Oligonucleotide microarray analysis of human lens epithelial cells: TGFbeta regulated gene expression.

PURPOSE: Transforming growth factor beta (TGFbeta), a pro-fibrotic cytokine has been proposed a causative factor in the progression of lens pathologies including posterior capsule opacification (PCO), a condition that occurs after cataract surgery. This study employs oligonucleotide microarrays to provide a global profile of gene expression in FHL 124 cells, to identify changes in gene expression following treatment with TGFbeta1 and TGFbeta2, and to enable putative genes relating to TGFbeta regulation and PCO to be identified. METHODS: Routinely cultured FHL 124 cells maintained in serum free Eagle's Minimum Essential Medium (EMEM) were treated with either TGFbeta1 or TGFbeta2 at 10 ng/ml for 24 h then total RNA extraction was carried out. Total RNA (16 microg) was used to analyze gene expression by spotted oligonucleotide microarray hybridization. The spotted oligonucleotide microarrays employed contained 13,971 oligonucleotide probes, each designed to be specific for an individual gene. Array images were analyzed using GenePix Pro 3.0, followed by raw data import into GeneSpring 7.0 where a cross gene error model (CGEM) filter was applied. Data was subjected to LoWess normalization prior to comparison of the different treatment groups. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to validate the oligonucleotide microarray data, using a select number of genes exhibiting differential expression. RESULTS: A total of 301 genes were up-regulated by more than 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these up-regulated genes had biological functions relevant to lens epithelial cells including roles in contraction, transdifferentiation and as extracellular matrix (ECM) components. A total of 164 genes were down-regulated by more that 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these down-regulated genes have biological functions including roles in apoptosis, signaling, and as anti-oxidants. Following treatment with TGFbeta1 and TGFbeta2, QRT-PCR successfully validated the differential changes in gene expression detected by oligonucleotide microarrays. CONCLUSIONS: TGFbeta1 and TGFbeta2 regulate the gene expression of genes that have important roles in human lens epithelial cell biology. Most importantly, TGFbeta induces the gene expression of a number of fibrotic markers which may have a role in promoting the development of PCO such as transdifferentiation markers, contractile factors, and ECM components.

Calcium mobilisation modulates growth of lens cells.

The modulating effect of calcium cell signalling agonists on tissue growth was studied in a rabbit lens cell line (NN1003A). Calcium mobilisation was measured after Fura-2 incorporation and growth assayed either by direct Coulter counting or [3H]-thymidine incorporation. Transient increases in cytoplasmic calcium were elicited by rabbit serum, histamine, ATP and PDGF. Thapsigargin induced a prolonged increase and all of the above agonists failed to elicit a response after thapsigargin. Rabbit serum and PDGF both increased cell growth in a concentration-dependent manner. While histamine and ATP had little effect in serum-free medium, they reduced serum-stimulated growth. Acetylcholine and FGF did not produce a marked rise in cytoplasmic calcium and neither did they modulate growth. Both thapsigargin and caffeine greatly inhibited growth. These findings indicate that, in lens cells, agonists that mobilise calcium, whether by acting through G-protein or tyrosine kinase receptors, also modulate lens cell growth. Agents such as thapsigargin and caffeine that inactivate the same calcium store also inhibit growth.

Insulin growth factor and epidermal growth factor trigger mitosis in lenses cultured in a serum-free medium.

The mitogenicity of insulin, insulin growth factor (IGF), and epidermal growth factor (EGF) was evaluated on rabbit lenses cultured in medium KEI-4. IGF, the most highly purified of the insulin-like growth factors was a potent mitogen for mammalian lens epithelia cells. IGF and EGF triggered cell proliferation throughout the normally amitotic central and pre-equatorial region of the epithelium. The mitotic response elicited by IGF and EGF was dose dependent, was preceded by DNA synthesis, exceeded that engendered by equimolar insulin, and exhibited a chronology identical to that brought about by crystalline insulin. Lenses cultured in KEI-4 alone or in KEI-4 supplemented with growth hormone, proinsulin, the A and/or B chain of insulin, or MSA, another of the insulin-like growth factors belonging to the somatomedin family, did not show a mitotic response. Simultaneous exposure of the lens to IGF and EGF resulted in an increase in the total number of mitotic figures over that obtained with equimolar concentrations of IGF and EGF. Our results suggest that IGF or other insulin-like growth factors may be capable of regulating cell division in the mammalian lens in vivo. That the lens epithelium responded to IGF and EGF may indicate that lens epithelial cells are subject to multiple hormonal interaction. Since growth factors appear to be cell type specific, information obtained from the rabbit lens epithelium should be useful in delineating the factors and conditions required for the growth of cultured human lens cells.

