Effect of cytokines on regulation of the production of transforming growth factor beta-1 in cultured human Tenon's capsule fibroblasts

PURPOSE: Transforming growth factor-beta1 (TGF-beta1) is thought to play a pivotal role in the regulation of the wound healing process after glaucoma filtering surgery. The aim of the present study was to investigate whether platelet-derived growth factor isoforms (PDGF-AA, PDGF-BB), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) modulate the production of latent and/or active TGF-beta1 by cultured human Tenon's capsule fibroblasts (HTF). METHODS: Human Tenon's capsule fibroblasts were seeded at two different densities (30 cells/mm2 and 150 cells/mm2) and stimulated for five days with PDGF-AA, PDGF-BB, bFGF, EGF, IL-1beta and TGF-beta1. Control cells were treated with serum-free medium (WM/F12). The concentrations of latent and active TGF-beta1 in the medium were determined using an immunoassay before and after activation of TGF-beta1 by transient acidification. RESULTS: The concentration of latent TGF-beta1 in conditioned media from HTF seeded at high density (150 cells/mm2) significantly increased after stimulation with 5 ng/ml TGF-beta1 (151.5 +/-41.7 pg/ml) or 10 ng/ml IL-1beta (45.7+/-8.1 pg/ml). The concentration of active TGF-beta1 in conditioned media also significantly increased after stimulation of HTF with 5 ng/ml TGF-beta1 (48.4+/-27.5 pg/ml). CONCLUSIONS: The present results indicate that TGF-beta1 is the most potent inducer of its own synthesis in HTF. Activation of an autocrine TGF-beta1 loop may play a role in the wound healing response after glaucoma filtering surgery.     

Effect of growth factors on the activation of human Tenon's capsule fibroblasts

PURPOSE: To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1beta, TGF-beta1 and TGF-beta2 on the proliferation and myofibroblast transformation of cultured human Tenon's capsule fibroblasts and to characterize expression of PDGF- and TGF-beta-receptors in these cells. METHODS: To determine cell proliferation, cell number of 2nd passage cultured human Tenon's capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate alpha-smooth-muscle actin (alpha-SMA) expression. Expression of PDGF- and TGF-beta-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. RESULTS: A significant increase in proliferation (p < or = 0.05) was detected after exogenous stimulation with PDGF-AA (10 ng/ml and 100 ng/ml), PDGF-AB (10 ng/ml and 100 ng/ml), PDGF-BB (10 ng/ml and 100 ng/ml), bFGF (100 ng/ml), IL-1beta (1 ng/ml and 10 ng/ml), TGF-beta1 (0.5 ng/ml) and TGF-beta2 (0.5 ng/ml). Both TGF-beta1 and TGF-beta2 stimulated expression of alpha-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-beta1) and 500 ng/ml (TGF-beta2). Protein and mRNA of PDGF-receptor type alpha and type beta and TGF-beta-receptors type I, II and III are expressed in cultured human Tenon's capsule fibroblasts. CONCLUSIONS: The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon's capsule fibroblasts after glaucoma filtering surgery while TGF-beta-isoforms are essential for the transformation of Tenon's capsule fibroblasts into myofibroblasts.     

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。     

Extracellular matrixes influence alpha-smooth muscle actin expression in cultured porcine lens epithelial cells.

PURPOSE. To determine whether extracellular matrix (ECM) influences the expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in cultured porcine lens epithelial cells (LECs). METHODS. Porcine LECs were cultured for 6 days in an F-12 nutrient mixture supplemented with 5% fetal bovine serum on wells that were coated with laminin, fibronectin, type I collagen, or type IV collagen. LECs cultured on uncoated wells served as a control. Alpha-SMA was detected immunocytochemically with a mouse monoclonal antibody, and the ratio of the number of alpha-SMA-positive cells to the total number of cells, the P/T ratio, was calculated. RESULTS. The P/T ratio of the LECs on the uncoated dishes was about 5%. LECs cultured on wells coated with laminin or type IV collagen significantly reduced the ratio, whereas fibronectin or type I collagen had no effect. CONCLUSIONS. The ECM influences alpha-SMA expression in cultured porcine LECs.     

