Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition

Posterior capsule opacification (PCO) is a common complication of cataract surgery caused by epithelial mesenchymal transition (EMT) and aberrant lens cell growth. One path to prevention depends on maintaining the quiescent lens epithelial phenotype. Here we report that lithium chloride (LiCl) is a potent stabilizer of the lens epithelial phenotype. In lens epithelial explants (controls), at low cell density, cells readily depolarized, spread out, and proliferated. By contrast, in the presence of LiCl, cells did not spread out or exhibit migratory behaviour. Using concentrations of 1-30 mM LiCl we also showed that cell proliferation is inhibited in a dose-dependent manner. Confocal microscopy and immunohistochemistry for ZO-1 and E-cadherin showed that LiCl treatment maintained tight junctions at the apical margins of cells. Taken together with measurements of cell heights, this showed that the cells in LiCl-treated explants maintained the apical baso-lateral polarity and cobblestone-like packing that is characteristic of lens epithelial cells in vivo. Significantly, the effects of LiCl also extended to blocking the potent EMT/cataract-promoting effects of transforming growth factor beta (TGFbeta) on lens epithelial cells. In TGFbeta-treated explants, cells progressively dissociated from one another, taking on various elongated spindle shapes and strongly expressing alpha-smooth muscle actin (alpha-SMA). These features are characteristic of PCO. In both rat and human capsulorhexis explants, LiCl treatment effectively blocked the accumulation of alpha-SMA and maintained the cells in a polarized, adherent, cobblestone-packed monolayer. These findings highlight the feasibility of applying molecular strategies to stabilize lens epithelial cells and prevent aberrant differentiation and growth that leads to cataract.     

ERK1/2信号传导通路介导碱性成纤维细胞生长因子诱导人晶状体上皮细胞中环氧合酶2的表达

背景：碱性成纤维细胞生长因子在各种白内障形成中都起着重要的作用,可促进晶状体上皮细胞的增殖并化生为纤维细胞,但其信号通路尚不清楚。 目的：探讨ERK1/2信号转导通路在碱性成纤维细胞生长因子诱导的人晶状体上皮细胞环氧合酶2表达中的作用。 方法：使用10 μg/L的碱性成纤维细胞生长因子干预培养的人晶状体上皮细胞0,1,3,6,12,24 h,RT-PCR检测刺激不同时间人晶状体上皮细胞环氧合酶2 mRNA的表达,Western blot检测细胞中环氧合酶2及磷酸化ERK1/2的表达。在阻断实验中应用特异性ERK1/2的阻断剂PD98059 阻断ERK信号转导通路1 h,再用10 μg/L碱性成纤维细胞生长因子刺激细胞6 h,Western blot检测细胞中环氧合酶2的表达。 结果与结论：碱性成纤维细胞生长因子刺激后的人晶状体上皮细胞中环氧合酶2 mRNA及其蛋白的表达显著增加(P < 0.01),同时碱性成纤维细胞生长因子诱导人晶状体上皮细胞磷酸化ERK1/2活性增强,表达水平随作用时间而增加,30 min时达到最高峰(P < 0.01),6 h后恢复至基线水平;PD98059可抑制人晶状体上皮细胞环氧合酶2的表达 (P < 0.01)。说明ERK1/2信号转导通路参与了碱性成纤维细胞生长因子诱导的人晶状体上皮细胞中环氧合酶2的表达,在后发性白内障的形成过程中起着重要作用。     

Development of an accommodating intra-ocular lens--in vitro prevention of re-growth of pig and rabbit lens capsule epithelial cells

Cataract surgery is routinely performed to replace the clouded lens by a rigid polymeric intra-ocular lens unable to accommodate. By implanting a silicone gel into an intact capsular bag the accommodating properties of the natural lens can be maintained or enhanced. The implantation success of accommodating lenses is hampered by the occurrence of capsular opacification (PCO) due to lens epithelial cell (LEC) growth. In order to prevent LEC proliferation, a treatment regime using actinomycin D, cycloheximide and water was developed. The effectiveness of treatment was analyzed using an in vitro, MTT-based cell culture system and an ex vivo pig eye model in which the implanted lens-in-the-bag is cultured as a whole. LEC were exposed to treatment solutions for 5 min, then the cells were allowed to recover and to re-colonize the substratum. MTT conversion by cells was transiently inhibited by cycloheximide dissolved in water and by water alone. Exposure to actinomycin D resulted in a lasting inhibition of MTT conversion and consequently cell proliferation. These in vitro data could not be fully reproduced in the ex vivo pig eye model due to essential differences between both models. Treatment with actinomycin D containing solutions, however, resulted in a nearly complete absence of cells on the capsular wall. The pig eye model is a promising approach to further evaluate the effects of peri-surgical treatment during the accommodating intra-ocular lens implantation.     

