Phosphoinositide 3-kinase as a therapeutic target in angiogenic disease.

Phosphoinositide 3-kinases (PI3Ks) generate lipids that control multitudinous intracellular cell signaling events which participate in cell survival and proliferation. In addition, PI3K signaling also contributes to metabolism, immunity, angiogenesis and cardiovascular homeostasis, and many diseases. The diverse actions of PI3K stem from the existence of their various isoforms and a variety of protein effectors. Hence, PI3K isoform-specific inhibitors have already achieved a wonderful effect on treating cancer. Herein, we summarize the molecular mechanism of PI3K inhibitors in preventing the permeability of vessels and neovascularization. Additionally, we briefly illustrate how PI3K signaling modulates blood vessel growth and discuss the different roles that PI3K isoforms play in angiogenesis.

Normal vitreous promotes angiogenesis via activation of Axl.

Vitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.

Normal vitreous promotes angiogenesi via the epidermal growth factor receptor.

Vitreous, a transparent tissue in our body, contains anti-angiogenesis factors. Our previous work reported that vitreous activates the signaling pathway of epidermal growth factor receptor (EGFR), which plays a critical role in angiogenesis. The aim of this study was to determine the role of EGFR in vitreous-induced angiogenesis-related cellular responses in vitro. Using a pharmacologic and molecular approach, we found that vitreous increased proliferation and migration via EGFR in human umbilical vein endothelial cells (HUVECs). Furthermore, we demonstrated that vitreous promoted tube formation via EGFR in HUVECs. Subsequently, depletion of EGFR using CRISPR/Cas9 and blockage with EGFR inhibitor AG1478 suppressed vitreous-induced Akt activation and cell proliferation, migration, and tube formation in HUVECs. The significance of the angiogenic effect derived from vitreous demonstrates the importance of vitreous in the ocular physiology and the pathobiology of angiogenesis-related ophthalmic diseases, such as proliferative diabetic retinopathy.

Aberrant DNA methylation of mTOR pathway genes promotes inflammatory activation of immune cells in diabetic kidney disease.

DNA methylation has been implicated in the pathogenesis of diabetic kidney disease (DKD), but the underlying mechanisms remain unclear. In this study, we tested the hypothesis that aberrant DNA methylation in peripheral immune cells contributes to DKD progression. We showed that levels of DNA methyltransferase 1 (DNMT1), a key enzyme for DNA methylation, were increased along with inflammatory activity of peripheral blood mononuclear cells in DKD patients. Inhibition of DNMT1 with 5-aza-2'-deoxycytidine (5-Aza) markedly increased the proportion of CD4+CD25+ regulatory T cells in peripheral blood mononuclear cells in culture and in diabetic animals. Adoptive transfer of immune cells from 5-Aza-treated animals showed beneficial effects on the host immune system, resulting in a significant improvement of DKD. Using genome-wide DNA methylation assays, we identified the differentially methylated cytosines in the promoter regions of mammalian target of rapamycin (mTOR) regulators in peripheral blood mononuclear cells of diabetic patients. Further, mRNA arrays confirmed the consistent induction of genes expressed in the mTOR pathway. Importantly, down-regulation of DNMT1 expression via RNA interference resulted in prominent cytosine demethylation of mTOR negative regulators and subsequent decrease of mTOR activity. Lastly, modulation of mTOR resulted in changes in the effect of 5-aza on diabetic immune cells. Thus, up-regulation of DNMT1 in diabetic immune cells induces aberrant cytosine methylation of the upstream regulators of mTOR, leading to pathogenic activation of the mTOR pathway and consequent inflammation in diabetic kidneys. Hence, this study highlights therapeutic potential of targeting epigenetic events in immune system for treating DKD.

Identifying circRNA-associated-ceRNA networks in retinal neovascularization in mice.

Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.

Application of CRISPR-Cas9 in eye disease.

The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease (Cas)9 is an effective instrument for revising the genome with great accuracy. This system has been widely employed to generate mutants in genomes from plants to human cells. Rapid improvements in Cas9 specificity in eukaryotic cells have opened great potential for the use of this technology as a therapeutic. Herein, we summarize the recent advancements of CRISPR-Cas9 use in research on human cells and animal models, and outline a basic and clinical pipeline for CRISPR-Cas9-based treatments of genetic eye diseases.

Subretinal Injection: A Review on the Novel Route of Therapeutic Delivery for Vitreoretinal Diseases.

