Protection from oxidative insult in glutathione depleted lens epithelial cells.

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2 and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2O2-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H2O2 when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15-20% of the control values without a concomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to an initial concentration of 0. 05 mm H2O2 (for 50,000 cells) or 2 doses of 0.5 mm H2O2 (for 800,000 cells). After exposure to H2O2, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3 and 6 and counted on day 7. When 50,000 or 800,000 cells with decreased glutathione were exposed to 0.05 or 0.5 mm H2O2 the H2O2 was cytotoxic, whereas cells treated with H2O2 alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H2O2 from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H2O2 exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EM level. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells were treated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine and then challenged with H2O2 cytotoxicity was prevented and the cells were capable of growth. The data show that H2O2 was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the generation of reactive oxygen species. In addition, the results indicate that GSH has an important protective role independent of its ability to decompose H2O2 via glutathione peroxidase.

Heme oxygenase synthesis is induced in cultured lens epithelium by hyperbaric oxygen or puromycin.

We showed previously that treatment of cultured rabbit lens epithelial cells (LECs) with hyperbaric oxygen (HBO) produced DNA strand-breaks, caused reversible inhibition of protein synthesis and induced the synthesis of a 32 kD protein. In the present work, we employed immunostaining procedures to identify the 32 kD protein as heme oxygenase-1 (HO-1). Increased synthesis of the enzyme was observed as early as 12 hr after HBO-treatment, reached a maximum at 18 hr and was not detectable at 36 hr. Exposure of the cells to hemin also increased the synthesis of HO-1. An HBO-induced inhibition of protein synthesis and the subsequent induction of HO-1 was also observed in the capsule-epithelium of cultured rabbit lenses. For both LECs and the cultured lens, only HO-1 and not heme oxygenase-2 was HBO-inducible. Use of the antioxidant dimethylthiourea with HBO-treated lenses or LECs did not alter the observed effects on protein synthesis or the induction of HO-1. In contrast to results obtained with 50 atm O2, a pressure of 25 atm O2 inhibited protein synthesis only slightly and failed to induce synthesis of the 32 kD protein (although, as shown previously, identical exposure of LECs to 25 atm O2 significantly damaged DNA). Inhibition of protein synthesis in LECs and cultured lenses with the use of puromycin also induced synthesis of HO-1. Both hemin (10 micron), a source of iron, and 50 atm O2 produced a three-fold increase in the concentration of ferritin, a natural iron chelator, in LECs two days after exposure; no effects on ferritin levels were observed after 1 or 3 days. The finding that the increase in ferritin concentration occurred in the cells significantly after hemin- or HBO-induced synthesis of heme oxygenase indicates that chelatable iron rather than the heme molecule itself may have been the primary agent responsible for inducing ferritin synthesis. The data suggest that HBO-induced synthesis of HO-1 in the lens epithelium may be the result of an inhibition of protein synthesis, possibly leading to an accumulation of heme, rather than a direct protective response against oxidative stress.

Regional differences in the distribution of catalase in the epithelium of the ocular lens.

Oxidative stress is thought to play a major role in cataract formation. The present experiments are aimed at gaining a better understanding of the systems that protect the lens from damage by reactive oxygen species. The aqueous humor normally contains hydrogen peroxide (H2O2), a compound capable of generating reactive oxygen species. The systems protecting the ocular lens from oxidative damage are primarily confined to the epithelium, a single layer of cells on the anterior side of the organ directly beneath the lens capsule. When cultured rabbit lenses were challenged with a single dose of 0.2 mM H2O2, cells in the peripheral region of the epithelium survived; those in the central region died. Here we investigate the histochemical and immunoperoxidase distributions of catalase, an enzyme which detoxifies H2O2, in cells from the peripheral and central regions of the epithelium on flat mount preparations of the epithelium. In a flat mount, the entire population of lens epithelial cells can be viewed on one preparation. The reaction product for catalase activity and its immunoperoxidase localization were more intense in peripheral epithelial cells than in cells throughout the central epithelium. Treatment of cultured lens epithelial cells or rabbit lenses with 3-aminotriazole or potassium cyanide, inhibitors of catalase, reduced or abolished the histochemical reaction product. Ultrastructural cytochemistry confirmed the presence of catalase in microperoxisomes of the epithelial cells from whole lenses. The decreased level of catalase throughout the central epithelium may account for the increased susceptibility of these cells to H2O2-induced cell death.

