A Study of Growth Factor Receptors in Human Lens Epithelial Cells and their Relationship to Fiber Differentiation

Recent studies have demonstrated that several growth factors enhance fiber differentiation in cultured human lens epithelial (HLE) cells in early passages. However, these effects gradually decrease in cells of later passages. The purpose of this investigation is to test the hypothesis that the decreasing effect of growth factors on fiber differentiation in later passages may be due to a decrease or the inactivation of growth factor receptors as a function of serial subcultures. Specimens of HLE cells were obtained from infants. First through to fourth passage cells were treated with 10 ng ml⁻¹of epidermal growth factor, basic fibroblast growth factor or insulin-like growth factor-I. Fiber differentiation was determined from spontaneous lentoid formation by phase-contrast and transmission electron microscopy. Growth factor binding to the receptor on the cell surface was determined by transmission electron microscopy using the conjugates of colloidal gold and growth factors, and the number of receptors on the cell surface were also quantified by immunocytochemistry. Spontaneous lentoid formation was enhanced by all of the growth factors studied in the first passage. However, in the second and third passage only double layering of cells without characteristic fiber differentiation was observed while in the fourth passage, growth factors had no effect on differentiation. The number of growth factor bindings as well as the number of growth factor receptors gradually decreased with the number of passages. The loss of effect of growth factors on fiber differentiation with increasing number of passages correlated with the decrease in receptor number.     

Differential Expression of Photoreceptor-Specific Proteins During Disease and Degeneration in the Progressive Rod-Cone Degeneration (prcd) Retina

Progressive rod-cone degeneration (prcd) is a late-onset hereditary retinal degeneration characterized by normal development of photoreceptors prior to degeneration and death of visual cells. We reported previously that expression of opsin mRNA and protein decreases prior to visual cell degeneration. To examine the specificity of this reduction, we have used immunocytochemistry to correlate photoreceptor-specific protein expression with visual cell disease progression. Eyes from light-adapted age-matched control andprcd-affected dogs were fixed in paraformaldehyde, embedded in diethylene glycol distearate (DGD) wax, and reacted with antibodies specific to interphotoreceptor retinoid-binding protein (IRBP), S-antigen, opsin, phosducin, γ-phosphodiesterase (γ-PDE), and β₁-transducin. While IRBP expression did not change with disease progression, immunoreactivity to other proteins varied. For S-antigen and opsin, immunoreactivity decreased dramatically with the transition from photoreceptor disease to degeneration; γ-PDE immunolabeling in rods also decreased, but the reduction was less abrupt. However, for two other proteins (phosducin and β₁-transducin), immunoreactivity increased initially and was redistributed (particularly to the rod outer segment) in early disease (stage 1). Our results show that there is a differential expression of photoreceptor-specific proteins with disease and degeneration that is not uniform for all the gene products examined; expression can be decreased, altered in distribution or remain unchanged. It is clear that the decrease of opsin expression described previously is not an isolated phenomenon in the progression ofprcd, but is part of a more generalized degenerative process which eventually culminates in cell death.     

Pax6基因在视网膜母细胞瘤中的表达及临床意义

目的:探讨Pax6基因在视网膜母细胞瘤(retinoblastoma,Rb)中的表达及临床意义。方法:选择我院2001-01/2012-12收存在眼科病理室的15例Rb组织切片设为观察组,再选取15例正常视网膜组织切片设为对照组。应用Western-Blot及RT-PCR(逆转录酶链反应)法分别对正常视网膜组织及Rb组织中的Pax6蛋白和Pax6 mRNA的表达进行检测,同时应用Western-Blot法对Pax6基因下游的BRN3b及MATH5促分化基因在蛋白水平的表达进行检测,最后进行组间比较,进而对Pax6基因在Rb中的表达及临床意义进行探讨。结果:观察组Pax6基因mRNA表达平均值为0.99±0.03,Pax6基因蛋白表达平均值为2.07±0.15,BRN3b蛋白表达平均值为0.195±0.016,MATH5蛋白表达平均值为0.190±0.031,均明显高于对照组,差异有统计学意义(P<0.05)。结论:异常表达的Pax6基因可能对Rb的出现起到促进作用。     

Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

PurposeHeat shock protein B8 (HspB8) can be upregulated rapidly in many pathologic processes, but its role in traumatic optic neuropathy remains unclear. In this study, we investigated the involvement of autophagy in the effects of HspB8 by using the optic nerve crush (ONC) model.MethodsMale C57BL/6J mice were intravitreally injected with recombinant adeno-associated virus type 2 (AAV2-shHspB8 or AAV2-GFP) and subsequently received ONC by a self-closing tweezers. Western blot and immunohistochemistry staining were used to evaluate the expression of HspB8. We conducted retinal flat-mount immunofluorescence to measure the quantities of retinal ganglion cells (RGCs), and full-field flash electroretinogram (ff-ERG) and optomotor response (OMR) were used to evaluate retinal function. The autophagy level was reflected by western blot, immunohistochemistry staining, and transmission electron microscope (TEM) images. We also applied 3-methyladenine (3MA) and rapamycin (Rapa) to regulate autophagy level in optic nerve injury.ResultsONC stimulated the expression of HspB8. Declines of RGCs and ff-ERG b-wave amplitudes resulting from ONC can be alleviated by HspB8 downregulation. Increased autophagy activity after ONC was observed; however, this change can be reversed by intravitreal injection of AAV2-shHspB8. Furthermore, application of autophagy inhibitor 3MA had the same neuroprotective effects as AAV2-shHspB8, as illustrated by ff-ERG and quantities of RGCs. Also, protection of AAV2-shHspB8 was compromised by the autophagy activator Rapa.ConclusionsInhibition of HspB8 in mice optic nerve injury had neuroprotective effects, which may be derived from its downregulation of autophagy.     

Elevation of Intracellular Ca<Superscript>2+</Superscript> Concentration Induced by Hypoxia in Retinal Ganglion Cells

Purpose To analyze the mechanism of hypoxia-induced changes of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in retinal ganglion cells (RGCs). Methods Fluo-3 was applied to the cut edge of the optic nerve of 6-week-old rats. The retina was sliced, and the Ca images were captured. A hypoxic condition was created by superfusing the retinal slice with an oxygen/glucose-deprived solution. Results The retrograde staining method filled the RGCs selectively. Fifteen minutes of hypoxic conditions induced an increase in [Ca²⁺]_i in the RGCs (Δ0.13 ± 0.03, n = 23). Application of 60 μM dl-2-amino-5-phosphonovaleric acid partially blocked the hypoxia-induced [Ca²⁺]_i increase in dendrites (Δ0.03 ± 0.02, n = 4, P < 0.05) but not in the somata (Δ0.12 ± 0.02, n = 9). The RGC dendrites showed a further increase in [Ca²⁺]_i after being switched back to an oxygenated solution (Δ0.14 ± 0.04, n = 4). Neither 6-cyano-7-nitroquinoxaline-2,3-dione disodium, dl-threo-β-benzyloxyaspartate, nifedipine, nor bepridil inhibited the hypoxia-induced [Ca²⁺]_i increase. A Ca²⁺-free superfusion prevented the hypoxia-induced [Ca²⁺]_i increase in the somata (Δ0.07 ± 0.02, n = 5, P < 0.05) but not in the dendrites (Δ0.16 ± 0.005, n = 4). Conclusions The mechanism of the hypoxia-induced increase in [Ca²⁺]_i differs between somata and dendrites. The N-methyl-d-aspartate channel of dendrites seems to be the main route of Ca²⁺ influx. Jpn J Ophthalmol 2007;51:175–180 ? Japanese Ophthalmological Society 2007     

Comparison of Ganglion Cell and Retinal Nerve Fiber Layer Thickness in Pigment Dispersion Syndrome,Pigmentary Glaucoma,and Healthy Subjects with Spectral-domain OCT

Purpose: To evaluate the ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL) thickness in pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) with RTVue spectral domain optical coherence tomography (SD-OCT). Methods: A total of 102 subjects were enrolled: 29 with PDS, 18 with PG, and 55 normal subjects. Full ophthalmic examination including visual field analysis was performed. SD-OCT was used to analyze GCC superior, GCC inferior, and average RNFL thickness. To compare the discrimination capabilities, the areas under the receiver operating characteristic curves were assessed. Results: Superior GCC, inferior GCC, and RNFL thickness values of patients with PG were statistically signicantly lower than those of patients with PDS (p?p?p?>?0.05). Conclusions: The SD-OCT-derived GCC and RNFL thickness parameters can be useful to discriminate PG from both PDS and normal subjects.     