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.

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.

Hepatocyte growth factor function and c-Met expression in human lens epithelial cells.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor c-met perform a multitude of functions. However, despite the significant degree of study of HGF and c-met in numerous tissues and cell types, relatively few investigations have been performed on the lens. In the current study, therefore, the role of HGF and the receptor c-met in human lens epithelial cells was investigated. METHODS: Anterior epithelium and capsular bags were prepared from human donor eyes and maintained in Eagle's minimum essential medium (EMEM) in a 5% CO(2) atmosphere at 35 degrees C. In addition, the human lens cell line FHL124, was routinely cultured and seeded onto glass coverslips (c-met immunodetection), 12-well plates (DNA and protein synthesis), and tissue culture dishes (migration). c-Met was detected by immunocytochemistry and fluorescence-activated cell scanning (FACS). HGF was measured using enzyme-linked immunosorbent assay (ELISA) techniques. Proliferation and protein synthesis were determined by [(3)H]thymidine and (35)S-methionine incorporation into DNA and proteins, respectively. Migration was assessed using a scratch-wound assay and time-lapse video microscopy. RESULTS: HGF was detected at all stages of culture of capsular bags in protein-free medium. Moreover, c-met was present on the native epithelium and after mechanical trauma was seen to be upregulated. Immunolocalization and FACS analysis demonstrated c-met expression on FHL124 cells throughout the whole population. Furthermore, FACS analysis showed that serum-maintained cells sustained a higher level of receptor expression relative to serum-deprived cells. Additionally, HGF was found to stimulate proliferation, protein synthesis, and migratory responses. CONCLUSIONS: c-Met receptors are expressed in native epithelium, capsular bag cultures, and FHL124 cells. Receptor is distributed across the entire cell population; however, this expression is environmentally and mechanically sensitive. HGF is also present in capsular bags at all stages of culture. In addition, HGF can stimulate migration, proliferation, and protein synthesis. It therefore appears that a multifunctional autocrine loop involving HGF and c-met is in place and could be important in the development of posterior capsule opacification.

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.

Action spectrum for cytotoxicity in the UVA- and UVB-wavelength region in cultured lens epithelial cells.

PURPOSE: This study was done to quantitate the biologic effects of different wavelengths of radiation in the UVA- and UVB-wavelength region on cultured rabbit lens epithelial cells. METHODS: An action spectrum for UV-induced cytotoxicity as measured by colony-forming ability was determined using six different monochromatic wavelengths from 297 to 405 nm in rabbit lens epithelial cell line N/N1003A. Cell survival was determined by clonogenic assay. Fluence rates were monitored with a calibrated radiometer. RESULTS: Survival curves show that cell killing was most efficient at 297 nm. After quantum correction, the efficiency of 297-nm radiation in cell killing was 7 times greater than was 302-nm radiation. Radiation at 297 nm was more than 170, 340, 560, and 2000 times as effective in cell killing as 313-, 325-, 334-, and 365-nm radiation, respectively. The action spectrum had a shape similar to the DNA absorption spectrum in the UVB region, suggesting that DNA may be one of the critical targets for damage to the cells. At wavelengths longer than 313 nm, the shape of the action spectrum deviated from the DNA absorption spectrum. CONCLUSIONS: Cytotoxicity of UV radiation in cultured lens epithelial cells varies greatly with wavelength within the UVA and UVB regions. Different mechanisms may predominate in the two wavelength regions. Cultured cells may provide a suitable system for investigating the mechanisms by which UV radiation damages lens epithelial cells and leads to cataract formation.

Modulation of lens epithelial cell proliferation by enhanced prostaglandin synthesis after UVB exposure.

