Retinoids enhance the number of EGF receptors in corneal endothelial cells

Corneal endothelium appears to be an important target tissue for retinoids and epidermal growth factor (EGF). We report here that retinoic acid, or its synthetic analogue CBS-211 A (10(-8)-10(-7) M) dose-dependently enhances the mitogenic effect of EGF on cultured bovine corneal endothelial cells (BCEC). Furthermore, retinoid treatment, especially with CBS-211 A, increases the EGF-binding capacity of BCEC without any modification of EGF receptor affinity. The addition of cycloheximide (0.5 microgram ml-1) to cultures simultaneously with retinoids suppressed the retinoid effect on EGF-binding, whereas this protein synthesis inhibitor was ineffective when added 24 hr later. These results suggest that retinoids could induce the expression of EGF receptors on BCEC as an early event. This study, which demonstrates a cooperation between retinoids and EGF in corneal endothelium, could explain the beneficial effect of retinoic acid previously reported on this tissue repair in vivo. Our data could offer clues for new pharmaceutical strategies in ophthalmology therapy.     

Growth factors and corneal endothelial cells: I. Stimulation of bovine corneal endothelial cell DNA synthesis by defined growth factors.

Peptide growth factors and other physiological growth modifiers were evaluated for their ability to stimulate DNA synthesis in early passage cultures of bovine corneal endothelial cells (BCEC). Increasing concentrations of newborn bovine serum (0.5-10%) causes a progressive increase in DNA synthesis, which approached a plateau at 10% serum. Supplementing medium with 10% serum from different lots of newborn bovine serum or fetal bovine serum stimulated significantly different levels of DNA synthesis by BCEC. Addition of epidermal growth factor (EGF) (2 nM) to medium containing 10% newborn or fetal bovine serum further increased DNA synthesis. Dose-response curves for EGF, transforming growth factor-alpha, basic fibroblast growth factor (bFGF), and insulin-like growth factor I showed that each significantly stimulated high levels of DNA synthesis (200-700% increase) compared with BCEC cultured in serum-free medium. Vaccinia growth factor, insulin, and transforming growth factor-beta each significantly stimulated lower levels of DNA synthesis (30-200% increase), whereas nerve growth factor, multiplication stimulating activity, and platelet-derived growth factor all failed to significantly stimulate DNA synthesis above the level of serum-free medium. Other physiological growth modifiers were tested for their effects on DNA synthesis of BCEC. Transferrin and low levels of 3',5'-cyclic monophosphate (cAMP) stimulated very low levels of DNA synthesis (50% increase) whereas linoleic acid, high levels of selenium, or cAMP each inhibited DNA synthesis 25-75% below the level of BCEC cultured in serum-free medium. A series of eight formulations containing various combinations of EGF, FGF, insulin, transferrin, selenium, linoleic acid, retinoic acid, cAMP, heparin, and endothelial cell growth factor were tested for their mitogenic action on BCEC cultures. A formulation containing EGF, insulin, transferrin, selenium, and linoleic acid (EGF + ITSL) stimulated the highest level of DNA synthesis of BCEC, which was approximately 25% higher than the increase stimulated by addition of 10% newborn bovine serum. The formulation consisting of EGF + ITSL was also evaluated as a supplement to corneal storage media. Addition of EGF + ITSL to three corneal storage media (McCarey-Kaufman, K-Sol, CSM) significantly stimulated increases in cell numbers of approximately 50% above the unsupplemented corneal storage media. These results demonstrate that BCEC respond selectively to different defined peptide growth factors and physiological growth modifiers, and suggest that supplementation of corneal storage media with a defined formulation (EGF + ITSL) may enhance corneal endothelial cell density.     