Evaluation of the Ganglion Cell Complex and Retinal Nerve Fiber Layer in Low,Moderate, and High Myopia: A Study by RTVue Spectral Domain Optical Coherence Tomography

Purpose: To assess the effect of low, moderate, and high myopia on the thickness of the retinal nerve fiber layer (RNFL) and Ganglion cell complex (GCC) measured by Spectral Domain Optical Coherence Tomography (SD-OCT) in non-glaucomatous subjects. Methods: The subjects were divided into three groups: low (n = 81, 35.6%), moderate (n = 79, 34.8%), and highly myopic eyes (n = 67, 29.5%). The RNFL thickness profile, including the average, superior, nasal, inferior, and temporal quadrant and each of the eight directional thicknesses, was measured. GCC parameters, including the average, superior, and inferior values, the focal loss volume (FLV), and the global loss volume (GLV), were measured. The correlation between the OCT measurements and the axial length was evaluated. Results: The average, superior, inferior, and nasal RNFL thicknesses of low and moderate myopic eyes were found to be significantly higher than those of highly myopic eyes. The temporal RNFL thicknesses were not different among the three groups. The average, superior, and inferior ganglion cell complex values of low and moderate myopic eyes were significantly higher than those of highly myopic eyes. The FLV and GLV of low and moderate myopic eyes were significantly higher than those of highly myopic eyes (p = 0.001 for all). In the moderate and high myopia groups, the average RNFL thickness and GCC thickness were both negatively correlated with the axial length. Conclusion: Highly myopic subjects tend to have thinner RNFL and GCC thicknesses than subjects with low and moderate myopia.     

Neurotrophic Factors, Cytokines and Stress Increase Expression of Basic Fibroblast Growth Factor in Retinal Pigmented Epithelial Cells

Basic fibroblast growth factor (bFGF) and FGF receptors have been localized to photoreceptors and retinal pigmented epithelium (RPE), but the function of bFGF in adult retina and RPE is unknown. Exogenous bFGF has a neuroprotective effect in retina and brain and its expression is increased in some neurons in response to cytokines or stress. In this study, we investigated the effect of light, other types of stress, neurotrophic factors, and cytokines on bFGF levels in cultured human RPE.Some agents that protect photoreceptors from the damaging effects of constant light, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, and interleukin-1β, increase bFGF mRNA levels in RPE cells. Intense light and exposure to oxidizing agents also increase bFGF mRNA levels in RPE cells and cycloheximide blocks the increase. An increase in bFGF protein levels was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that bFGF is modulated in RPE cells by stress and by agents that provide protection from stress and support the hypothesis that bFGF functions as a survival factor in the outer retina.     

Influence of IL-1β and TNF-α on Fas Expression of Human Retinal Pigment Epithelial Cells in Vitro

Purpose: To observe Fas expression change of cultured human retinal pigment epithelial (RPE) cells by IL-lβ and TNF-α. Methods: With flow cytometry, immunohischemistry, and color imaging system, Fas expressions by exposure to IL-1β and/or TNF-α were measured. Results:The gray degree values of Fas expression were 67.5 ±6.1 in IL-1β+TNF-α-treated group, 80.1 ±9.2 in IL-1 β-treated group, and 70.4±6.4 in TNF-α-treated group, respectively. There were significant differences (P ＜ 0.005) compared with control group (107.0±10.2). Flow cytometry showed that 15.0% cultured human RPE cells expressed Fas. Fas-positive in IL-lβ, TNF-α, and IL-lβ+TNF-α-treated groups expressed was 28.1%, 34.5%, and 65.2%, respectively. Conclusion: IL-1 β, TNF-α, and combining both of them can up-regulate Fas protein expression, which may contribute to more Fas (+) cells in proliferative vitreoretinopathy PVR). Minimizing this process by means of inducing apoptosis of Fas (+) proliferative cells of Fas/FasL pathway is a future preventive and therapeutic possibility for PVR. Eye Science 2004;20:39-41.     

EGR1对晶状体上皮细胞炎症反应及EMT的早期调控作用

目的：探究早期生长反应基因1（EGR1）对小鼠白内障术后晶状体上皮细胞（LECs）炎症反应以及上 皮-间质转化（EMT）的早期调控作用。方法：实验研究。选取10～16周龄EGR1敲除小鼠（EGR1-/-） 和野生型小鼠（WT）行白内障手术造模,于造模后0 h,3 h,24 h,48 h,72 h,4 d和5 d处死相应小 鼠并取眼球做冰冻切片。免疫荧光染色观察晶状体囊袋中EGR1蛋白、CD11b、LCN2、CXCL1和 αSMA的表达情况,Image J定量荧光强度。EGR1-/-小鼠和WT小鼠组间比较采用配对t检验,组内不 同时间点比较采用单因素方差分析。结果：WT小鼠白内障术后LECs中EGR1蛋白表达上调、炎症 反应指标CD11b、LCN2、CXCL1表达升高。EGR1-/-小鼠白内障术后LECs中EGR1蛋白表达无变化, CD11b、LCN2、CXCL1表达量明显小于WT小鼠（均P<0.01）。此外,EGR1-/-小鼠白内障术后EMT特 征性标记物αSMA的表达量、囊袋内有形细胞数量亦明显小于WT小鼠（72 h、4 d和5 d,均P<0.05）。 结论：白内障手术可早期激活LECs中的EGR1,激活的EGR1参与调控LECs的炎症反应和EMT,敲 除EGR1可以减轻上述炎症反应和EMT。     