PURPOSE: The goals of this investigation were to examine the synthesis of prostaglandins after UVB exposure of lens epithelial cells and to investigate their role in cell proliferation. METHODS: Cultured rabbit lens epithelial cells (cell line N/N1003A) were exposed to low levels of UV radiation. Prostaglandins were assayed by radioimmunoassay; products of arachidonic acid metabolism were analyzed by thin-layer chromatography and mass spectroscopy. Cell proliferation was measured by [3H]thymidine incorporation and proliferative autoradiography. RESULTS: Cultured lens epithelial cells exposed to UVB radiation showed a dose-dependent increase in basal prostaglandin synthesis measured 24 hours after UV exposure. At an optimal dose (250 J/m2) of UVB, prostaglandin E2 (PGE2) synthesis was enhanced tenfold. Product identity was confirmed using thin-layer chromatography and mass spectroscopy with authentic standards. Incubation of irradiated cells with exogenous arachidonic acid followed by extraction and thin-layer chromatography revealed that the cultures produced PGE2, prostaglandin I2 (measured as 6-keto-prostaglandin F1 alpha), prostaglandin F2 alpha, and hydroxyeicosatetraenoic acid. The synthesis of all of these products was enhanced threefold in cells exposed to 250-J/m2 UVB. Indomethacin pretreatment eliminated the synthesis of prostaglandins, further confirming their identity. To discover the relationship between PGE2 synthesis and irradiation-induced cell proliferation, [3H]thymidine incorporation into DNA was determined 24 or 48 hours after exposure. These experiments revealed a fivefold increase in incorporation induced by UVB exposure. UVB-enhanced incorporation of thymidine was eliminated by pretreatment of cultures with indomethacin to eliminate PG synthesis. However, when 100 nM PGE2 was added to the indomethacin-treated irradiated cultures, incorporation of the label was restored toward the level detected in the UVB-stimulated cells. Addition of other prostaglandins to the cultures was ineffective. CONCLUSIONS: The results indicate that the synthesis of PGE2 is enhanced by exposure of lens epithelial cells to UVB radiation. PGE2 seems to play a specific role in cell proliferation after UV exposure. This increase in PGE2 synthesis may be important in posterior subcapsular cataract formation in humans and in animals exposed to UVB radiation in vivo.

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.

Enhanced prostaglandin synthesis after ultraviolet-B exposure modulates DNA synthesis of lens epithelial cells and lowers intraocular pressure in vivo.

PURPOSE: To study the functional significance of prostaglandin synthesis after ultraviolet-B (UVB) exposure of cultured human lens epithelial cells and rabbit eyes in vivo. METHODS: Prostaglandin E2 (PGE2) was assayed using a radioimmunoassay (RIA) and mass spectroscopy. An immortalized human lens epithelial cell line (HLE-B3) was exposed to UV irradiation, and the synthesis of PGE2 was compared with the rabbit lens epithelial cell line N/N1003A. Intact human lenses were exposed to UVB in organ culture. [3H]Thymidine incorporation was measured in cultured lens epithelial cells by incubation with the radiolabel. The effects of isobutyl methyl xanthine (IBMX), an inhibitor of phosphodiesterase and of dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, on PGE2 synthesis and DNA synthesis, were determined. Rabbit eyes were exposed to UVB radiation in vivo. Intraocular pressure was measured at specific times after exposure. Aqueous humor was remove from rabbit eyes, and its PGE2 content was measured by RIA. RESULTS: Cultured human lens epithelial cells (HLE), like rabbit lens epithelial cells (RLE), showed a dose-dependent increase in basal PGE2 synthesis 24 hours after UVB exposure. However, the amount of PGE2 synthesis was 2000-fold higher in the rabbit cells. Ultraviolet-B radiation enhanced the incorporation of [3H]thymidine in lens epithelial cells. Pretreatment of cells with indomethacin reduce PGE2 synthesis and [3H]thymidine incorporation. The human and rabbit cells responded in a similar manner to changes in DNA synthesis after UVB exposure. The addition of IBMX or dbcAMP to indomethacin-treated, UVB-exposed cells restored DNA synthesis toward the levels observed in the UVB-exposed cells. An increase in the concentration of cAMP was observed in lens epithelial cells exposed to exogenous PGE2. PGE2 synthesis in intact human lenses also increased twofold 24 hours after UVB exposure. Exposure of the rabbit eye in vivo to an optimal dose of UVB produced an increase in the PGE2 levels of the lens and the aqueous humor. Measurements of the intraocular pressure (IOP) of the animals showed a decrease in IOP by 2.21 +/- 0.66 and 6.45 +/- 0.79 mm Hg (mean +/- SEM, P = 0.004, t-test) at 6 and 24 hours after UVB exposure, respectively. The decrease in IOP was prevented by pretreatment with indomethacin. Exposure of the rabbit lens to UVB radiation in vivo enhanced [3H]thymidine incorporation twofold into the lens. Pretreatment of rabbits with indomethacin before exposure reduced this response. CONCLUSIONS: Results indicate that UVB exposure enhances PGE2 synthesis in HLE cultures as well as in rabbit lenses irradiated in vivo. This increased PGE2 synthesis is related to the increase in DNA synthesis observed after UVB treatment. The modulation of DNA synthesis in cultured lens epithelial cells after UVB exposure may be mediated by a cAMP-dependent mechanism.