Retinal pigment epithelium rescues vascular endothelium from retinoic acid-induced apoptosis

PURPOSE: To determine whether retinoids are capable of inducing vascular endothelial cell apoptosis and whether the presence of an intact RPE monolayer can block retinoid-induced vascular endothelial cell death. METHODS: Confluent fetal bovine aortic endothelial (FBAE) cells were incubated with various concentrations of all-trans or 9-cis retinoic acid (an analogue of 11-cis retinoic acid). Apoptosis rates were determined at 24 hours, and the effect of inhibition of protein synthesis and activation of protein kinase C on apoptosis was investigated by supplying culture medium with 0.1 mg/mL cycloheximide and 10 nM phorbol myristate acetate. To investigate the impact of RPE on retinoid-induced apoptosis, confluent FBAE cells were cultured with a confluent layer of RPE in inserts where retinoids were added to the upper compartment. A confluent bovine corneal endothelium monolayer was used as the control. The permeabilities of the RPE and bovine corneal endothelium monolayers to fluorescein (20 microg/mL) and 9-cis retinoic acid (3 x 10(-4) M) were also determined. RESULTS: 9-cis Retinoic acid induced higher rates of apoptosis in FBAE cells than did all-trans retinoic acid and the control (P = 0.004). This effect was dose-dependent, with an ED(50) of 1.4 microM (r = 0.99, P = 0.004). Cycloheximide did not inhibit 9-cis retinoic acid-induced apoptosis, but phorbol myristate acetate significantly decreased the apoptosis rate (P = 0.005). The presence of a confluent RPE monolayer reduced the 9-cis retinoic acid-induced apoptosis rate (P = 0.002), but the presence of a bovine corneal endothelial monolayer did not (P > 0.05). Both cell types established a similar diffusion barrier against fluorescein and 9-cis retinoic acid. CONCLUSIONS: 9-cis Retinoic acid is an important mediator of vascular endothelial apoptosis. A confluent monolayer of RPE can prevent endothelial cell apoptosis, and this effect is not due simply to establishment of a diffusion barrier by the RPE.     

Growth factors and corneal endothelial cells: II. Characterization of epidermal growth factor receptor from bovine corneal endothelial cells.

Epidermal growth factor (EGF) is a potent mitogen for corneal endothelial cells and may play a role in endothelial wound healing. To further characterize the interaction of EGF with endothelial cells, we measured biochemical parameters of 125I-EGF binding to cultured bovine corneal endothelial cells (BCEC), determined the pattern of EGF-induced protein phosphorylation, and investigated the influence of retinoic acid (RA) and transforming growth factor beta (TGF-beta) on EGF-induced DNA synthesis and receptor levels. Binding of 125I-EGF to BCEC was dependent on time, reaching a plateau after approximately 2 h at 37 degrees C, was specific for EGF, and had high affinity (Kd = 100 pM) with approximately 21,000 receptors per cell. Cellular substrates for the EGF receptor kinase, which may function as initial second messengers for EGF, were detected by autoradiography of sodium dodecyl sulfate polyacrylamide gels of 32P-labeled BCEC proteins. EGF stimulated phosphorylation of 170, 37, 21 and 20-kDa proteins. Addition of 1 nM, 100 nM, and 10 microM RA to BCEC cultured in serum-free medium for 24 h progressively inhibited DNA synthesis by up to 80% compared with control cultures. However, when added in combination with 5 nM EGF, 1 nM and 100 nM RA synergistically stimulated DNA synthesis by up to 80% above the level of EGF stimulation without altering EGF receptor levels or binding affinity. Thus, short-term exposure of BCEC to RA potentiated EGF-stimulated DNA synthesis, most likely by acting at a postreceptor step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of conjunctival transdifferentiation by topical retinoic acid