卵黄样黄斑营养不良一家系的BEST-1基因突变分析

目的：通过分子遗传学分析,确定中国东北地区一个先天性卵黄样黄斑营养不良家系在BEST-1基因的突变位点。方法： 采集一先天性卵黄样黄斑营养不良家系2例患者及5例健康成员和100个正常对照者的外周静脉血,提取基因组DNA。应用聚合酶链反应（PCR）扩增BEST-1基因的10个编码外显子,直接测序确定致病的基因突变,并与100名正常对照者的基因筛查结果进行比较。结果：直接测序后发现该先天性卵黄样黄斑营养不良家系BEST-1基因的外显子中,未发现任何突变。结论：BEST-1基因的外显子不存在该先天性卵黄样黄斑营养不良家系的致病突变。     

Variations in the rheostat model of apoptosis: What studies of retinal ganglion cell death tell us about the functions of the Bcl2 family proteins

Studies of the functions of members of the Bcl2 gene family suggested that apoptosis was controlled by a rheostat in which anti-apoptotic proteins like BCL2 bound and sequestered pro-apoptotic proteins like BAX. Our current understanding of these proteins suggests that this is a simplistic model. The new rheostat model predicts that BH3-only peptides act as neutralizing ligands for the anti-apoptotic proteins, thus allowing molecules like BAX to become activated and initiate mitochondrial dysfunction - a critical step in the intrinsic apoptotic program. Studies of retinal ganglion cell apoptosis indicate that a threshold of BAX expression is required for its successful activation, which is independent of the overall concentration of anti-apoptotic proteins in these cells.     

疏肝通窍法对青光眼视神经损害大鼠视网膜神经节细胞凋亡及视网膜、视神经超微结构的影响

目的：通过观察视网膜神经节细胞（RGCs）计数和视网膜、视神经超微结构及形态学变化,研究疏肝通窍法保护高眼压损害的视神经的作用机制,为开发保护青光眼视神经的有效中药方剂提供参考。方法：实验研究。以SD大鼠为实验动物,右眼前房注射复方卡波姆溶液建立慢性高眼压模型（90只）。将不同时间窗（1周、2周、3周）的慢性高眼压大鼠模型分别分为模型组（5只）、阴性对照组（5只）、阳性对照组（5只）、低剂量通窍明目4号治疗组（低剂量治疗组） （10 gkg-1d-1,5只）、中剂量治疗组（20 gkg-1d-1,5 只）和高剂量治疗组（40 gkg-1d-1,5 只）,以具有疏肝通窍作用的通窍明目4 号灌胃为干预手段,运用CMIAS系列数码医学图像分析系统观察RGCs计数,电镜观察视网膜、视神经超微结构,采用one-way ANOVA法和LSD法进行数据分析。结果：①RGCs计数：随着高眼压持续的时间延长,RGCs计数逐渐减少（F=87.67、29.69、33.38、38.03、33.67、23.36,P<0.001）,经药物治疗后,高眼压持续1周、2周和3周各组的高中剂量治疗组RGCs的存活量明显增加,与阴性对照组和阳性对照组比较差异均有统计学意义（P<0.001）。②模型组和阴性对照组视网膜结构排列紊乱,厚度变薄,空泡变性,细胞萎缩坏死,各治疗组视网膜的结构紊乱减轻,各层厚度略增加,空泡变性减少,细胞萎缩程度减轻。③模型组和阴性对照组视神经轴突排列紊乱,密度降低,微丝溶解,空泡样变,细胞器肿胀破坏,髓鞘变性,各治疗组视神经髓鞘的水肿程度减轻,髓索的变性有所修复,线粒体的水肿程度也减轻。结论：通窍明目4 号可以改善高眼压大鼠模型RGCs生存的微环境,保护未受损的细胞,修复轻度受损的RGCs,延缓或阻止部分受损细胞的下行性改变,减少高眼压大鼠模型RGCs的凋亡。疏肝通窍法对青光眼视神经损害具有保护作用。     

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases

Calcium-independent phospholipase A₂, group VIA (iPLA₂-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA₂-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA₂-VIA. iPLA₂-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA₂-VIA was primarily found in the cytosol. Inhibition of iPLA₂-VIA decreased the rate of proliferation, whereas over expression of iPLA₂-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA₂-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA₂-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA₂-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA₂-VIA in the regulation of RPE proliferation suggesting that iPLA₂-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.     