Hydrogen peroxide affects specific epithelial subpopulations in cultured rabbit lenses.

PURPOSE: To investigate the effect of hydrogen peroxide on the epithelial cells of cultured rabbit lenses. METHODS: Lenses were cultured in minimum essential medium containing a single dose of 0.03, 0.1, or 0.2 mM H2O2. Three hours later the medium was replaced with peroxide-free minimum essential medium. Lenses were also treated with 0.5 mM 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU) to lower the activity of glutathione reductase and then exposed to 0.03 mM H2O2 maintained nearly constant by glucose oxidase. After H2O2 treatment, lenses were fixed and whole mounts of the epithelium were prepared or lenses were processed for electron microscopy. RESULTS: Cells exposed to a single dose of 0.03 mM H2O2 appeared normal; 0.1 mM H2O2 was not cytotoxic. Exposure to 0.2 mM H2O2 elicited swelling in cells in the pre-equatorial region (30 minutes) followed by the formation of islands of cells in the pre-equatorial region at 1 hour. Central epithelial cells appeared normal at 1 hour, were swollen at 3 hours and dead at 24 hours. By 48 hours, dead cells were found in the pre-equatorial and central regions. Cells in the peripheral region of the epithelium did not exhibit cytotoxicity. If lenses were pretreated with BCNU and then challenged with a maintained level of 0.03 mM H2O2, cytotoxicity was induced in the central and pre-equatorial regions. Cells in the peripheral region survived BCNU-H2O2 treatment. CONCLUSIONS: Cells in the peripheral region of cultured lenses were more resistant to H2O2 cytotoxicity than cells in the central and pre-equatorial regions. The antioxidant defense or repair systems for H2O2-induced damage do not appear to be uniformly distributed in subpopulations of the lens epithelium.

Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.

PURPOSE: To determine if lens epithelial lines can be established from cryopreserved whole rabbit lenses and from cryopreserved capsule-epithelial preparations (CEPs). METHODS: Lenses or freshly isolated CEPs were cryopreserved and subsequently thawed. Thawed whole lenses were cultured for 48 hours in growth medium and fixed, and whole mounts were examined for mitosis. In addition, CEPs were peeled from cryopreserved lenses and placed in tissue culture. Viability of cryopreserved cells was assessed measuring attachment efficiency and growth. RESULTS: Whole mounts from cryopreserved lenses that were thawed and placed in organ culture in a serum-containing medium exhibited numerous mitotic figures. Freshly isolated CEPs that were cryopreserved and CEPs from cryopreserved lenses generated cell lines. Attachment efficiency was 90% within 3 hours of plating. When 50,000 cells from cryopreserved CEPs were cultured in growth medium, 10(6) cells were noted after 7 days of culture. The cells completed 27 population doublings and showed no sign of senescence. CONCLUSIONS: Rabbit lens epithelial cell lines can be initiated from cryopreserved lenses or CEPs.

Lens epithelium: a primary target of UVB irradiation.

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.

The superoxide dismutase mimic TEMPOL protects cultured rabbit lens epithelial cells from hydrogen peroxide insult.