After resurfacing a total corneal epithelial defect extending 2-3 mm beyond the limbus, conjunctival epithelium gradually loses goblet cells and transforms into a corneal-like epithelium. We examined the effect of topical retinoic acid on the reversal of transdifferentiation on nonvascularized corneas. Four months after total denudation of corneal epithelium using n-heptanol, rabbit corneas without vascularization received topical drops of 0.1% (wt/vol) all-trans retinoic acid in corn oil 3 times a day. Before treatment, the transdifferentiation was complete, as evidenced by the absence of goblet cells on the corneal surface using a topographical assay and routine histology. After treatment for 15 days, goblet cells reappeared 3 mm into the peripheral cornea, and extended in a centripetal density to 4.5 mm after 32 days. To prove that retinoic acid was not angiogenic, retinoid-bearing Elvax-40 pellets were implanted into normal corneal stroma. Taken together, these data indicate that vitamin A or retinoids may be an important factor in the modulation of conjunctival epithelial transdifferentiation.     

碱性成纤维细胞生长因子和表皮生长因子对角膜中期保存液保存人角膜内皮细胞的影响

目的探讨碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)对中期保存角膜内皮细胞活性的影响。方法角膜中期保存液保存人角膜环,对照组为角膜中期保存液,实验组分为A、B、C、D组,分别为角膜中期保存液中加入bFGF(5ng/ml)、bFGF(20ng/ml)、EGF及bFGF+EGF,在保存角膜第3、7、14天后分别取保存角膜环复温观察,锥虫蓝茜素红联合染色检测角膜内皮活细胞率,电镜检测细胞超微结构的改变。结果保存角膜环第14天角膜内皮活细胞率,对照组为(10.35±1.32)%,A组为(62.18±1.56)%,B组为(92.57±0.90)%,C组为(71.01±2.67)%,D组(82.59±1.45)%,与对照组比较,各保存时间各实验组活细胞率均高,差异有统计学意义(P<0.01)。保存第14天,对照组保存的角膜环明显水肿、混浊不透明、内皮细胞形态结构不完整,超微结构显示角膜内皮细胞Y型连接断裂;而实验B、D组,保存的角膜环轻度水肿、后弹力层皱褶少、角膜透明度高、内皮细胞形态结构完整,超微结构显示保存角膜内皮细胞连接紧密,Y型连接无明显断裂,细胞表面可见较丰富的微绒毛,胞体大,核突起明显。结论bFGF和EGF在角膜中期保存液中均有促进细胞增殖、保持角膜细胞活性的作用,以bFGF作用更明显。     

Distribution of epidermal growth factor (EGF) receptors in rabbit corneal epithelial cells, keratocytes and endothelial cells, and the changes induced by transforming growth factor-beta 1.

Three kinds of rabbit corneal cells (epithelial cells, keratocytes and endothelial cells) were cultured, and the growth promoting effects of epidermal growth factor (EGF) were examined in these cells. It was found that the sensitivity of the epithelial cells to EGF was the highest, that of the endothelial cells was a little lower, and that of the keratocytes was the lowest. Transforming growth factor-beta 1 (TGF-beta 1) did not influence growth of the three kinds of corneal cells. It was found that TGF-beta 1 enhanced the growth promoting effect of EGF in the keratocytes, but not in the epithelial and endothelial cells. EGF receptors in the corneal cells were labelled with [125I]EGF and analysed by Scatchard plot. In the epithelial and endothelial cells, both high and low affinity receptors were found, while the keratocytes had only low affinity receptor. In the epithelial cells, the number and the association constant of the high affinity receptors were, respectively, 2.79 x 10(4) per cell and 0.034 nM, and those of the low affinity receptors were, respectively, 13.4 x 10(4) per cell and 0.700 nM. In the endothelial cells, the number and the association constant of the high affinity receptors were 1.27 x 10(4) per cell and 0.086 nM, respectively, and those of the low affinity receptors were 8.91 x 10(4) per cell and 1.536 nM. In the keratocytes, the number of receptors and the association constant were 9.49 x 10(4) per cell and 1.535 nM, respectively. The corneal cells were treated with TGF-beta 1 for 24 hr and its influence on EGF receptors was examined. The results showed that TGF-beta 1 induced the high affinity receptors in the keratocytes, although TGF-beta 1 did not influence EGF receptors in the epithelial and endothelial cells. The number of high affinity receptors in keratocytes treated with TGF-beta 1 was 1.02 x 10(4) per cell and the association constant was 0.171 nM (this was approximately tenfold higher than that of the receptor of keratocytes not treated with TGF-beta 1).     