Effect of growth factors on bovine retinal pigment epithelial cell migration and proliferation

BACKGROUND: To examine the effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and transforming growth factor beta 2 (TGF(beta2)) on bovine retinal pigment epithelial (RPE) cell migration and proliferation. MATERIALS AND METHODS: Cultured bovine RPE cells were treated with 10 ng/ml PDGF, bFGF, aFGF, IGF-1, EGF, or TGF(beta2). RPE cell migration studies were performed in multiwell plates confluently covered with RPE cells. After inhibition of proliferation and denudation of half of each well, cells were incubated with various growth factors. Migration was measured as the number of cells that had entered the denuded area after 20 h. RPE cell proliferation was determined by [(3)H]thymidine incorporation after growth factor stimulation for 24 h. RESULTS: RPE cell migration was significantly enhanced after incubation with PDGF (stimulation of 213% compared to the negative control, p = 0.002), bFGF (206%, p = 0.003), aFGF (175%, p = 0.003), IGF-1 (150%, p = 0.003), and EGF (144%, p = 0.003). RPE cell proliferation was stimulated by bFGF (322% compared to the negative control, p < 0.005), PDGF (119%, p < 0.005), aFGF (121%, p < 0.005), and EGF (94%, p < 0.005). IGF-1 showed no significant effect on RPE cell proliferation; TGF(beta2) displayed no effect on RPE cell migration nor on proliferation. CONCLUSIONS: The peptide growth factors PDGF, bFGF, aFGF, IGF-1, and EGF play an important role in initiating RPE cell migration. Basic FGF, PDGF, aFGF, and EGF stimulate RPE cell DNA synthesis.     

Critical role of the Rho-kinase pathway in TGF-beta2-dependent collagen gel contraction by retinal pigment epithelial cells

Retinal pigment epithelial cells (RPEs) are thought to be one of the main components of fibrous membrane observed in eyes with proliferative vitreo-retinopathy. We investigated the signalling mechanisms of TGF-beta2-dependent collagen gel contraction by RPEs.An in vitro type I collagen gel contraction assay was performed to evaluate the effect of TGF-beta2 on gel contraction. The expression of alpha-smooth muscle actin (alpha-SMA) and the phosphorylation state of myosin light chain (MLC) were analyzed by Western blotting. The involvement of protein kinases such as p44/42 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), p38 MAPK and phosphatidylinositol-3 kinase was investigated. The contribution of Rho-kinase and/or MLC-kinase was also evaluated using respective kinase inhibitors (Y27632, hydroxyfasudil and ML7). Additionally, RPEs were immunostained to examine whether the expression of alpha-SMA detected in our western blotting correlated to the stress fiber formation within the cells. TGF-beta2 caused time (0-5 days)-and dose (0 10 ng ml(-1))-dependent gel contraction associated with overexpression of alpha-SMA and phosphorylation of MLC (p < 0.01, respectively). PKC inhibitor (GF109203X, 5 microM) and p38 MAPK inhibitor (SB203580, 10 microM) significantly attenuated TGF-beta2-elicited gel contraction via partial downregulation of both alpha-SMA expression and MLC phosphorylation (p < 0.01, respectively). The gel contraction was prominently inhibited in the presence of Y27632 (10 microM) or hydroxyfasudil (10 microM) with strong suppression of MLC phosphorylation but had no significant effect on alpha-SMA expression. Treatment with ML7, in contrast, resulted in a marginal inhibition of MLC phosphorylation and gel contraction. Finally, pretreatment of the cells with Y27632 or hydroxyfasudil prevented the formation of stress fiber within the cells. These results indicate that TGF-beta2-dependent myofibroblastic transdifferentiation and MLC phosphorylation by RPEs involve both PKC and p38 MAPK pathways at least in part. Myofibroblastic transdifferentiation of RPEs appears to be independent of the Rho-kinase pathway, and the presence of alpha-SMA does not necessarily reflect the contractile potential of a cell. While Rho-kinase inhibitors are incapable of preventing myofibroblastic transdifferentiation itself, this pathway could be one of the critical targets of cell-mediated contraction of the tissue containing fibrillar collagens by transdifferentiated RPEs.     