Compared to intravitreal injection, subretinal injection has more direct effects on the targeting cells in the subretinal space, which provides a new therapeutic method for vitreoretinal diseases, especially when gene therapy and/or cell therapy is involved. To date, subretinal delivery has been widely applied by scientists and clinicians as a more precise and efficient route of ocular drug delivery for gene therapies and cell therapies including stem cells in many degenerative vitreoretinal diseases, such as retinitis pigmentosa, age-related macular degeneration, and Leber's congenital amaurosis. However, clinicians should be aware of adverse events and possible complications when performing subretinal delivery. In the present review, the subretinal injection used in vitreoretinal diseases for basic research and clinical trials is summarized and described. Different methods of subretinal delivery, as well as its benefits and challenges, are also briefly introduced.

Mx1 of black carp functions importantly in the antiviral innate immune response.

Mx (myxovirus resistance) is an important antiviral protein in the innate immune responses of vertebrates to microbial pathogens. In this study, we cloned and characterized Mx1 of black carp (Mylopharyngodon piceus). The full-length cDNA of black carp Mx1 (bcMx1) consists of 2781 nucleotides and the predicted bcMx1 protein contains 631 amino acids. bcMx1 contains a GTPase domain at the N-terminnus, a "central interactive domain" in the middle and a GTPase effector domain at the C-terminus. bcMx1 mRNA was constitutively transcribed in all tissues tested, including the heart, liver, spleen, kidney, intestine, muscle, skin and gill; and bcMx1 mRNA levels increased in all but the gill after grass carp reovirus (GCRV) or viraemia of carp virus (SVCV) infection. Quantitative PCR analysis of Mylopharyngodon piceus fin (MPF) cells indicated that bcMx1 mRNA levels increased after GCRV or SVCV infection at different multiplicities of infection (MOI). Western blotting demonstrated that the molecular weight of bcMx1 is ∼75 kDa and immunofluorescent staining data of both HeLa cells and EPC cells showed that bcMx1 is a cytosolic protein. EPC cells transfected with plasmid expressing bcMx1 showed increased antiviral activity against SVCV and GCRV. All our data suggest that bcMx1 is an antiviral protein in the innate immune response of the black carp.

9-cis-retinoic acid improves sensitivity to platelet-derived growth factor-BB via RXRα and SHP-1 in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. But few efficient therapeutic methods have been reported. This study discussed the functions of 9-cis-retinoic acid (9-cis-RA) in sensitizing retinal pericytes to platelet-derived growth factor (PDGF)-BB. Using streptozotocin (STZ)-induced diabetic mice and high glucose-treated bovine retinal pericytes (BRPC), we analyzed the impacts of 9-cis-RA by detecting cell apoptosis via DNA fragmentation assay and detecting related factors through adenovirus or lentivirus infection and western blot. Results showed that in retinas of STZ-induced diabetic mice, 9-cis-RA significantly inhibited expression of SHP-1 (P < 0.01), thus promoting p-AKT and p-ERK1/2, which reflected the improved sensitivity to PDGF-BB. In BRPC, 9-cis-RA also improved sensitivity to PDGF-BB and suppressed cell apoptosis (P < 0.01) via down-regulating SHP-1. Further mechanism analyses showed that the efficient functioning of 9-cis-RA relied on the existence of its receptor, retinoic X receptor α (RXRα), independent of the previous reported protein kinase C delta (PKCδ)/SHP-1 axis. Because 9-cis-RA could not inhibit SHP-1 or improve sensitivity to PDGF-BB when RXRα was knocked down, while it still suppressed SHP-1 after overexpression of PKCδ. Taken together, these results indicated the vital roles of 9-cis-RA in improving sensitivity to PDGF-BB of retinal pericytes in DR, and provided basic evidences of new therapeutic targets like RXRα for further DR treatment.

[Application of Icare rebound tonometer in children after congenital cataract surgery].