Effects of epidermal growth factor, fibroblast growth factor, and transforming growth factor-beta on corneal cell chemotaxis.

The effects of recombinant basic fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta) on migration of human and bovine corneal cells were determined using checkerboard analysis in Boyden chambers. EGF, FGF, and TGF-beta each stimulated high levels of chemotactic migration. Each growth factor, however, induced a different dose-response pattern. Migration stimulated by FGF reached a plateau at a concentration between 100 and 200 ng/ml for endothelial, epithelial, and stromal fibroblasts. By contrast, chemotactic responses to EGF peaked between 10 and 50 ng/ml, then decreased at higher concentrations. TGF-beta also stimulated a peak in migration in all three corneal cells, but the peak of migration occurred at an approximately 1000-fold lower concentration (1 pg/ml) than for EGF. Checkerboard analysis demonstrated that FGF and EGF, but not TGF-beta, stimulated chemokinesis of bovine, stromal, and endothelial cells. These results demonstrate that FGF, EGF, and TGF-beta induce migration in pure populations of bovine and human corneal cells and support the concept that these growth factors may play key roles in corneal wound healing by regulating migration of corneal cells.     

Down-regulation of androgen receptor expression and inhibition of lacrimal gland cell proliferation by retinoic acid

Androgens and retinoids are known to be involved in control of lacrimal gland function. Because retinoids generally antagonize androgen function it was the purpose of this study to investigate interactions of retinoic acid and androgens in rabbit lacrimal acinar cells in culture by determining effects of retinoic acid on androgen receptor (AR) mRNA expression, AR protein levels and androgen-stimulated cell proliferation. Experiments were conducted using primary rabbit lacrimal acinar cells and a transformed rabbit lacrimal acinar cell line. Exposure of primary lacrimal acinar cells in culture to 10(-10)-10(-6)M all-trans retinoic acid for 4-24hr causes an approximately 50% decrease in AR mRNA expression. Expression of AR protein in primary and transformed rabbit lacrimal acinar cells was confirmed by immunohistochemistry. Exposure of the primary cells to 10(-6)M retinoic acid for 24hr caused a 40% decrease in AR protein levels as determined by measurement of binding of(3) [H]-dihydrotestosterone (DHT) to cells in culture and Scatchard analysis. Exposure to 10(-9)-10(-6)M DHT stimulates proliferation of transformed rabbit lacrimal acinar cells. This effect is receptor mediated since it is blocked by the AR antagonist, flutamide. Proliferation of the lacrimal acinar cells is inhibited by retinoic acid, as compared to control, and retinoic acid also completely inhibits androgen stimulation of cell proliferation. This study supports the hypothesis that androgens play a supportive role in lacrimal gland function. The antagonistic influences of androgens and retinoic acid suggests that, under physiologic conditions there is a balance between the effects of androgens and retinoids in the lacrimal gland. A decrease in androgen levels in a dry eye patient may alter the balance between the effects of these important controllers of gene expression. The antagonistic effect of retinoids on androgens in the lacrimal gland must also be considered when devising pharmaceutical treatments for dye eye.     

Factors modulating the effect of retinoids on cultured retinal pigment epithelial cell proliferation.