Expression of Crystallins, Pax6, Filensin, CP49, MIP, and MP20 in lens-derived cell lines.

PURPOSE: Cell lines are the systems of choice to analyze cellular functions related to the particular organ system. For lens research, three cell lines are widely used: N/N1003A (derived from rabbit lenses), alpha TN4, and NKR-11 (both of murine origin). The aim of the current study was to characterize these particular cell lines with respect to their expression of genes that are considered to be lens specific or expressed preferentially in the lens, such as crystallins, Pax6, Filensin, CP49, MIP, and MP20. METHODS: alpha A- and alpha B-crystallin cDNA from rabbit lenses were sequenced. The expression of various genes was analyzed by reverse transcription-polymerase chain reaction using specific primers and mRNA from three lens-derived cell lines. For control, the expression of the selected genes was compared in nonlenticular tissues of mouse as well as in non-lens-derived murine cell lines (EF43, NIH-3T3, and L929). RESULTS: None of the transcripts for beta B2-crystallin, gamma-crystallins, MIP, MP20, filensin, and CP49 could be detected in the lens-derived cell lines. Transcripts for alpha A-crystallin were amplified in alpha TN4, but not in N/N1003A and NKR-11 cells. Pax6, a master control gene of eye development, is expressed in all three lens-derived cell lines and, additionally, in cell lines of neuronal origin, but not in corneal endothelial cells and in the currently used control cell lines. CONCLUSIONS: Three cell lines of lenticular origin were tested for expression of genes that were found abundantly in the lens. The observed expression of Pax6 in all lens-derived cell lines allows their use in the analysis of corresponding signal chains.

Effect of ultraviolet-B radiation on protein synthesis in cultured lens epithelial cells.

Exposure of cultured rabbit lens epithelial cells to repetitive doses of UV-B radiation delays their growth and alters the synthesis of specific proteins. Irradiated cells on the shoulder of the survival curve exhibited a dose-dependent decrease in growth when subcultured in serum-supplemented medium. UV-B irradiation did not affect the subsequent attachment efficiency of the cells. Control and UV-B irradiated cells were incubated with [35S]methionine and the pattern of protein synthesis in the cells was analyzed by SDS-PAGE and autoradiography. Analysis of the labeled proteins from cells exposed to UV-B radiation showed the induction of a 32 kD polypeptide and the loss of a 26 kD polypeptide compared with controls. Analysis of the proteins released by the UV-B irradiated cells into the culture medium revealed the 50% loss of a 37 kD radiolabeled protein compared with controls. The alteration of protein synthesis in lens epithelial cells by UV-B radiation may contribute to cataract formation.

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.

Incorporation of 14C acetate into the phospholipids and fatty acids of rabbit lens in organ culture.