Immunohistologic study of interleukin-1, transforming growth factor-beta, and alpha-smooth muscle actin in lens epithelial cells in diabetic eyes

PURPOSE: To assess the effects of the cytokines interleukin-1 (IL-1), transforming growth factor-beta (TGF-beta), and alpha-smooth muscle actin (alpha-SMA) in lens epithelial cells (LECs) in normal and diabetic eyes. SETTING: Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan. METHODS: Ten eyes of 10 patients with diabetic mellitus and 20 normal eyes of 20 patients with senile cataract were studied. The anterior lens capsules with LECs obtained by capsulotomy during cataract surgery were cultured. The LECs obtained immediately after surgery and on the third day of culture were immunohistologically studied to assess the activities of the cytokines. RESULTS: Interleukin-1 and TGF-beta staining showed a low level activity in some LECs in diabetic eyes but only a minimum level of activity in those in normal eyes. During culture, LECs in diabetic eyes became small and transformed into fusiform and fibroblast-like cells, and these cells were strongly stained for IL-1 and TGF-beta. Normal eyes showed little changes in cell morphology and were weakly stained for IL-1 and TGF-beta. Both with culture and with no culture, alpha-SMA showed only minimal activity in both diabetic and normal eyes, with no difference. CONCLUSION: Lens epithelial cells after cataract surgery had low IL-1 and TGF-beta activities, and these activities increased during culture. Diabetic eyes showed higher cytokine activities and more marked morphologic changes than normal eyes, suggesting that increased proliferative activity and increased cytokine activity of LECs contribute to strong anterior capsule contraction in diabetic eyes.     

Effect of the cytokines on the prostaglandin E2 synthesis by lens epithelial cells of human cataracts.

BACKGROUND--Lens epithelial cells (LECs) derived from human cataracts have been reported to produce various cytokines and prostaglandin E2 (PGE2) in culture. The effects of IL-1, TGF-beta, and b-FGF on the PGE2 synthesis by LECs have been studied. METHODS--A circular piece of the anterior capsule with attached LECs was obtained by capsulotomy during cataract surgery and cultured. The primary, almost confluent, cultures were used for the study. The PGE2 concentration of the culture media for 24 h was measured after the addition of recombinant human IL-1 alpha, TGF-beta 2, or b-FGF at various concentrations. The PGE2 concentration was also measured in the media to which each cytokine and rabbit polyclonal anti-human antibodies against the corresponding cytokine had been added. RESULTS--The PGE2 concentration of the culture media after addition of IL-1 alpha at the concentration of 100 or 500 pg/ml (1765 (768) and 3071 (1121) pg/10(4) cells) or TGF-beta 2 at the concentration of 10 or 100 ng/ml (689 (264) and 750 (189) pg/10(4) cells) was significantly increased compared with that in the controls (67 (20) pg/10(4) cells). These effects were suppressed by the corresponding anticytokine antibodies. Basic FGF and anti-human b-FGF showed no significant effect on the PGE2 concentration. IL-1 and TGF-beta increased but b-FGF did not affect the PGE2 synthesis by LECs in culture. CONCLUSION--IL-1 and TGF-beta may participate in postoperative inflammation after cataract surgery by increasing PGE2 synthesis by residual LECs.     

Suppression of human lens epithelial cell proliferation by proteasome inhibition, a potential defense against posterior capsular opacification

PURPOSE: Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the remaining lens epithelial cells (LECs) after cataract surgery. Studies have shown that proteasome inhibition interferes with EMT and remodeling of the extracellular matrix. This study was conducted to investigate suppression of LEC proliferation by proteasome inhibition and its signaling pathway. METHODS: HLE B-3 cells and human lens epithelium explants from 17- to 20-week fetal lenses were cultured and treated with TGF-beta2 (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL) and 5 or 10 muM MG132. LEC proliferation was determined using both the WST-1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by Western blot analysis. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition. RESULTS: TGF-beta2 suppressed proliferation of HLE B-3 cells, whereas FGF-2 and HGF enhanced proliferation. Proliferation suppression by TGF-beta2 was blocked by adding FGF-2 or HGF. Proteasome inhibitor (MG132) treatment strongly inhibited the proliferation of LECs, either alone or in the presence of TGF-beta2, FGF-2, or HGF. These findings were confirmed by observing PCNA expression. Similar results were obtained with primary human LECs. Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. MG132 caused a significant increase in p21 and p27 protein and decrease in CDK2, but no change in p53, p57, CDK4, or CDK6 protein. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, which reduced p21 and p27 protein expression to very low levels that remained below basal control levels, even after treatment with MG132. CONCLUSIONS: Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-beta2, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking development of PCO.     

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。     

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1

PURPOSE: To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. METHODS: Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. RESULTS: Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. CONCLUSIONS: These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.     