The effect of several naturally-occurring retinoids and 13-cis-retinoic acid on the proliferation of cultured bovine retinal pigment epithelial (RPE) cells was investigated. None of the retinoids tested were toxic to the cultures and all, except retinylpalmitate, inhibited cell proliferation when given for more than 3 days. The relative potencies of the retinoids were; all-trans-retinoic acid greater than 13-cis-retinoic acid greater than all-trans-retinol approximately equal to all-trans-retinaldehyde. Uptake of retinoic acid by cultured RPE cells was 10-fold less than the uptake of retinol. Although retinoic acid-treated cultures showed strong density-dependent growth inhibition, cellular proliferation was inhibited more in sparse cultures than in dense ones. Retinoic acid did not significantly inhibit the proliferation of first passage bovine or rabbit RPE cells, but partially inhibited the proliferation of first passage human RPE cells. The sensitivity of all these cultures to growth inhibition by retinoic acid increased in subsequent subcultures, yet there was no effect of passage number on retinoic acid uptake. This study demonstrates that RPE cell proliferation can be inhibited by retinoic acid but the sensitivity of these cells to the retinoid's effects are modulated by incubation time, in vitro aging, and cell density.     

EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells

Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.     

bFGF、EGF和NGF对人角膜内皮细胞生长调控的实验研究

目的：探讨碱性成纤维细胞生长因子（Basicfibroblastgrowthfacfor，bFGF）、表皮细胞生长因子（Epidermalgrowthfactor，EGF）和神经细胞生长因子（Nervegrowthfactor，NGF）对体外培养的人角膜内皮细胞的生长调控作用。方法：将相同数量的人角膜内皮细胞接种于96孔板。加入浓度分别为0ng／ml、1ng／ml、3ng／ml、10ng／ml、30ng／ml、100ng／ml的EGF、bFGF和NGF进行培养。5天后MTT法用检测增殖情况。结果：在0ng／ml、1ng／ml、3ng／ml、10ng／ml、30ng／ml、100ng／ml浓度下bFGF组的平均OD值分别为：0．224±0．045、0．239±0．040、0．262±0．0342、0．278±0．0319、0．281±0．0324、0．260±0．0310。EGF组的平均OD值分别为：0．228±0．0304、0．245±0．0418、0．267±0．0454、0．275±0．0347、0．271±0．0449、0．250±0．0253。NGF组的平均OD值分别为：0．216±0．0187、0．228±0．0226、0．231±O．0225、0．242±0．0279、0．245±0．0294、0．247±0．0349。结论：bFGF在30ng／ml范围内对内皮细胞生长有促进作用，并具有剂量依赖性。高于100ng／ml时促生长作用降低。EGF在10ng／ml范围内对内皮细胞生长有促进作用，并具有剂量依赖性。高于30ng／ml时促生长作用降低。NGF本次实验剂量范用内对角膜内皮细胞生长无明显作用。     

Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells.

The authors tried to determine whether human corneal endothelial cells in primary culture synthesize messenger RNA (mRNA) coding for epidermal growth factor (EGF), EGF receptor, basic fibroblast growth factor (FGFb), transforming growth factor beta-1 (TGFb1), and interleukin-1 alpha (IL-1 alpha). Oligodeoxythymidine-primed complementary DNA (cDNA) was generated from total cellular RNA extracted from eight independent primary corneal endothelial cell cultures. Four of these cultures, maintained 18-51 days, had obvious increases in cell numbers and mass over the 2 weeks before RNA extraction and were populated primarily with cells that were small, uniform, and mononuclear (proliferative cultures). The morphology of the cells in other four cultures, maintained 47-78 days, was predominantly large, irregular, vacuolated, and occasionally multinucleated. These cells were identical to senescent cells found in previous studies, and the cell number did not increase in these cultures over the 2 weeks preceding RNA extraction (senescent cultures). The polymerase chain reaction (PCR) was used to amplify the growth factors (EGF, FGFb, TGFb1, and IL-1 alpha), EGF receptor, and beta actin sequences from each of the cDNA samples. The EGF receptor, FGFb, and beta actin mRNAs were present in all eight cDNA samples. The EGF mRNAs were detected by PCR alone in four of the samples from proliferative cultures, TGFb1 mRNAs in three, and IL-1 alpha mRNAs in three. In the samples from senescent cultures, 0, 1, and 0 mRNAs were detected, respectively. Southern blots of the PCR products were probed with oligonucleotides complementary to sequences in each of the amplified products. This technique showed that the appropriately sized amplification products were specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