The effect of the haptic portion of intraocular lens on the development of posterior capsular opacification in rabbit.

Using a white rabbit model, the effect of the haptic portion of the intraocular lens (IOL) and intracapsular ring on the development of posterior capsular opacification (PCO) after extracapsular cataract extraction (ECCE) with phacoemulsification was studied. Implantation of both the intracapsular ring and IOL developed less PCO than implantation of the IOL alone. ECCE followed by implantation of the intracapsular ring alone also developed less PCO than ECCE alone. Through this experimental work in a rabbit model, it could be conceived that the haptic portion of IOL and the intracapsular ring can prevent the development of PCO.     

RAC1 overexpression promotes the proliferation,migration and epithelial-mesenchymal transition of lens epithelial cells

Cataract is a main cause of blindness worldwide. RAC1 has been reported to have a close relationship with the proliferation and migration of cells. However, the relationship between RAC1 and cataract is not yet clear. The proliferation and migration of lens epithelial cells are key factors in the formation of cataract as well as in the complication of cataract surgery. In this study, the effect of RAC1 overexpression on the proliferation and migration of lens epithelial cells was explored. Results showed that RAC1 overexpression promoted the proliferation of lens epithelial cells and increased the protein level of proliferating cell nuclear antigen. RAC1 overexpression also promoted migration and invasion of lens epithelial cells and had an influence on the epithelial-mesenchymal transition process. These results indicate that RAC1 may become a therapeutic target of cataract and inhibition of RAC1 may become a promising way for the therapy of cataract.     

The efficacy of an acrylic intraocular lens surface modified with polyethylene glycol in posterior capsular opacification

To investigate if the surface modification of intraocular lens (IOL) is efficient in the prevention of posterior capsular opacification (PCO), the acrylic surface of intraocular lens (Acrysof) was polymerized with polyethylene glycol (PEG-IOL). The human lens epithelial cells (1 x 10(4) cells/mL) were inoculated on PEG grafted or unmodified acrylic lenses for the control. The adherent cells on each IOL surface were trypsinized and counted. The every PEG-IOL was implanted in 20 New Zealand rabbits after removal of crystalline lens. The formations of PCO were checked serially through retroilluminated digital photography, and the severity scores were calculated using POCOman. The cell adherence patterns on each IOL were examined by scanning electron microscopy. As a result, the mean number of adherent cells of PEG-IOL (3.2+/-1.1 x 10(3)) tended to be smaller than that of the acrylic controls (3.6+/-1.9 x 10(3)) without a statistical significance (p=0.73). However, the mean severity of PCO formation in PEG-IOL was significantly lower than that in the control during the third to sixth weeks after surgery. Scanning electron microscopy revealed that the more patch-like cells were found firmly attached to the IOL surface in control than in the PEG-IOL. Conclusively, PEG polymerization to the acrylic IOL would possibly lessen the formation of PCO after cataract removal.     

Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.     

Echistatin prevents posterior capsule opacification in diabetic rabbit model via integrin linked kinase signaling pathway

Purpose: To investigate the effect of disintegrin echistatin on integrin linked kinase (ILK) and subsequent PI3-K/Akt and ERK1/2 signaling pathways in the posterior capsule opacification (PCO) model of diabetic rabbit. Methods: 56 rabbits were injected alloxan to model diabetic. Then they accepted lens extraction surgery and randomly and intraoperatively injected distilled water (control group; n = 28) or 10.0 mg·L^-1 echistatin (echistatin-treated group; n = 28) into the anterior chamber. Each group was subdivided into ten days group (n = 14) and six weeks group (n = 14) respectively. The PCO severity was evaluated with a slit lamp microscope and light microscope for 10 days and 6 weeks postoperatively. The levels of ILK in the posterior capsule were determined by Q-PCR, Western blotting and Immunohistochemistry. Akt and ERK1/2 phosphorylation were analyzed by Western blotting. Results: 10 days and 6 weeks after surgery, the grades of PCO in the echistatin-treated group were lower than the control group. The lens epithelial cells (LECs) in the posterior capsule of echistatin-treated eyes had decreased degrees of proliferation and migration than the control group. And no significant side effects appeared after treated with echistatin. Echistatin could significantly reduce the expression of ILK in terms of both mRNA and protein levels. The phosphorylation levels of Akt and ERK1/2 were decreased in the echistatin-treated group compared with the control group. Conclusions: Echistatin could inhibit postoperative PCO occurrence and development in diabetic rabbit eyes, which may be related to down-regulation the expression of ILK and inhibition the PI3-K/Akt and ERK1/2 pathways.     