The superoxide dismutase mimic, 4-hydroxy TEMPO (TEMPOL), was used to investigate the mechanism by which H2O2 damages cultured rabbit lens epithelial cells and to identify some of the targets of H2O2 insult. Most studies aimed at determining the mechanism by which H2O2 exerts its cytotoxic effect have used iron chelators to prevent the generation of the damaging hydroxyl radical. Since TEMPOL does not chelate transition metals, we were afforded an additional means of investigating the mechanism by which H2O2 exerts its cytotoxicity. Cells at low or high density were cultured in MEM containing 5 mM TEMPOL and exposed to a single sub-lethal dose of 0.05 or 0.5 mM H2O2, respectively. Analysis of EPR spectra indicated that TEMPOL was stable in MEM, did not destroy H2O2 and penetrated the intracellular fluid. TEMPOL prevented or curtailed the H2O2-induced inhibition of cell growth, blebbing of the cell membrane, the decrease in NAD+, the activation of poly ADP-ribose polymerase, an enzyme involved in DNA repair, and limited the induction of single strand breaks in DNA normally brought about by H2O2. TEMPOL did not prevent the H2O2-induced decrease in reduced glutathione, lactate production, and the activity of glyceraldehyde 3-phosphate dehydrogenase, or the H2O2-induced increases in oxidized glutathione and hexose monophosphate shunt activity. Addition of TEMPOL 1-15 min after exposure of cells to H2O2 offered partial protection from the inhibition of cell division. TEMPOL at 5 mM did not inhibit cell growth. These results, coupled with our other findings suggest that some of the H2O2-induced damage in cultured rabbit LECs is mediated by intracellular redox-active metals involved in the Haber-Weiss cycle. Cellular changes not protected by TEMPOL, including attack of H2O2 on the thiol groups of GSH (mediated through glutathione peroxidase) and G3PDH, are likely brought about by H2O2 itself and not by reactions of oxygen free-radicals generated from H2O2.

Hyperbaric oxygen inhibits the growth of cultured rabbit lens epithelial cells without affecting glutathione level.

Studies on human patients and experimental animals indicate that hyperbaric O2 can opacify the lens nucleus and damage the lens epithelium in vivo. Here we investigate the effects of hyperbaric O2 on cultured rabbit lens epithelial cells (LECs). When the cells were exposed to 50 atm O2 (99% O2 + 1% CO2) for 3 hr there were no immediate effects on morphology, viability and transport processes (uptake of 86Rb and 14C-alpha AIB). In addition, the O2 treatment did not lower the high level of reduced glutathione or increase the low level of oxidized glutathione. However, 50 atm O2 did produce a near doubling in the glycolytic rate which maintained ATP at levels only slightly lower than normal. Although the 3-hr O2 treatment was not lethal, it completely inhibited cell division for 2 days. After 2 days, growth was initiated and, at day 7 the rate of growth was faster than the controls (control cells were treated with ambient air or 50 atm N2 for 3 hr). Cells treated with 8 atm O2 for 3 hr exhibited a slowed rate of growth, relative to controls, while exposure to 2 atm O2, did not inhibit mitosis. Changes in morphology (multilayering and elongation) of cells exposed to 50 atm O2, but not the controls, were evident 7 days after the 3-hr exposure. The incorporation of [35S]methionine into individual polypeptides and [3H]thymidine into DNA was significantly inhibited immediately following a 3-hr treatment with 50 atm O2, but both parameters recovered within 2 days. DNA strand breaks were observed in LECs following hyperbaric O2 treatment as low as 4 atm O2 for 3 hr and increased with higher pressures of O2, but not N2. Treatment with 50 atm O2 nearly doubled the activity of the DNA repair enzyme, poly-ADP-ribose polymerase, and decreased the level of its substrate NAD+; the latter effect was reduced by 3-aminobenzamide, an inhibitor of the enzyme. Thus, although LECs tolerated brief exposures to high pressures of O2 without cell death, DNA damage occurred at relatively low pressures of O2. All of the effects of hyperbaric O2 on LECs occurred without any alteration of the normal levels of reduced and oxidized glutathione. It appears that GSH is important in maintaining cell viability during exposure to an elevated level of O2, but that it is incapable of preventing O2-induced effects on growth and DNA.

The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 by cultured rabbit lens epithelial cells.