生长因子与角膜内皮细胞

角膜内皮细胞是维持角膜透明的关键细胞成分,角膜内皮细胞密度降低和形态异常可导致内皮细胞功能失代偿而降低视力。在伤口愈合过程中,生长因子可增加角膜内皮细胞密度槿刺激内皮细胞再生,促进伤口愈合。肽类生长因子影响着多种细胞生理过程,包括细胞增殖,分化,移行和存活。     

Mitotic activity of corneal endothelial cells in organ culture with recombinant human epidermal growth factor

Recombinant human epidermal growth factor (EGF) was assessed for its capacity to stimulate proliferation of human corneal endothelial cells in vitro in organ culture with 87 human corneas. The EGF in defined serum-free (S-F) media was able to stimulate endothelial cell mitosis (t test P less than 0.01) in matched transected human corneas after a four-day incubation period as judged by histologic studies. Clearly defined endothelial mitotic figures were seen in all stages of cell division throughout the endothelial cell layer. The implication of increasing corneal endothelial cell numbers in donor corneas before transplantation using a human growth factor, potentially available in pure form in unlimited quantities, is discussed.     

The effect of retinoic acid on thymidine incorporation and morphology of corneal stromal fibroblasts

The effect of retinoic acid on DNA synthesis and cell morphology was studied using corneal stromal fibroblasts in culture. All-trans retinoic acid induces an increase in DNA synthesis after 24 hours of exposure. Autoradiographic studies of 3H-thymidine incorporation into corneal stromal cells exposed to 10(-6) M retinoic acid for 24 hours showed an increase in labeling which ranged from 19.2% to 67.6% over control cultures. Scintillation analysis of labeled cultures also showed an increase in incorporation of 3H-thymidine into cells treated with 10(-6) M retinoic acid, with increases ranging from 21.8% to 114.7% above control cultures. Exposure of cultured corneal stromal cells to 10(-6) M retinoic acid resulted in a dramatic change in cell morphology such that they changed from spindle-shaped to round, flattened cells which were epithelioid in appearance. These data demonstrate that biological activity of retinoic acid in stromal fibroblasts and imply a role for vitamin A in maintenance of stroma structure and function.     

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.     

Factors affecting bovine corneal endothelial cell density in vitro.

AIMS: To examine factors influencing the density and contact inhibition of bovine corneal endothelial cells cultured in vitro. METHODS: Cell counts were performed on bovine corneal endothelial cells cultured for various times in the presence of 10% fetal calf serum, with or without varying concentrations of growth factors, 5% dextran T-500, or 2% chondroitin sulphate, at 32 degrees C or 37 degrees C, and after treatment with beta galactosidase. RESULTS: Both basic fibroblast growth factor (FGFb) and retinal crude extract (RCE), but neither epidermal growth factor (EGF) nor acidic fibroblast growth factor (FGFa), increased endothelial cell density in vitro (p < 0.05). Continuous exposure to RCE resulted in a higher cell density than did a 24 hour pulse (p < 0.01), and higher cell densities were achieved at 37 degrees C than at 32 degrees C (p < 0.0001). In the absence of RCE, dextran T-500 increased cell density modestly (p < 0.05); in the presence of RCE, the addition of dextran T-500 had no effect on final cell density, whereas chondroitin sulphate significantly decreased final cell density (p < 0.01). In the absence of exogenous growth factors, beta galactosidase treatment resulted in a 50% increase in final cell density compared with controls (p < 0.0001). CONCLUSIONS: Bovine corneal endothelial cell growth can be augmented under conditions different from those used in corneal preservation systems. The final cell density in a confluent monolayer can be increased by treatment with beta galactosidase, suggesting that corneal endothelial cells may be contact inhibited through a beta galactosidase sensitive receptor system.