Transforming growth factor-beta-induced epithelial-mesenchymal transition in the lens: a model for cataract formation

The vertebrate lens has a distinct polarity and structure that are regulated by growth factors resident in the ocular media. Fibroblast growth factors, in concert with other growth factors, are key regulators of lens fiber cell differentiation. While members of the transforming growth factor (TGFbeta) superfamily have also been implicated to play a role in lens fiber differentiation, inappropriate TGFbeta signaling in the anterior lens epithelial cells results in an epithelial-mesenchymal transition (EMT) that bears morphological and molecular resemblance to forms of human cataract, including anterior subcapsular (ASC) and posterior capsule opacification (PCO; also known as secondary cataract or after-cataract), which occurs after cataract surgery. Numerous in vitro and in vivo studies indicate that this TGFbeta-induced EMT is part of a wound healing response in lens epithelial cells and is characterized by induced expression of numerous extracellular matrix proteins (laminin, collagens I, III, tenascin, fibronectin, proteoglycans), intermediate filaments (desmin, alpha-smooth muscle actin) and various integrins (alpha2, alpha5, alpha7B), as well as the loss of epithelial genes [Pax6, Cx43, CP49, alpha-crystallin, E-cadherin, zonula occludens-1 protein (ZO-1)]. The signaling pathways involved in initiating the EMT seem to primarily involve the Smad-dependent pathway, whereby TGFbeta binding to specific high affinity cell surface receptors activates the receptor-Smad/Smad4 complex. Recent studies implicate other factors [such as fibroblast growth factor (FGFs), hepatocyte growth factor, integrins], present in the lens and ocular environment, in the pathogenesis of ASC and PCO. For example, FGF signaling can augment many of the effects of TGFbeta, and integrin signaling, possibly via ILK, appears to mediate some of the morphological features of EMT initiated by TGFbeta. Increasing attention is now being directed at the network of signaling pathways that effect the EMT in lens epithelial cells, with the aim of identifying potential therapeutic targets to inhibit cataract, particularly PCO, which remains a significant clinical problem in ophthalmology.     

Prevention of capsular opacification after accommodative lens refilling surgery in rabbits

Silicone gel-like polymers have been proposed to replace the cataractous lens and therewith restore both vision and accommodation. Lens replacement is associated with opacification of the capsular bag due to the lens epithelial cell response. In this study, the in vivo effectiveness of a 5 min treatment with actinomycin D and/or cycloheximid to prevent the development of capsular opacification after filling the capsular bag with a silicone polymer as an accommodating lens was studied. It was found that treating the inside of the capsular bag with a solution containing actinomycin D reduced the development of visible capsular opacification for three months. In some animals, the lens capsules were completely clear, indicating the potential of this method. Side effects of the treatment in the form of visible cornea opacification occurred and ranged from mild to severe in some animals, while in other animals no toxicity occurred. This indicates that a safe application of the cytotoxic substances is feasible. In view of the side effects and the fact that not all lens capsules of the animals treated with actinomycin D were clear, improvements in the methods used are necessary and seem to be possible.     

Delayed postoperative opacification of foldable hydrophilic acrylic intraocular lenses

Phacoemulsification and implantation of a foldable intraocular lens for cataract is almost a routine procedure in ophthalmic practice. Late postoperative opacification of hydrophilic acrylic (hydrogel) intraocular lenses (IOLs) has been reported, but the appearance differs from our findings. We report clinical, pathologic, ultrastructural, and spectroscopic analyses of foldable hydrogel IOLs undergoing late opacification resulting in visual disturbances. This retrospective study comprised 486 patients (550 eyes) who had implantation of a foldable hydrogel posterior IOL. Three cases (three eyes) had been found to have opacification of the IOLs. The three lenses were exchanged because of significant visual disturbances, and were examined using light microscopy, scanning electron microscopy and energy dispersion X-ray (EDX) spectroscopy. Incisional biopsies of the capsular membrane in contact with the opacified IOL were performed in one patient. The mean interval between implantation of the hydrogel IOLs and the diagnosis of opacification was 35.3 months (range, 34-36 months). All three of the cases had quiet anterior chamber. No obvious posterior capsular opacification were found. The best corrected visual acuity of the three cases who had exchanged IOLs were all 20/25 or better. Microscopic analysis revealed multiple, fine, granular structures both on the surface and matrix of the opacified IOLs. EDX analysis showed the presence of calcium and phosphorous were within the granular structures. Histological examination of capsular membrane did not reveal any evidence of calcium salts or special pathological exchanges. Further studies should be undertaken to determine the incidence and possible mechanisms of this phenomenon.     