联合共培养系统诱导骨髓间充质干细胞分化为视网膜色素上皮样细胞

目的 探讨添加脑源性神经生长因子（BDNF）、表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）的培养基联合人类视网膜色素上皮细胞（HRPECs）共培养对骨髓间充质干细胞（BMSCs）定向诱导分化的影响.方法 实验研究.实验分三组：HRPECs＋BDNF、EGF、bFGF＋BMSCs共培养组、BDNF、EGF、bFGF＋BMSCs共培养组和对照组（单独BMSCs）.第一组取第3代HRPECs接种在双层培养板的上层,将第3代人BMSCs接种于下层培养板中,在双层六孔板的每孔中加入混合有20 ng/mlbFGF、20 ng/ml EGF、20 ng/ml BDNF及10%胎牛血清（FBS）的DMEM-LG培养液（需做免疫细胞化学染色应同时在下层放入18 mm×18 mm盖玻片进行细胞爬片）.第二组取第3代人BMSCs接种在六孔培养板中,每孔中加入含20 ng/ml bFGF、20 ng/ml EGF、20 ng/ml BDNF及10%FBS的DMEM-LG培养液.第三组将第3代人BMSCs接种于六孔培养板中,加入10%FBS的DMEM-LG培养液.在倒置相差显微镜下观察细胞形态学变化.2周后停止培养,采用免疫细胞化学染色法和RT-PCR检测角蛋白18、RPE65蛋白存诱导细胞中的表达.数据采用Holm-Sidak法进行分析.结果 诱导2周后,第一组BMSCs细胞呈圆形、类圆形、不规则形、短棒状外观,细胞内有色素颗粒形成,其他两组没有类似改变.三组间免疫细胞化学染色法检测RPE65蛋白、角蛋白18,光密度值结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65：t=37.416、36.236,P＜0.05;角蛋白18：t=38.611、37.532,P＜0.05）.而第二组和第三组间差异没有统计学意义（RPE65：t=1.180,P＞0.05;角蛋白18：t=1.079,P＞0.05）.RT-PCR检测相对mRNA表达量,结果显示第一组和第二组间、第一组和第三组间差异有统计学意义（RPE65/β-actin：t=176.110、174.820,P＜0.05;角蛋白18/β-actin：t=243.230、241.560,P＜0.05）.而第二组和第三组间差异无统计学意义（RPE65/β-actin：t=1.283.P＞0.05;角蛋白18/β-actin：t=1.670,P＞0.05）.结论 利用BDNF、EGF、bFGF联合HRPECs共培养可以使BMSCs分化为视网膜色素上皮样细胞.     

The Aqueous Levels of TGF-β2 in Patients with Glaucoma

PURPOSE: To evaluate the aqueous levels of transforming growth factor beta-2 (TGF-beta2) in open angle glaucoma patients. METHODS: The aqueous levels of active TGF-beta2 were detected in 17 eyes of 17 patients using ELISA method. The control group consisted of 6 cataract extracted patients. RESULTS: Mean age of the patients (60.8 +/- 8.8 years) was similar to that of the controls (57.5 +/- 9.8 years) (p = 0.516). Levels of TGF-beta2 in aqueous samples of glaucoma patients (2.74+/- 1.23 ng/ml) were found to be elevated when compared to those of controls (1.67 +/- 0.32 ng/ml) (p = 0.020). CONCLUSION: We might suggest that the elevated levels of TGF-beta2 in the aqueous of glaucoma patients could play a role in the pathogenesis of glaucoma.     

Differences of bFGF gene expression in lens epithelial cells between fetuses and cataract patients

AIM: To study the differences of basic fibroblast growth factor (bFGF) gene expression in lens epithelial cells (LECs) between fetuses and cataract patients. · METHODS: In situ hybridization was used to detect bFGF mRNA in the LECs that were cultured and in tissue sections from fetuses and in the LECs from the anterior capsule of cataract patients. Image analysis was used for the relative quantitative analysis of bFGF mRNA. · RESULTS: bFGF gene existed in the LECs that were cultured and in tissue sections from fetuses and in the LECs from the anterior capsule of cataract patients. The integral absorbance for the fetal cultured cells, the fetal tissue sections and the capsule membrane cells of cataract patients were 627.1±268.7, 131.5±42.8 and 79.2±26.3 respectively. The integral absorbance of fetal cultured LECs was significantly higher than that of fetal section LECs (P <0.01). The integral absorbance of cataract LECs was significantly lower than that of fetal LECs (P <0.01). · CONCLUSION: The in vitro culture of LECs can improve bFGF gene expression. The bFGF gene expression in fetal LECs is significantly higher than that in cataract LECs.     

