Application of sustained delivery microsphere of cyclosporine A for preventing posterior capsular opacification in rabbits

AIM:To explore the inhibitory effect of a sustained cyclosporin A (CsA) delivery microsphere (CsA-MS) on posterior capsular opacification (PCO) in rabbit eyes after cataract extraction. ·METHODS:Twenty New Zealand white rabbits accepted cataract extraction plus intraocular lens implantation and their left eyes were intraoperatively injected CsA-MS prepared using polymer polylactioglycolic acid (PLGA) as a carrier and their right eyes were injected with empty MS. The changes in cornea, anterior chamber reaction, intraocular pressure, PCO and CsA concentration in aqueous humor were examined postoperatively and all the eyes were enucleated 3 months after surgery for histopathological and morphological examination with light microscopy and electron microscopy. · RESULTS:Conjunctival hyperemia, corneal edema, intraocular pressure and anterior chamber response of experimental and control eyes were similar, while PCO in CsA MS injected eyes was greatly improved compared with that in control eyes. Posterior capsules in CsA-MS injected eyes were smooth and lens epithelial cells (LEC) did not proliferate significantly (P 】0.05), while LEC in posterior capsule of control eyes had different degrees of proliferation and cortical regeneration. LEC in CsA-MS injected eyes were not functionally active and underwent apoptosis, whereas LEC in control eyes were functionally active (F-test, P =0.025). In addition, the cornealultrastructure showed no differences between CsA-MS and MS injected eyes. CONCLUSION:CsA-MS has high bioavailability in rabbit eyes and could inhibit postoperative PCO occurrence and development during the study period, suggesting that CsA-MS may be a promising, effective and safe administration route to prevent PCO in clinic.

“NIR-triggered ROS storage”photodynamic intraocular implant for high-efficient and safe posterior capsular opacification prevention

Posterior capsular opacification(PCO)is the leading cause of vision loss after cataract,mainly caused by the adhesion,proliferation and trans-differentiation of post-operative residual lens epithelial cells(LECs).Effective PCO prevention remains a huge challenge to ophthalmologists and researches for decades.Herein,we developed a“NIR-triggered ROS storage”intraocular implant(CTR-Py-Pp IX)based on capsular tension ring(CTR),which is concurrently linked with photosensitizer protophorphyrin IX(Pp IX)and energy storage2-pyridone derivative(Py),to guarantee instantaneous and sustainable ROS generation for LECs killing,aiming to achieve more efficient and safer photodynamic therapy(PDT)to effectively prevent PCO.The silylated Pp IX-Si and Py-Si were covalently conjugated to the plasma activated CTR surface to obtain CTR-Py-Pp IX.Results demonstrated that CTR-Py-Pp IX had dual functions of PDT and battery,in which Pp IX could generate ROS extracellularly under irradiation,with one part directly inhibiting LECs by lipid peroxidation(LPO)induction of cell membranes.Meanwhile,the excess ROS stored in Py could be continuously released to amplify LPO levels after the irradiation was removed.Ultimately,the proliferation of LECs in capsular bag was completely inhibited under mild irradiation conditions,achieving a sustainable and controlled PDT effect for effective PCO prevention with good biocompatibility.This NIR-triggered ROS storage intraocular implant would provide a more efficient and safer approach for long-term PCO prevention.

Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

Cataract is the leading cause of visual impairment,and posterior capsular opacification(PCO)is the most common long-term complication of modern cataract surgery,which can cause severe visual impairment after surgery.The proliferation,migration,and epithelial-mesenchymal transition(EMT)of residual lens epithelial cells(LECs)stimulated by growth factors and cytokines,are the key pathological mechanisms involved in the development of PCO.This study demonstrated that non-steroidal anti-inflammatory drug(NSAID),bromfenac,was capable of effectively inhibiting cell migration,overexpression of EMT markers,such as fibronectin(FN),matrix metalloproteinase 2(MMP2),α-smooth muscle actin(α-SMA),and transcription factor Snail,and extracellular signal-regulated kinase(ERK)/glycogen synthase kinase-3β(GSK-3β)signaling induced by transforming growth factor-β2(TGF-β2)in vitro.The inhibitory effect of bromfenac on TGF-β2-induced EMT was also verified on a primary lens epithelial cell model using human anterior capsules.Furthermore,based on ultrasonic spray technology,we developed a drug-eluting intraocular lens(IOL)using poly(lactic-co-glycolic acid)(PLGA)with sustained bromfenac release ability for the prevention of PCO development.In the rabbit models of cataract surgery,bromfenac-eluting IOL exhibited remarkable PCO prevention and inflammation suppression effects with excellent biocompatibility.In conclusion,bromfenac can inhibit TGF-β2-induced cell migration and the EMT of LECs via ERK/GSK-3β/Snail signaling.The present study offers a novel approach for preventing PCO through PLGA-based drug sustained-release IOLs.