Lenses were cultured in Medium 199 containing 14C acetate for 3, 6, 12 and 20 hours. There was a linear increase in incorporation into the total lipids with time. The total lipid extract was subjected to two-dimensional chromatography and incorporation into the individual classes of lipids was measured. For all time periods approximately 70% of all incorporated radioactivity was seen in the sterol and phosphatidyl choline (PC) fractions. Incorporation was observed in phosphatidyl ethanolamine (PE), aldehydes of PC and PE plasmalogens, phosphatidyl inositol (PI), PC plasmalogens, PE plasmalogens, sphingomyelin (SM), phosphatidic acid (PA) and phosphatidyl serine (PS). The fatty acids of the total lipids were methylated and subjected to gas chromatography, and individual fatty acids were collected. The greatest incorporation occurred in palmitic acid (16:0) and myristic acid (14:0) with minor amounts in lauric acid (12:0). These are the major products of de novo synthesis. The remainder of the radioactivity appeared in fatty acids 18 carbons or longer, which are the products of an elongation mechanism.

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.

Electron microscopy of cultured mammalian lenses. II. Changes preceding and accompanying insulin-induced mitosis.

Morphological changes preceding insulin-induced mitosis in cultured rabbit lenses were characterized. The fine structure of these lenses was compared with that of lenses exposed to culture conditions in which a mitotic response failed to materialize. Lenses were cultured in: (1) medium KEI-4, i.e., a completely defined medium, which retains the central epithelium in a nonproliferating state, (2) KEI-4 plus insulin, and (3) KEI-4 plus insulin, theophylline, and dibutyryl adenosine 3':5'-cyclic monophosphate (I-DBcAMP-T). The insulin-induced mitotic response was inhibited by dibutyryl cAMP and theophylline or papaverine or 8-bromoadenosine cAMP. At one hour, lenses cultured in KEI-4 alone or in KEI-4-I-DBcAMP-T showed a moderate increase in free ribosomes relative to zero hour controls. In contrast, lenses exposed to KEI-4-insulin exhibited a pronounced increase in the number of free ribosomes and had prominent nucleoli. An increase in the degree of folding of the cell membrane was detected in all lenses at three hours but was decidedly more prominent in lenses cultured in KEI-4-insulin. At seven hours, lenses exposed to KEI-4-insulin showed an increase in the size and apparent number of mitochondria. At all intervals, ribosomes were more numerous in lenses cultured in KEI-4-insulin. Lenses cultured in medium KEI-4 alone for 24 and 52 hours displayed a morphology ostensibly similar to that of zero hour controls. At 52 hours the epithelial cells of lenses exposed to insulin, DBcAMP and theophylline were multilayered and markedly elongated. The increase in ribosomes (one hour), changes in the cell membrane (three hours), and the heightened mitochondria profile all precede the initiation of DNA synthesis and mitosis which commenced at 24 and 40 hours, respectively.

TGFbeta2 influences alpha5-beta1 integrin distribution in human lens cells.

TGFbeta plays a central role in posterior capsule opacification, in which cell proliferation and matrix deposition, accompanied by capsular wrinkling, are largely responsible for the increased light scatter involved. Human FHL124 cells were plated onto uncoated glass coverslips to form circular patches so that the central cells reached confluency while the peripheral cells grew outwards. Cell patches were exposed to serum free (SF) EMEM (control) or TGFbeta supplemented (10 ng ml(-1)) EMEM. Fibronectin (Fn), alpha5beta1 integrin and F-actin were localized by immunofluorescence techniques and analysed by confocal microscopy. In the confluent, central cells in SF medium alpha5beta1 showed a punctate distribution while Fn was present in strongly staining fibres. TGFbeta had no effect on integrin or Fn distribution in confluent cells. In the peripheral, motile cells of the patches in SF conditions alpha5beta1 was localized in well-defined focal adhesion plaques at the ends of actin stress fibres, while Fn was distributed in a punctate perinuclear pattern. TGFbeta had a profound dispersing effect on the integrin causing a widespread distribution of alpha5beta1 in the membrane with no apparent association with the actin filaments. The cells had a more fibroblastic morphology with increased deposition of Fn near the nucleus. All the TGFbeta-induced changes were inhibited by the TGFbeta antibody CAT152 (Cambridge Antibody Technology). Culture with a function-blocking alpha5 antibody or Fn antibody resulted in detachment of the peripheral cells from the patches, but the central cells remained intact. The patch culture method therefore provides a convenient means of investigating the differences between confluent and growing lens cells both in terms of the patterns of alpha5beta1 integrin and Fn and also in the response of the molecular arrangements of both to TGFbeta2.