Cytokine regulation of hyaluronate production by human Tenon's capsule fibroblasts

PURPOSE: The high-molecular weight glycosaminoglycan hyaluronate (HA), a component of the extracellular matrix, has been shown to play important roles in many biological processes including cell proliferation, migration and differentiation. In the present study, the effect of cytokines on production of hyaluronate (HA) by human tenon's capsule fibroblasts (HTF) was determined. METHODS: HTF (2(nd) passage) were seeded at a cell density of 30 cells/mm(2) and stimulated by six different cytokines (platelet-derived growth factor (PDGF)-AA, PDGF-BB, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin (IL)-1beta and transforming growth factor (TGF)-beta1). Controls were treated with aliquots of serum-free medium only. Concentrations of HA were determined using a radiometric assay based on the specific binding of HA to HA binding proteins. RESULTS: The concentration of HA in conditioned medium of HTF was significantly increased only after stimulation with PDGF-AA [10 and 100ng/ml], IL-1beta [1 and 10ng/ml] and TGF-beta1 [5ng/ml]. CONCLUSIONS: Production of HA by HTF is regulated by PDGF-AA, IL-1beta and TGF-beta1 and is speculated to be involved in the wound healing reaction after glaucoma filtration surgery.     

碱性成纤维细胞生长因子基因在胎儿与白内障患者晶状体上皮细胞内表达的比较

目的:研究碱性成纤维细胞生长因子基因在胎儿和白内障患者晶状体上皮细胞内表达的区别。方法:采用原位杂交方法,用cDNA探针检测胎儿培养及组织切片中的晶状体上皮细胞和白内障患者前囊中的晶状体上皮细胞的bFGF的mRNA,并用图像分析进行相对定量,比较胎儿培养细胞、组织切片细胞及患者囊膜细胞的积分光吸收度值。结果:胎儿的培养及组织切片中晶状体上皮细胞和白内障患者前囊中的晶状体上皮细胞都存在bFGF基因表达。胎儿体外培养晶状体上皮细胞、胎儿组织切片晶状体上皮细胞和白内障患者晶状体上皮细胞积分光吸收度值分别为627.1±268.7,131.5±42.8和79.2±26.3。胎儿体外培养晶状体上皮细胞积分光吸收度值显著高于胎儿组织切片晶状体上皮细胞(P<0.01);白内障患者晶状体上皮细胞积分光吸收度值显著低于胎儿晶状体上皮细胞(P<0.01)。结论:晶状体上皮细胞体外培养可增加bFGF基因表达;胎儿晶状体上皮细胞bFGF基因表达显著高于白内障患者晶状体上皮细胞。     

Presence of alpha smooth muscle actin in lens epithelial cells of aphakic rabbit eyes.

AIMS: To determine whether alpha smooth muscle actin (alpha-SMA), a marker for myofibroblastic cells, is present in lens epithelial cells (LECs) in rabbit aphakic eyes. METHODS: Phacoemulsification was performed in rabbit eyes, which were enucleated after surgery. Immunohistochemical methods were used to detect alpha-SMA in LECs. RESULTS: Five days after surgery, the presence of alpha-SMA positive LECs was observed mainly around the adhesive portion of the anterior capsule margin and the posterior capsule. The posterior capsule was wrinkled at the adhesive portion. The alpha-SMA positive LECs were flattened with spindle-shaped cross sections. Seven days after surgery, the alpha-SMA positive LECs covered most of the central posterior capsule. They disappeared 10 days after surgery. On the other hand, the cuboidal LECs in the capsular bag were negative for alpha-SMA. CONCLUSION: The flattened LECs with spindle-shaped cross sections observed 5 days after cataract surgery contained alpha-SMA. Such LECs were distinguished biochemically from the cuboidal LECs, which lacked alpha-SMA.     

Epidermal growth factor receptor in cultured human retinal pigment epithelial cells