Augmented cellular uptake and homologous targeting of exosome-based drug loaded IOL for posterior capsular opacification prevention and biosafety improvement

Posterior capsular opacification(PCO),the most common complication after cataract surgery,is caused by the proliferation,migration and differentiation of residual lens epithelial cells(LECs)on the surface of the intraocular lens(IOL).Although drug-loaded IOLs have been successfully developed,the PCO prevention efficacy is still limited due to the lack of targeting and low bioavailability.In this investigation,an exosome-functionalized drug-loaded IOL was successfully developed for effective PCO prevention utilizing the homologous targeting and high biocompatibility of exosome.The exosomes derived from LECs were collected to load the anti-proliferative drug doxorubicin(Dox)through electroporation and then immobilized on the aminated IOLs surface through electrostatic interaction.In vitro experiments showed that significantly improved cellular uptake of Dox@Exos by LECs was achieved due to the targeting ability of exosome,compared with free Dox,thus resulting in superior anti-proliferation effect.In vivo animal investigations indicated that Dox@Exos-IOLs effectively inhibited the development of PCO and showed excellent intraocular biocompatibility.We believe that this work will provide a targeting strategy for PCO prevention through exosome-functionalized IOL.

Consilience and unity in ocular anterior segment research

In his beautiful book,Consilience:The Unity of Knowledge,the eminent biologist Edward O Wilson,advocates the need for integration and reconciliation across the sciences.He defines consilience as“literally a‘jumping together’of knowledge with a linking of facts…to create a common groundwork of explanation”.It is the premise of this paper that as much as basic biomedical research is in need of data generation using the latest available techniques–unifying available knowledge is just as critical.This involves the necessity to resolve contradictory findings,reduce silos,and acknowledge complexity.We take the cornea and the lens as case studies of our premise.Specifically,in this perspective,we discuss the conflicting and fragmented information on protein aggregation,oxidative damage,and fibrosis.These are fields of study that are integrally tied to anterior segment research.Our goal is to highlight the vital need for Wilson’s consilience and unity of knowledge which in turn should lead to enhanced rigor and reproducibility,and most importantly,to greater understanding and not simply knowing.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca^(2+)-dependent AMPK/mTOR pathway

●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

A new technique for Nd:YAG laser posterior capsulotomy

AIM:To investigate the effects of a new opening pattern in neodymium:yttrium-aluminum-garnet(Nd:YAG)laser posterior capsulotomy on visual function.METHODS:This technique was conducted along a circular pattern.The energy ranged between 0.8 and1.2 mJ/pulse was consumed and mean total energy levels were 74±21 mJ(mean±standard deviation:SD,from 40 to167)and laser shots aimed at 150μm away behind a datum point and went along an imaginary line which extends 0.5 mm inside from optic margin and into the circular en bloc pattern.Vitreous stands were attached with fragment and then they were cut off by the laser after circular application.The circular fragment was completely separated from vitreous,and then this fragment was quickly sunk in intravitreal space.RESULTS:The follow-up period ranges from at least a week to 40mo,making 15.8mo on average.The procedural outcome showed 96%(74 eyes out of the 77eyes)enhancement in patients’visual acuity.Cystoid macular edema or retinal detachment was not observed in any of the patients during follow-up periods.CONCLUSION:This new technique is expected to improve the weaknesses that the conventional procedures have by adding the process to cut off vitreous stands attached with the fragment by the laser to the circular application.

Comparison of design of intraocular lens versus the material for PCO prevention

AIM: To evaluate the influence of different intraocular lens(IOL) designs made of PMMA on posterior capsular opacification(PCO) and compare with foldable designs.· METHODS: Phacoemulsification and IOL implantation was done in one eye of 24 New Zealand White rabbits, with IOL of two different designs (Square edged or round edge) and two different materials(PMMA or HEMA). After three months, the animals were sacrificed and enucleated. Evaluation of PCO included posterior view, migration of anterior capsular epithelial cells to the posterior capsule following epithelial-mesenchymal transition were assessed by staining the histological sections of posterior capsule by hematoxylin-eosin(HE) and Periodic acid- Schiff (PAS). The IOLs were extracted and stained with HE to evaluate the presence of adherent cells on the lens surface. · RESULTS: PCO was highest with round edged rigid lens. There was no significant difference in the PCO between the square edged PMMA and square edged foldable lens.· CONCLUSION: It is the design of the IOL not the material that offers protection on PCO formation.