Adaptive evolution of cone opsin genes in two colorful cyprinids, Opsariichthys pachycephalus and Candidia barbatus

Opsariichthys pachycephalus and Candidia barbatus are two phylogenetically related freshwater cyprinids that both exhibit colorful, yet quite different nuptial coloration. This study was designed to test the hypothesis that differences in nuptial coloration between two species could reflect differences in color perception ability and the opsin genes that coded for it. Genes encoding the visual pigments of these two species were cloned and sequenced, lambda(max) of cone photoreceptors and the reflectance spectra of their body coloration were measured to test the hypothesis. The 14-nm spectral shift between green-light-sensitive photoreceptors of these two cyprinids is found to correlate well with differences in their reflective spectra. The spectral shift could result from differential expression of opsin genes and the interactive effects of the amino acid replacements in various minor sites. These results support our hypothesis that nuptial coloration is tied to color perception ability and opsin genes.     

翼状胬肉组织中CD34、AC133、STRO-1、C—KIT的表达

目的评估源白骨髓的干细胞与翼状胬肉发病的相关性,探寻翼状胬肉的发病机制。方法选择翼状胬肉头部达到瞳孔缘的病例12例（12眼）,其中男6例,女6例;年龄51～66岁;原发性翼状胬肉8例,复发性翼状胬肉4例,均长于鼻侧。对照组材料来自同一眼球、距翼状胬肉边缘约10mm的正常球结膜组织。采用EnVision免疫组织化学法检测骨髓干细胞标志物来源的CD34、AC133、STRO-1、C-KIT的表达。结果光镜下可见翼状胬肉组织中有大量带有骨髓干细胞或祖细胞阳性标志物来源的细胞,翼状胬肉组织头部阳性表达更强。4种阳性细胞的分布及细胞形态类似。C—KIT阳性表达细胞聚集于翼状胬肉上皮基底组织和基质组织。上皮基底部阳性细胞呈圆形或椭圆形,而在基质组织中呈纺锤形且主要分布于血管周围。CD34阳性表达细胞主要分布于翼状胬肉组织上皮基底层,在血管内皮也有阳性表达,类似于C—KIT,但比C—KIT上皮层组织阳性细胞少。细胞形态在上皮基底部呈圆形或椭圆形。AC133阳性表达细胞主要分布于上皮层,基质和血管内皮中也有阳性表达,形态类似CD34和C-KIT。STRO-1阳性表达细胞的分布不同,主要见于基质层,阳性细胞形态呈纺锤形,而上皮层中未见表达。各种因子的表达在原发性和复发性翼状胬肉间无明显差别。对照组切片中标志物的表达均呈阴性。结论带有源白骨髓干细胞阳性标志物的细胞在翼状胬肉的发病和术后复发过程中可能起重要作用。     

Expression of TNF-α, IL-1β, and IFN-γ in Staphylococcus epidermidis slime-positive experimental endophthalmitis is closely related to clinical inflammatory scores

PURPOSE: The purpose of this study was to investigate the expression of inflammatory cytokines TNF-alpha, IL-1beta, and IFN-gamma in the vitreous after experimentally induced endophthalmitis by a Staphylococcus epidermidis slime-producing strain. METHODS: Seventy-two experimental Lewis rats received an intravitreal injection of 7000 viable organisms of Staphylococcus epidermidis slime-producing ATCC strain 35983, while 72 control rats received an intravitreal injection of sterile normal saline. Eyes were graded daily for signs of clinical inflammation and were removed 6, 12, 24, 48, 72 h, and 7 days after injection. Vitreous was obtained and titers of TNF-alpha, IL-1beta, and IFN-gamma were measured with established enzyme-linked immunosorbent assays. RESULTS: In the experimental group, the clinical inflammatory score reached maximum (4+) within 24 h, while inflammation was almost abolished by day 7 (score 0-0.5+). Statistically increased levels of TNF-alpha and IL-1beta were detected in the experimental vitreous with maximum levels observed at 12 h. IFN-gamma was also detected in the experimental vitreous and reached maximum levels at 48 h. None of the cytokines examined was detected in sera at any time point in experimental or control rats. CONCLUSIONS: The results of this study suggest that Staphylococcus epidermidis experimental endophthalmitis induces the expression of cytokines TNF-alpha, IL-1beta, and IFN-gamma in the vitreous. The time course of those cytokine expression levels is closely associated to the clinical presentation of this endophthalmitis model.