Long term following up of G lens of phaco model

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柔红霉素、骆驼蓬总碱对兔视网膜的毒性观察

目的 探讨药物柔红霉素(daunorubicin,DNR)、骆驼蓬总碱(total alkaloid Of Harmaline,TAH)眼内注射防治后发性白内障对视网膜的毒性作用.方法 于兔右眼晶体囊摘除(extracapsular lens extraction,ECLE)术中分别前房内注射0.2mg/mlTAH和0.2mg/ml DNR溶液0.1ml,通过眼底镜检查、视网膜电图及眼组织病理学等研究对象兔眼视网膜的影响.结果 对照眼与15只注入TAH眼眼底无明显的病理变化;TAH眼手术前后视网膜电图b波的振幅和潜时与对照眼无差别.而10只注入DNR眼出现严重的内皮性角膜水肿混浊,前房大量纤维索性渗出特等明显的炎性反应眼底及视网膜电图无法检查.组织病理学检查显示TAH组眼前节结构及视网膜各层结构正常,细胞排列规律;透射电镜检查TAH眼的视网膜感光细胞外节盘膜结构清晰.排列整齐.视网膜细胞排列紊乱,部分明显呈核固缩,表现出明显的毒性反应.结论 TAH眼内注射对兔视网膜的毒性较小,有可能用于后发性白内障的防治研究,而DNR对兔视网膜及其它组织表现出明显毒性,应进一步研究其使用的安全剂量和剂型.     

MicroRNA-23b-3p promotes the proliferation,migration, and epithelial–mesenchymal transition of lens epithelial cells by targeting Sprouty2

Cataract, opacification of the lens, is one of the most important reasons of visual impairment and blindness. Though microRNAs (miRNAs) have been demonstrated to play important roles in cataractogenesis, the underlying molecular mechanisms in this progress remain obscure. In the present study, microRNA-23b-3p (miR-23b) overexpression promoted the proliferation, migration and epithelial-mesenchymal transition (EMT), whereas miR-23b knockdown markedly inhibited the proliferation, migration and TGF-β-induced EMT of lens epithelial cells (LECs). In TGF-β-induced LECs, the expression of miR-23b was markedly upregulated and the expression of Sprouty2 (SPRY2) was markedly downregulated, furthermore the mRNA and protein levels of SPRY2 were markedly decreased in miR-23b inhibitor-transfected LECs. We then performed a Dual-luciferase reporter assay to confirm that miR-23b directly targeted SPRY2. The promoted migration and EMT of LECs by enforced expression of miR-23b were suppressed by SPRY2 overexpression. The findings present the first evidence indicating that miR-23b can promote the proliferation, migration, and EMT of LECs by targeting SPRY2 and the inhibition of miR-23b may possess the therapeutic potential for cataract.     

ADV-miR-184体外转染对人晶状体上皮细胞移行的影响

目的 探讨携带miR-184的重组腺病毒(ADV-miR-184)体外转染对人晶状体上皮细胞移行的影响.方法 ADV-miR-184在293细胞中扩增、纯化并滴定病毒滴度;ADV-miR-184体外感染人晶状体上皮细胞(HLE-B3),采用细胞划痕法测定ADV-miR-184对HLE-B3移行距离的影响.结果 测定ADV-miR-184的病毒滴度为1.6×109 puf/mL;分别将ADV-miR-184以MO110、MO150、MOI100、MOI200、MOI500转染HLE-B3 72 h后,细胞移行距离随着ADV-miR-184的浓度增加而减少,MOI100组与MOI50组、MOI10组相比有明显差异(P＜0.05).但与MOI200、MOI500实验组相比变化不明显.与对照组比较,MOI100感染细胞的移行距离在转染后24 h、48 h、72 h、96 h明显缩短(P＜0.05).结论 ADV-miR-184可成功转染人晶体上皮细胞,并可抑制细胞的移行,提示miR可能参与后发性白内障的形成过程.     