Effects of transforming growth factor β2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

AIM:To Investigate the effects of transforming growth factorβ2(TGF-β2)and connective tissue growth factor(CTGF)on transdifferentiation of human lens epithelial cells(HLECs)cultured in vitro and synthesis of extracellular matrix(ECM).METHODS:HLECs were treated with TGF-β2(0,0.5,1.0,5,10μg/L)and CTGF(0,15,30,60,100μg/L)for different times(0,24,48,72h)in vitro and the expression ofα-smooth muscle actin(α-SMA),the main component of the extracellular matrix typeⅠcollagen(Col-1)and fibronectin(Fn)were measured by using real-time polymerase chain reaction(PCR)and western-blot.RESULTS:TGF-β2 and CTGF significantly increased expression ofα-SMA mRNA and protein(P【0.05,P【0.001),Fn mRNA and protein(P【0.001),Col-1 mRNA and protein(P【0.001).TGF-β2 could induce HLECs expression of CTGF mRNA and protein in dosedependent manner(P【0.05,P【0.001).TGF-β2 and CTGF could induce HLECs to expressα-SMA,Fn and Col-1 in time-dependent manner.Each time of TGF-β2and CTGF induced HELCs expression ofα-SMA,Fn,Col-1 mRNA and protein was significant increase compared with control(P【0.05,P【0.001).CONCLUSION:TGF-β2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth

AIM： To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein（Gen-NLC） to inhibit human lens epithelial cells（HLECs） proliferation.·METHODS： Gen-NLC was made by melt emulsification method. The morphology, particle size（PS）, zeta potentials（ZP）, encapsulation efficiency（EE） and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier（NLC）, genistein（Gen） and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8（CCK-8） assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction（RT-q PCR） and immunofluorescence analyses.·RESULTS： The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was-7.14 ±0.38 m V and the EE of Gen in the nanoparticles was 92.3% ±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72 h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The m RNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group.·CONCLUSION： Sustained drug release by Gen-NLCs may impede HLEC growth.

Inhibitory Effects of NO-Fluvastatin on Proliferation of Human Lens Epithelial Cells in vitro by Modulating Cell Cycle Regulatory Proteins

The effects of NO-Fluvastatin on proliferation of human lens epithelial cells （HLECs） and the action mechanism were investigated. Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. The expression of cell cycle regulatory proteins CyclinE mRNA and P21waf1 mRNA was detected by reverse transcription polymerase chain reaction （RT-PCR）. MTT staining colorimetry showed that HLECs proliferation was markedly inhibited by NO-Fluvastatin and the effect was dependently related to time （24, 48 and 72 h） and dosage （1, 5 and 20 μmol/L）. Flow cytometry revealed that NO-Fluvastatin could significantly block HLECs in the G0/G1 phase, resulting in the increased cells in the G0/G1 phase and decreased in the S phase （P〈0.05）. RT-PCR showed that NO-Fluvastatin could obviously inhibit the CyclinE mRNA expression and induce the P21waf1 mRNA expression as compared with the negative control groups （P〈0.05）. This experiment suggested that NO-Fluvastatin could suppress the proliferation of HLECs by regulating cell cycle regulatory proteins （inhibiting the expression of CyclinE mRNA and inducing the expression of P21waf1 mRNA）, resulting in the arrest of HLECs in the G0/G1 phase, which can offer theory basis for NO-Fluvastatin in treating posterior capsular opacification in clinic practice.

Knockdown of lncRNA XIST prevents the epithelial-mesenchymal transition of TGF-β2-induced human lens epithelial cells via miR-124/Slug axis

Posterior capsular opacification(PCO)is linked to the pathological process of lens epithelial cells,which includes proliferation,migration,and epithelial-mesenchymal transition(EMT).Our goal was to investigate whether long noncoding RNA(lncRNA)XIST contributes to EMT via targeting miR-124/Slug axis in TGF-β2-induced SRA01/04 cells.EMT was induced by stimulating SRA01/04 cells with 10 ng/mL TGF-β2 for 24 h,and PCO microenvironment was simulated.The expressions levels of lncRNA XIST,miR-124,and Slug were measured by realtime polymerase chain reaction(RT-PCR)and western blot.The role and mechanism of lncRNA XIST in promoting EMT of TGF-β2-treated SRA01/04 cells were investigated by using cell transfection,cell counting kit-8(CCK-8),immunofluorescence staining,transwell assay,wound-healing assay,RT-PCR,western blot and dual-luciferase reporter assay.The expression of Slug and lncRNA XIST was markedly increased,while miR-124 was downregulated in TGF-β2-treated SRA01/04 cells compared with the control group.Knockdown of lncRNA XIST reduced EMT,migration,and cell viability in TGF-β2-induced SRA01/04 cells;moreover,it adversely modulated miR-124 and adjusted the expression of Slug in SRA01/04 cells,while these effects were diminished by co-transfection with AMOmiR-124.Our data demonstrated that lncRNA XIST functioned as a competitive endogenous RNA(ceRNA)of miR-124 to modulate the expression level of Slug,thereby modulating EMT,migration,and cell viability in SRA01/04 cells.In conclusion,lncRNA XIST has a crucial role in promoting TGF-β2-induced EMT via modulating the miR-124/Slug axis in SRA01/04 cells,and it may provide a novel therapeutic option for PCO treatment.