The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 were investigated in cultured rabbit lens epithelial cells. Exposure of cells to H2O2 was carried out following inhibition of either of the two antioxidant systems. Two different procedures were used to expose the cells to extracellular H2O2, one in which a low, steady state level of 0.025 mM H2O2 was maintained in the culture medium with the use of glucose oxidase and the other in which H2O2 was added to the medium as a single pulse at levels ranging from 0.03 to 0.5 mM. When lens cells were treated with a low, steady state level of H2O2, the glutathione redox cycle was the primary means of defense against oxidative damage. Cells with fully active catalase but with inhibited glutathione reductase were not able to resist the cytotoxic effects of a 0.025 mM level of extracellular H2O2. Under these conditions the cells were nearly completely depleted of reduced glutathione within 15 min. The cellular damage observed after 1.5 hr of culture included loss of cell-to-cell contact, rounding up of the cells and formation of numerous blebs. In contrast, cells with completely inhibited catalase but with an unimpaired glutathione redox cycle suffered few damaging effects from a 3-hr exposure to 0.025 mM H2O2. When lens cells were pulsed with a single challenge of 0.5 mM H2O2, both the glutathione redox cycle and catalase were found to be essential for survival of the cells. While control cells were able to withstand the pulse of H2O2, cells with impaired activities of either the glutathione redox cycle or catalase were killed. Control cells treated with 0.5 mM H2O2 may have been protected from damage by the fact that the cellular level of GSH never dropped below 35% of normal. The cause of cell death following inhibition of catalase appeared to be related to an inability of the cells to remove peroxide from the culture medium, at a rapid rate, following the H2O2-pulse. Although cells with impaired glutathione reductase activity removed H2O2 from the medium at a rate comparable to that of control cells (due to uninhibited catalase activity), they did not survive the challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Susceptibility of lens epithelial membrane SH groups to hydrogen peroxide.

Although membrane SH groups are thought to be targets of oxidative insults, no measurement of lens epithelial membrane SH groups following exposure to potentially damaging oxidants has been reported. Here we investigate the effect of hydrogen peroxide, an oxidant found in the aqueous humor, and of p-chloromercuriphenylsulfonic acid (p-chloromecuribenzene-sulfonic acid) (PCMBS), a relatively impermeant sulfhydryl probe, on membrane SH groups and ion homeostasis in cultured lens epithelial cells. Exposure to PCMBS caused a 10% loss of membrane SH groups, an increase in sodium and calcium levels, and a decrease in potassium, but did not affect the intracellular level of glutathione (GSH). After 5 min of exposure to an initial concentration of 1.0 mM hydrogen peroxide, GSH declined from 14.1 mM to 3 mM, there was a 20% loss of membrane SH groups and within 1 hr, potassium declined from 132 to 116 mM. Cells that were exposed to 0.1 or 0.5 mM peroxide did not exhibit significant loss of membrane SH groups and did not show a decrease in GSH comparable to that found in cells treated with 1 mM peroxide. The peroxide induced loss of membrane SH groups and subsequent change in ion homeostasis occurred only when there was a rapid and sustained loss of intracellular glutathione. Thus lens epithelial cell membrane SH groups are not only important in ion regulation but are targets of hydrogen peroxide when the intracellular level of GSH is significantly diminished.

Establishment of lens epithelial cell lines from Emory and cataract resistant mice and their response to hydrogen peroxide.

We determined the conditions required for the establishment of lens epithelial cell lines from individual Emory and age matched cataract-resistant (CR) mice, and investigated the response of these cells to hydrogen peroxide. The technique described here permits the establishment of mouse lens epithelial cell lines from individual animals and provides an opportunity to study changes in epithelial function that precede and accompany cataract formation and aging. Capsules with lens epithelial cells were isolated from 1, 6 month, and 1.5-2 year old Emory and CR mice and cultured in minimal essential medium (MEM) containing 4% heat inactivated fetal bovine serum and 4% heat inactivated rabbit serum. Seeding efficiency at 3 hours was approximately 83% for all lines, doubling time was 22-24 hours, and the shape of the growth curves was comparable for Emory and CR mice from each age group. A three hour exposure of Emory and CR mouse lens epithelial cells from older animals to a constant level of 0.02 mM hydrogen peroxide or to an initial concentration of 0.01, 0.03, or 0.05 mM hydrogen peroxide resulted in a delay in growth. The delay in cell proliferation was decidedly more pronounced in lens epithelial cells from Emory mice. Lens epithelial cells from cataractous mice appear to be more sensitive to oxidative insult than their CR counterparts.