BACKGROUND: Migration and proliferation of retinal pigment epithelial (RPE) cells play an important role in proliferative vitreoretinopathy. Epidermal growth factor receptor (EGFR) is a cell surface receptor with intrinsic tyrosine kinase activity. The engagement of the receptor by its ligand can induce intracellular mitogenic signal transduction pathways and stimulate proliferation, migration and differentiation of cells. This experiment aimed to investigate the activation and role of EGFR signal transduction pathway in proliferation of human RPE cells. METHODS: Cultured human RPE cells of the 3rd to 6th passages were studied by colorimetric assay for cellular growth and survival (MTT assay) to test the effects of EGF (0.1, 1, 10, 50, and 100 ng/ml) and fetal bovine serum (FBS) on proliferation of human RPE cells. An in vitro wound healing model was also set up, and the number of cells that had entered the denuded area was counted. The human RPE cells were cultured for 3 days with 0.1% FBS, 10% FBS, 10 ng/ml EGF + 0.1% FBS and a combination of EGF and 10% FBS, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and mRNA, respectively. Activation of mitogen-activated protein kinase (MAPK) was detected by immunohistochemical method with specific antiphosphorylated extracellular signal-regulated kinase (ERK)1/2 antibody. RESULTS: EGF stimulated proliferation and migration of cultured human RPE cells in a concentration-dependent manner. The maximum of the proliferation rate of RPE cells was 81.8% with EGF at a concentration of 10-100 ng/ml of EGF in serum-free Dulbecco's modified essential medium (DMEM) and 122.7% at a concentration of 1-10 ng/ml of EGF in 5% FBS DMEM (p < 0.001); there was a significant difference between serum-free DMEM groups and 5% FBS DMEM groups. The maximum of the migration rate of the cells was 438.9% at a concentration of 10-100 ng/ml of EGF in 10% FBS DMEM, 147% with 10% FBS, and only 36% with EGF in 0.1% FBS at the concentration of 10 ng/ml (p < 0.001). EGF promoted the expression of EGFR protein and mRNA in RPE cells. FBS cooperated with EGF in the stimulation of EGFR expression, and it had a stronger effect in the process than EGF alone. After 3 days of incubation with EGF, phosphorylated ERK1/2 was detectable in the nucleus of RPE cells, whereas cells presented immunostaining positive for phosphorylated ERK1/2 in the cytoplasm before stimulation, indicating that EGF could induce MAPK nuclear translocation. CONCLUSION: EGF could induce EGF-EGFR-MAPK signal transduction pathway in human RPE cells in a concentration-dependent manner in vitro, which may play a key role in the activation of human RPE cell proliferation and migration.     

Einfluss von Octreotid in Kombination mit Wachstumsfaktoren auf die Proliferation von RPE-Zellen in vitro

BACKGROUND: The long-acting somatostatin analogue octreotide is used as a therapeutic option for patients with diabetic retinopathy and age-related macular degeneration. Growth factors, such as EGF, bFGF, VEGF, and PDGF, have been implicated in the pathogenesis of these diseases. The aim of this study was to investigate the effect of octreotide on the growth-factor-induced proliferation of bovine retinal pigment epithelial (RPE) cells in vitro. METHODS: RPE cells were assayed for proliferation as measured by (3H)-thymidine uptake (ccpm). Bovine RPE cells at passage 3-8 were seeded in a 96-well plate and incubated for a 24-h period with a minimum medium followed by a 24-h incubation with the different growth factors at a concentration of 10 ng/ml with and without 5 x 10(-5) M octreotide. In a different assay the cells were incubated with octreotide at 5 x 10(-10)-5 x 10(-4) M. Afterwards the cells were pulsed with 5 microCi/ml (3H)-thymidine for 12 h, and the incorporated radioactivity was measured on glass fiber filters in a beta-counter (mean +/- SEM ccpm). The Wilcoxon Signed Rank test and the student's t-test were used for statistical analyses. RESULTS: There was a biphasic effect of octreotide on RPE cell proliferation. Exposure of RPE cells to PDGF (20,225 +/- 3304 ccpm) and bFGF (3441 +/- 539 ccpm) resulted in a significantly higher proliferation compared to control medium (1543 +/- 352 ccpm) (p < 0.05). No difference was found for EGF (2385 +/- 383 ccpm). For VEGF (776 +/- 83 ccpm) a significant reduction in RPE cell proliferation was found (P < 0.05). There was a significant reduction in proliferation of RPE cells with PDGF, bFGF, and EGF in combination with octreotide versus growth factors alone (octreotide in combination with PDGF 308 +/- 82 ccpm, with bFGF 229 +/- 88 ccpm, with EGF 2362 +/- 91 ccpm) (p < 0.005). VEGF in combination with octreotide resulted in a significant inhibition of RPE cell proliferation compared to VEGF alone (p < 0.0005). CONCLUSION: The data suggest that there is an inhibitory effect of octreotide on RPE cell proliferation of bovine RPE cells and on the increased proliferation of bovine RPE cells induced by PDGF and bFGF. An enhanced inhibitory effect is found for the combination of octreotide and VEGF.