanism of initial attachment of corneal epithelial cells to polymeric surfaces

The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: ‘tissue culture’ polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)—80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85–95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture neophytes can employe endogenous mechanisms for the initial attachment to TCPS, whereas cells established in culture are dependent on exogenous vitronectin in order to attach.

Cyclic AMP Activates Anion Channels in Cultured Bovine Corneal Endothelial Cells

Ion coupled fluid transport by the corneal endothelium is stimulated by adenosine through a cAMP dependent mechanism. This study examines if anion conductance is enhanced by cAMP and, hence by adenosine. Cl−fluxes, measured by changes in fluorescence of the Cl−sensitive dye SPQ, following removal or re-addition of Cl−Ringer, could be accelerated by 20 μMforskolin or 10 μMadenosine. The cAMP cocktail (20 μMforskolin+100 μMIBMX+100 μMcpt-cAMP) had no effect on resting [Cl−]i. However, when Cl−influx was inhibited by 100 μMfurosemide, net Cl−efflux was observed in response to the cAMP cocktail. Exposure to the cAMP cocktail alone depolarized the resting membrane potential. Conversely, the cAMP cocktail caused a relative hyperpolarization in cells which had been previously depolarized beyond the equilibrium potential for Cl−(ECl−), by application of 1 μMGramicidin D. cAMP dependent changes in membrane potential could be inhibited by 50 μMNPPB, but not by 200 μMDPC, 100 μMH2DIDS or 50 μMglibenclamide. Taken together, these results are consistent with NPPB-sensitive, cAMP activated Cl−channels. To examine if these channels are permeable to HCO−3, changes in pHi in response to the cAMP cocktail were measured in acidified and depolarized cells in the absence of Na+. The cAMP cocktail caused an increase in pHi only when HCO−3was present, consistent with HCO−3influx. In control HCO−3Ringer, the cAMP cocktail caused a transient decrease in pHi, which could not be accounted for by inhibition of Na+:nHCO−3cotransport or stimulation of Cl−/HCO−3exchange. These results are consistent with conductive HCO−3efflux through cAMP activated channels. We conclude that cultured bovine corneal endothelial cells possess cAMP activated anion channels.

Demonstration of the sodium-hydrogen exchanger NHE-1 in corneal endothelial cells

Purpose: Maintenance of corneal hydration and transparency requires the presence of many membrane transporter proteins in the corneal endothelium, including, it is suggested, a sodium-hydrogen (Na+/H+) exchanger. This study investigates the presence of the Na+/H+ exchanger (NHE) in bovine and sheep corneal endothelium. Methods: A polyclonal antibody, specific for type 1 Na+/H+ exchanger (NHE-1), was used to screen bovine and sheep corneal endothelial cells for the presence of NHE activity by the Western blotting technique. Using immunoassay quantification techniques (ELISA), the quantity of NHE-1 present was measured. Results: Western blot analysis, using the NHE-1 antibody, recognized proteins with a mass of 70 kDa and 56 kDa in bovine and sheep corneal endothelial cells respectively. From the measured activity of NHE-1 detected using ELISA and the previously measured cell density, an estimate of the number of NHE-1 molecules present in each corneal endothelial cell was calculated to be 40 000 per cell. Conclusion: Bovine and sheep corneal endothelium contain type I Na+/H+ exchanger (NHE-1). Future immunochemical studies will confirm its precise membrane location. This data provides direct evidence of its existence and the quantitative estimate of its concentration in the membrane provides further opportunity to resolve its role in transendothelial bicarbonate ion transport. Acknowledgement: The polyclonal antibody was a generous gift from Prof. L. Fliegel, Department of Biochemistry and Pediatrics, University of Alberta.