Study of cultured bovine capsular bag in pure ocular tissue

The proliferation, differentiation and fibrosis of lens epithelia cells （LECs） is mainly responsible for posterior capsular opacification （PCO）. From the primary culture of LECs to the culture of lens capsular bag, the models of posterior capsular opacification have been developed. At present, the most commonly used model is cell culture in medium with serum. But the culture in pure ocular tissue has not been reported. Therefore, we established a new model of posterior capsular opacification-culturing bovine lens capsular bag in pure ocular tissue to exclude the role of serum. Our study established a new culture method to investigate the proliferation, differentiation and apoptosis of lens epithelia cells in the aqueous humor with or without lens cortex and vitreous humor. The purpose of the study is to model posterior capsular opacification in vivo as closely as possible and to discuss the influence of ocular tissue on posterior capsular opacification.

Curcumin inhibits proliferation of human lens epithelial cells:a proteomic analysis

Objective: The incidence of after-cataracts [also known as posterior capsular opacification (PCO)] is between 30% and 50% three years following cataract surgery. Suppressing the proliferation of lens epithelial cells (LECs) is a primary goal in preventing PCO. Here, we investigated the proteomic regulation of the inhibitory effects of curcumin (Cur) on the proliferation of human lens epithelial B3 (HLE-B3) cells. Methods: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 20 mg/L Cur in a CO2 incubator for 24 h. Results: We found that the absorbance (A) value of rhbFGF group was significantly higher than the A value of the control group. Furthermore, the A value of the Cur group was significantly lower com- pared to the rhbFGF group, with an inhibition of 53.7%. Five different protein spots were obtained from proliferative HLE-B3 cells induced by rhbFGF. Eight different protein spots were obtained in HLE-B3 cells incubated with Cur. There were the common variational protein spots at mass/charge (m/z) ratios of 8 093 and 13 767 between rhbFGF group and control group as well as between the Cur group and rhbFGF group. Conclusions: These results show that Cur effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. The protein spots at m/z of 8 093 and 13 767 may be the targets of Cur-induced inhibition of HLE-B3 cell proliferation. Cur may be a reliable and effective drug for pre- vention and treatment of polymerase chain reaction (PCR).

Evaluation of long-term visual performance following AcrySof ReSTOR lens implantation

Background Multifocal lens has become popular in cataract surgery. Short-term outcome after AcrySof ReSTOR Lens implantation had been reported by many studies, but long-term visual performance and the effect of posterior capsular opacification （PCO） on visual performance need further investigation. Methods This retrospective study involved 54 eyes from 41 cataract patients implanted with ReSTOR lens, with a follow-up period of 12 to 31 months. Manifest refraction spherical equivalence （MRSE）, monocular uncorrected and best-corrected distance visual acuity, uncorrected and distance-corrected near and intermediate visual acuity, contrast sensitivity were assessed. The effect of PCO on visual performance was evaluated by comparing visual parameters between pre and post-capsulotomy. Results Uncorrected distance visual acuity of eyes with MRSE within ±0.5 diopter （D） was better than those with MRSE greater than ±0.5 D （P 〈0.05）. Uncorrected distance and near visual acuity （LogMAR） was 0.10 and 0.17 respectively. Best corrected distance visual acuity and best distance-corrected near visual acuity （LogMAR） was 0.00 and 0.16, a significant improvement was noted after correction （P=0.000, P=-0.001, respectively）. Contrast sensitivity logarithm was comparable with the normal value at difference spatial frequencies except at 12 cpd. In 5 eyes with mild PCO, post-capsulotomy visual parameters were better than pre-capsulotomy （P 〈0.05）. Conclusion ReSTOR lens provides a good long-term distance and near vision, functional intermediate vision and contrast sensitivity. Mild PCO significantly affects visual performance and needs early intervention.