Influence of the activity of glutathione reductase on the response of cultured lens epithelial cells from young and old rabbits to hydrogen peroxide.

Our previous studies on cultured rabbit lens epithelial cells from 4-day-old rabbits showed that the glutathione redox cycle plays an important role in detoxifying H2O2, a potentially damaging oxidant present in the aqueous humor. Here we report the effect of donor age and cell density on the ability of cultured rabbit lens epithelial cells to detoxify H2O2. Lens epithelial cells (8 x 10(5] from a 4-day-old and an 8-year-old rabbit were cultured for 3 hr in minimal essential medium (MEM) or in MEM containing 0.01-0.1 mM H2O2 maintained with glucose oxidase. We determined the effect of H2O2 on the level of reduced glutathione (GSH), hexose monophosphate shunt activity, cell growth, and morphology. For growth studies, cells were exposed to the desired concentration of H2O2 for 3 hr and then cultured in MEM plus 10% rabbit serum for 7 days and counted. Young and old untreated cells contained high levels (30-40 nmol/8 x 10(5) cells) of GSH. Cells from 4-day-old rabbits tolerated 0.03 mM H2O2 with no effect on GSH and a minimal decrease in subsequent cell growth. However, in the older cells, GSH and growth were substantially diminished following treatment with 0.03 mM H2O2. Cells plated out at high density (8 x 10(5] were more tolerant of 0.03 mM H2O2 than cells plated out at low density (5 x 10(4]. Maximum shunt activity in the younger cells exposed to H2O2 was twice that of the older cells and occurred at a higher level of H2O2 (0.04 compared with 0.03 mM). Enzyme activities in untreated young and old cells were comparable for hexokinase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase. However, glutathione reductase activity was 50% lower in the cells from the 8-year-old rabbit. The toxicity of H2O2 to cultured lens epithelial cells was directly related to donor age and inversely related to cell density. The damage in the older lens epithelial cells at 0.03 mM H2O2 was apparently due, in part, to a diminished response of the glutathione redox cycle to oxidative challenge.

Retention of lens specificity in long-term cultures of diploid rabbit lens epithelial cells.

Rabbit lens epithelial cells from newborn animals exhibited limited growth when cultured under standard conditions. Cell lines were generated when explants from individual lenses were cultured in medium supplemented with conditioned medium or untreated rabbit serum. All lines exhibited a stable epithelial morphology. One line, N/N1003A, was examined extensively with respect to its growth, ploidy, and maintenance of lens-specific functions. Cells at population-doubling level (pdl) 120 exhibited a normal chromosomal banding pattern, were diploid, were non-tumorigenic in vivo, did not grow in suspension culture, and did not exhibit sustained growth in medium supplemented with low concentrations of serum. The shape of the growth curves and the final density for cells at pdl 24 and 181 exposed to various concentrations of serum were identical. The cells showed no diminution in growth as a function of in vitro age. The cells retained lens-specific functions. Proteins were isolated from cells at pdl 40 and 170, and were separated on polyacrylamide gels. Western immunoblot analysis using antiserum to alpha-crystallin, a tissue-specific protein found in lens epithelial cells in vivo, indicated the presence of alpha-A- and alpha-B-crystallin polypeptides. The cells also contained the transcription factors required for activating the murine alpha-A-crystallin gene promoter, which is known to function with precise tissue specificity. When an expression vector including the bacterial chloramphenicol acetyltransferase (CAT) gene controlled by the alpha-A-crystallin gene promoter was introduced into the lens epithelial cells, the CAT gene was expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Detoxification of H2O2 by cultured rabbit lens epithelial cells: participation of the glutathione redox cycle.