Demonstration of the sodium‐hydrogen exchanger NHE‐1 in corneal endothelial cells

Purpose: Maintenance of corneal hydration and transparency requires the presence of many membrane transporter proteins in the corneal endothelium, including, it is suggested, a sodium‐hydrogen (Na+/H+) exchanger. This study investigates the presence of the Na+/H+ exchanger (NHE) in bovine and sheep corneal endothelium.Methods: A polyclonal antibody, specific for type 1 Na+/H+ exchanger (NHE‐1), was used to screen bovine and sheep corneal endothelial cells for the presence of NHE activity by the Western blotting technique. Using immunoassay quantification techniques (ELISA), the quantity of NHE‐1 present was measured.Results: Western blot analysis, using the NHE‐1 antibody, recognized proteins with a mass of 70 kDa and 56 kDa in bovine and sheep corneal endothelial cells respectively. From the measured activity of NHE‐1 detected using ELISA and the previously measured cell density, an estimate of the number of NHE‐1 molecules present in each corneal endothelial cell was calculated to be 40 000 per cell.Conclusion: Bovine and sheep corneal endothelium contain type I Na+/H+ exchanger (NHE‐1). Future immunochemical studies will confirm its precise membrane location. This data provides direct evidence of its existence and the quantitative estimate of its concentration in the membrane provides further opportunity to resolve its role in transendothelial bicarbonate ion transport.Acknowledgement: The polyclonal antibody was a generous gift from Prof. L. Fliegel, Department of Biochemistry and Pediatrics, University of Alberta.

Human corneal epithelial cells reorient and migrate cathodally in a small applied electric field

PURPOSE. To test whether human corneal epithelial cells (HCECs) respond to small applied electric fields (EFs) in a similar manner to bovine corneal epithelial cells (BCECs), the orientation and directed migration in small EFs of both primary cultures and of a human corneal epithelial cell line were quantified. METHODS. Primary cultures of human corneal epithelial cells (PHCECs) and transformed human corneal epithelial cells (THCECs) were exposed to EFs (100 mV/mm–250 mV/mm) in different media. Cell migration was traced using an image analyser. RESULTS. PHCECs and THCECs reoriented and migrated towards the cathode (negative pole) when cultured in small direct current (dc) EFs. Both the reorientation and directional migration were voltage- and serum-dependent, as shown previously for bovine cells. PHCECs and THCECs showed significant perpendicular orientation in EFs at 150 mV/mm in medium with serum, while at the same voltage, no significant orientation was found in serum free medium. PHCECs started to show perpendicular reorientation around 30 min after onset of EF at 150 mV/mm. They showed significant directional migration at 150 mV/mm, with directedness of 0.35 ± 0.07 and a migration rate of 9.1 ± 0.7 µm/h (n = 90), both significantly higher than that of cells in serum free medium. Addition of EGF-induced significant reorientation and directional migration of THCECs at 100 mV/mm. Additionally, as for BCECs, which remained viable and responsive to electric fields for at least 75 h at 150 mV/mm, THCECs also remained viable and showed responsiveness during long periods of exposure to EFs (at least 20 h). CONCLUSIONS. Cultured human primary CECs and a human corneal epithelial cell line both responded to small EFs with perpendicular reorientation and cathodally-directed migration. Cell responses were qualitatively similar to those reported previously for bovine CECs. The endogenous EFs generated by wounded cornea may play an important role in promoting cell shape changes and directed migration of CECs during the healing process.

Culture of cells from human tumors of the nervous system on an extracellular matrix derived from bovine corneal endothelial cells.

Cell culture has become an integral part of the daily routine of most biological laboratories. Many genetic tests, chemo-resistance testing for chemotherapy, and the study of deregulated cell proliferation in cancer, cannot be performed without cultured cells. In endocrinology and developmental biology, elaborate cell culture techniques allow the investigation of regulatory processes and cellular interactions in in vitro systems, in which the experimental conditions can be defined as in no other experimental paradigm.

In-vitro development of corneal epithelial cells on a new hydrogel for epikeratoplasty.

This study demonstrates the adhesion and growth of bovine corneal epithelial cells on the surface of a new hydrogel. The hydrogel, containing 78% of water and 22% of AN-69 polymer (poly(acrylonitrile-sodium methallyle sulfonate)), was obtained by phase inversion of polymer-dimethylformamide solution in physiological saline (0.9% NaCl). Experiments were also carried out using hydrogel treated with arginine, human albumin and collagen IV. Covering of hydrogel samples by epithelial cells was completed within ten days, with good cell viability. The epithelial cells spread out and formed a consistent cell layer, confirmed by immunocytochemistry experiments against cytokeratins. Transmission electron micrographs showed numerous desmosomes between cells and the presence of some membrane differentiations at the cell/hydrogel interface. This study suggests therefore that the hydrogel might be suitable for the development of artificial epikeratoplasty grafts.