Vitronectin is significant in the adhesion of lens epithelial cells to PMMA polymers

A major complication of intraocular lens surgery is diminished visual acuity caused by the regrowth of lens epithelial cells (secondary cataract). Polymethylmethacrylate (PMMA) is a commonly used intraocular lens material. This study addresses the mechanisms underlying the initial adhesion of lens epithelial cells to PMMA and a functionalized PMMA-based terpolymer known to inhibit cell proliferation. Rabbit lens epithelial cells were cultured on the test polymer surfaces in medium containing serum depleted of either fibronectin or vitronectin (or both) to identify the role of these proteins in the initial process of cell adhesion. Adherent cells were quantitated after 60 min, and the actin cytoskeleton and focal contact formation were compared in each serum treatment on both polymers. Vitronectin was significantly more effective for initial cell attachment to both polymers than fibronectin. Normal cell spreading on PMMA required vitronectin and was independent of fibronectin, whereas cell spreading on the terpolymer was abnormal and required the presence of fibronectin and vitronectin together. Together, these results help to explain the inhibition of cell proliferation previously shown on the functionalized PMMA. This work contributes to the design of a polymer for use in intraocular lenses that inhibits proliferation of the target cells.     

羟基喜树碱抑制体外培养兔晶体上皮细胞的生长

为研究羟基喜树碱（HCPT）对体外培养兔晶体上皮细胞（RLECs)生长的作用,取2－3代的RLECs在96孔板中培养24h后,用不同浓度的HCPT分别作用24及72h,用甲基噻唑四唑（Methyl thiazolyal tetrazolium,MTT)比色测定法观察其对RLECs增殖的影响,结果显示HCPT能抑制RLECs的增殖,24h的半数抑制量（ID50）为96．041mg/L,72h的ID50为1．34mg/L;且HCPT还可抑制RLECs的贴壁,提示HCPT可能在用于降低后发性白内障的发生方面有一定作用。     

Ultraviolet A exposure induces reversible disruption of gap junction intercellular communication in lens epithelial cells

Gap junction intercellular communication (GJIC) is essential for the proper function of many organs including the lens. Disruption of GJIC can cause lens metabolic disorder and can induce cataracts. The purpose of this study was to investigate the signal transduction pathways involved in GJIC disruption following ultraviolet A (UVA) exposure in lens epithelial cells. Following exposure of human lens epithelial cells to UVA, connexin 43 (Cx43), the main component of gap junctions, was down-regulated at both the mRNA and protein levels. Furthermore, we observed that UVA exposure can increase protein kinase C activity and stimulate reactive oxygen species generation and lipid peroxidation. Using scrape load dye transfer technique, we found that the GJIC is compromised by UVA exposure. In addition, we demonstrated that UVA-induced modulation of GJIC was associated with p38 mitogen-activated protein kinase activation. More importantly, at non-lethal doses (10 J/cm2), the UVA-induced GJIC disruption and the consequent alterations were reversible. Collectively, our data revealed a new signaling pathway in GJIC disruption following UVA exposure, suggesting that UVA-compromised gap junction activity may sensitize human lens to photoaging and cataract formation.     

Molecular genetics of congenital nuclear cataract

A cataract is defined as opacification of the normally transparent crystalline lens. Congenital cataract (CC) is a type of cataract that presents at birth or during early childhood. CC is one of the most common causes of visual impairment or blindness in children worldwide. Approximately 50% of all CC cases may have a genetic cause which is quite heterogeneous. CC occurs in a variety of morphologic configurations, including polar/subcapsular, nuclear, lamellar, sutural, cortical, membranous/capsular and complete. Nuclear cataract refers to the opacification limited to the embryonic and/or fetal nuclei of the lens. Although congenital nuclear cataract can be caused by multiple factors, genetic mutation remains to be the most common cause. It can be inherited in one of the three patterns: autosomal dominant, autosomal recessive, or X-linked transmission. Autosomal dominant inheritance is the most frequent mode with high penetrance. There may be no obvious correlation between the genotype and phenotype of congenital nuclear cataract. Animal models have been established to study the pathogenesis of congenital nuclear cataract and to identify candidate genes. In this review, we highlight identified genetic mutations that account for congenital nuclear cataract. Our review may be helpful for genetic counseling and prenatal diagnosis.