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4-6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 10(5) cells)-1 3 hr-1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0.01 mM. Participation of the glutathione redox cycle in the H2O2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1-14C]-labeled glucose. The oxidation of [1-14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0.04-0.05 mM H2O2, while the corresponding increase in oxidation of [6-14C]-labeled glucose was only 1.6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0.03-0.04 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 10(5) epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Thrombin induces cell division in rabbit lenses cultured in a completely defined serum-free medium.

Experiments were initiated to gain an understanding of the environmental factors that may regulate injury-induced mitosis and wound healing in the mammalian lens. The addition of thrombin or trypsin to a completely defined serum-free medium stimulated cell proliferation and migration in the cultured mammalian lens. A 30 min exposure of the rabbit lens to highly purified thrombin induced DNA synthesis and mitosis throughout the normally amitotic central region of the lens epithelium. Lenses exposed to thrombin for 24 or 52 hr exhibited cell migration and mitosis. The mitotic response brought about by thrombin was totally curtailed by hirudin and antithrombin III. Prothrombin, papain, or pepsin were not mitogenic toward the cultured lens. A 30 min exposure of the lens to trypsin induced cell division and migration, a response that did not occur in the presence of trypsin inhibitors. Lenses cultured in a trypsin-containing medium for 24 hr showed extensive cell death throughout the entire central region of the epithelium. In addition, an endogenous serine protease, plasminogen activator, was detected in cultured rabbit lens epithelial cells. Wound healing in the lens in vivo is accompanied by cellular migration and mitosis. The present experiments demonstrate that a highly purified serine protease, thrombin, which is present at the site of lenticular injury in vivo, is capable of inducing mitosis and migration in lens epithelia. The results suggest that thrombin or other exogenous and endogenous serine proteases might contribute to the process of wound healing in the ocular lens.

Establishment and characterization of a lens epithelial cell line from an eight year old rabbit.

Although information is available on the in vitro properties of lens epithelia of young adult animals from several species, few, if any reports document the conditions required for the initiation and long-term culture of lens epithelium from animals beyond their medium life-span. We report here on the conditions required for the culture of lens cells from an 8 year old rabbit. New Zealand White rabbits have a median life-span of approximately 7 years. Primary culture was initiated in MEM supplemented with 10% rabbit serum. Cells reached confluency within 25 days, responded to serum in a dose dependent manner and had an average doubling time of 23 h during the logarithmic growth phase. Cells increased in number in a dose dependent manner when insulin, insulin growth factor, epidermal growth factor (EGF), or fibroblast growth factor (FGF) was added to the culture medium. Thus, lens epithelia from this very old rabbit retained the ability to respond to highly purified growth factors. Cells exposed to a medium supplemented with insulin, EGF and FGF showed a five-fold increase in number at day 7 of culture, a value exceeding that brought about by the individual growth factors. An examination of chromosomal preparations indicated that the cells were aneuploid. Whether the aneuploidy was acquired in vitro or is a normal adjunct of aging in the lens in vivo is unknown. Proteins extracted from this line contained polypeptides that migrated to the position of and had apparent molecular weights of lens proteins.

Both human and newborn rabbit lens epithelial cells exhibit similar limited growth properties in tissue culture.

Human lens cells from 5-91-year old individuals were cultured in 8 different basal media containing fetal bovine, adult bovine, rabbit or human serum or human plasma or in a serum-containing medium supplemented with insulin, epidermal growth factor, fibroblast growth factor plus other hormones or trace elements. Cultures were initiated from explants of the capsule and epithelium or following enzymatic dissociation of cells from the capsule. Under all conditions the epithelial cells had a limited doubling potential. As a function of time in culture, cells enlarged, displayed numerous filaments and exhibited apparent in vitro senescence. Lens epithelia from 4-6 day old rabbits cultured under identical conditions mimicked the behavior of human lens cells. Lens epithelia from newborn rabbits may be a suitable model for investigating the basis of apparent in vitro senescence in this cell type and could help in defining the conditions required for the long-term growth of human lens cells. The limited growth of human lens epithelia suggests that these cells require tissue-specific nutrients or hormonal supplements not present in standard tissue culture media.