Growth, morphology, morphometry and keratin patterns of bovine corneal epithelial cells cultured in vitro

In this study, the effects of different culture systems on bovine corneal epithelial cells were analysed in order to better understand the influence of bovine keratocytes on epithelial cells. Growth, morphological, morphometrical analyses of cells and keratin patterns were evaluated. The aim was to improve the culture technique in order to obtain a good in vitro proliferation of these cells for their employment in clinical and toxicological situations. The bovine corneal epithelial cells were cultured under different conditions: on keratocyte or 3T3-J2 fibroblast feeder layers, with media conditioned either by the two feeder layers or with a basal medium. The epithelial cells cultured on a keratocyte feeder layer as compared to those grown under the other conditions, proved to have a higher growth rate as well as to be smaller in the cytoplasmic and nuclear area; moreover, after 21 days of culture they expressed 64-kDa keratin, designed as a marker for corneal epithelial cell differentiation. To sum up, the keratocyte feeder layer is the most effective for stimulating the growth and differentiation of corneal epithelial cells, resembling the in vivo situation. It might also be successfully employed for clinical and toxicological purposes.

In vitro study for staining and toxicity of rose bengal on cultured bovine corneal endothelial cells: Lee YC, Park CK, Kim MS, Kim JH: Cornea 1996; 15: 176–385

In vitro study for staining and toxicity of rose bengal on cultured bovine corneal endothelial cells: Lee YC, Park CK, Kim MS, Kim JH: Cornea 1996; 15: 176–385

Synergistic action of heparin and serum on basic fibroblast growth factor-modulated DNA synthesis and mitochondrial activity of cultured bovine corneal endothelial cells.

Basic fibroblast growth factor (bFGF) is a major mitogen and chemoattractant for many cell types. The synergistic role of fetal bovine serum (FBS) and heparin on the modulation of tissue-cultured bovine corneal endothelial cells by bFGF was studied. Cell modulation was assessed by DNA synthesis ([3H]thymidine incorporation), mitochondrial activity (MTT assay), mitochondrial volume (transmission electron microscope study), and cell shape in mitotically competent cells and cells mitotically inhibited by 5-fluorouracil, mitomycin C, and irradiation. The dose-dependent stimulation of bFGF was found to be different for DNA synthesis and mitochondrial activity, the maximal effective dose being 1 and 10 ng/ml, respectively. Supplementation of bFGF with FBS enhanced both DNA synthesis and mitochondrial activity and caused a shift in the dose response to lower bFGF concentrations for the DAN synthesis, but not for the mitochondrial activity. Supplementation of bFGF with heparin resulted in an additional response for the mitochondrial activity, but not for the DNA synthesis. In addition, bFGF increased mitochondrial volume and induced elongation of the cells. These processes were further enhanced by the addition of heparin to bFGF. These results suggest that serum and heparin have a differential effect on mitogenic and non-mitogenic cells processes modulated by bFGF.

In vitro study for staining and toxicity of rose bengal on cultured bovine corneal endothelial cells.

It has recently been reported that rose bengal (RB) is actually not a vital dye and that it has an intrinsic toxic effect. Therefore, we conducted a study to determine its staining capability and the presence of intrinsic toxicity. We also examined its effects on the proliferation of cultured bovine corneal endothelial cells. The cells stained despite short exposure (1 s) to rose bengal under a fluorescein photomicroscope. The cell nucleus stained first, followed by the cytoplasm; the intensity was directly dependent on the exposure time. Although the entire cell layer did not stain at an RB concentration of 0.001%, the cell layer stained diffusely above a concentration of 0.01%. At a 5% concentration, areas of cellular detachment and lysis were noted. The degree of cellular change was directly proportional to the concentration of RB and the exposure time. Cellular damage included cell swelling, intracytoplasmic vacuole formation, cell detachment, and lysis. The presence of light augmented the toxic effect of RB on cultured endothelial cells and was statistically more significant than RB's intrinsic toxic effect. Exposure to RB concentrations > 0.01% induced statistically significant concentration-dependent inhibition of cell proliferation. These results suggest that rose bengal may not actually be a vital dye on cultured bovine corneal endothelial cells and that it has intrinsic inhibitory and toxic effects with additional phototoxic effects on corneal endothelial cells. Furthermore, cell proliferation was markedly inhibited after exposure to RB.