OBJECTIVE: To compare the difference in intraocular pressure (IOP) readings as well as the tolerability between Icare rebound tonometer (Icare RBT) and Goldmann applanation tonometer (GAT), and to evaluate the application of Icare RBT in monitoring the intraocular pressure in children after congenital cataract surgery. METHODS: The IOP was measured with the Icare RBT and GAT respectively in 150 children (262 eyes) after congenital cataract surgery by two experienced ophthalmologists. Correlation and Bland-Altman analysis were used to assess the agreement in IOP readings between the two instruments. The influence of the central corneal thickness (CCT) adjusted for age on IOP readings was analyzed by linear regression analysis. The tolerance of the patients to Icare RBT and GAT measurement were surveyed. RESULTS: The mean age was (44.82 ± 11.56) months in 150 children, including 81 boys and 69 girls. The mean IOP readings by the Icare RBT and GAT were (16.08 ± 5.72) mmHg and (14.17 ± 5.05) mmHg, respectively. The mean difference between the Icare RBT and GAT was (1.91 ± 2.04) mmHg, which was significantly correlated with CCT (r=0.409, P<0.001). The IOP readings by Icare RBT was significantly correlated with that measured by GAT(r= 0.936, P<0.001). The 95% confidence interval of the difference between the two instruments was ?2.10 to 5.91 mmHg. The Icare RBT examination was well tolerated by the children compared to the GAT examination. CONCLUSION: The Icare RBT is easy to use and well tolerated by the children after congenital cataract surgery. Compared to GAT, the value measured by the IOPs trends to be overestimated. The difference in readings between the 2 tonometers will magnify with the increase in CCT.

Safety and efficacy of conbercept in neovascular age-related macular degeneration: results from a 12-month randomized phase 2 study: AURORA study.

PURPOSE: To assess the safety and efficacy of multiple injections of 0.5 and 2.0 mg conbercept using variable dosing regimens in patients with neovascular age-related macular degeneration (AMD). DESIGN: Randomized, double-masked, multicenter, controlled-dose, and interval-ranging phase 2 clinical trial divided into a 3-month loading phase followed by a maintenance phase. PARTICIPANTS: Patients with choroidal neovascularization secondary to AMD with lesion sizes of 12 disc areas or less and a best-corrected visual acuity (BCVA) letter score of between 73 and 24 were enrolled. METHODS: Patients were randomized 1:1 to receive either 0.5 or 2.0 mg intravitreal conbercept for 3 consecutive monthly does. After the third dose, each group was reassigned randomly again to monthly (Q1M group) or as-needed (pro re nata [PRN] group) treatment without changing the drug assignment. MAIN OUTCOME MEASURES: The primary end point was the mean change in BCVA from baseline to month 3, with secondary end points being the mean change in BCVA, mean change in central retinal thickness (CRT), and safety at month 12. RESULTS: We enrolled 122 patients. At the primary end point at month 3, mean improvements in BCVA from baseline in the 0.5- and 2.0-mg groups were 8.97 and 10.43 letters, respectively. At month 12, mean improvements in BCVA from baseline were 14.31, 9.31, 12.42, and 15.43 letters for the 0.5-mg PRN, 0.5-mg Q1M, 2.0-mg PRN, and 2.0-mg Q1M regimens, respectively. At month 12, mean reductions in CRT in the 4 regimens were 119.8, 129.7, 152.1, and 170.8 µm, respectively. There were no significant differences for the pairwise comparisons between all study groups. The difference in the number of injections between the 2 PRN groups was not statistically significant. Treatment with conbercept generally was safe and well tolerated. CONCLUSIONS: The significant gains in BCVA at 3 months were the same or better at 12 months in all conbercept dosing groups of neovascular AMD patients. During the 12 months, repeated intravitreal injections of conbercept were well tolerated in these patients. Future clinical trials are required to confirm its long-term efficacy and safety.

The effects of alpha lipoic acid in preventing oxidative stress-induced retinal pigment epithelial cell injury.

This study evaluated the protective effect of alpha lipoic acid (ALA) in human adult retinal pigment epithelium cells (ARPE-19). An RPE oxidative stress model was established in ARPE-19 cells. Cell apoptosis was detected by Annexin V/PI staining and Hoechst33342 staining. Autophagy activation was evaluated by formation of acidic vesicular organelles (AVO) as well as protein expression of Atg5 and LC3. Akt phosphorylation and Bax protein expression were measured using western blot. Exogenous H2O2 significantly increased intracellular ROS concentration and decreased the viability of ARPE-19 cells in a dose-dependent way. H2O2 (12.5 µmol/L) induced early stage apoptosis, significantly decreased Akt phosphorylation, and increased Bax protein level 4 h after stimulation. H2O2 (12.5 µmol/L) significantly increased AVO formation, mRNA and protein levels of Atg-5, and protein expression of LC3 I and II. Treatment of ARPE-19 cells with 37.5 µmol/L ALA significantly blocked increased intracellular ROS level, apoptosis, AVO formation, as well as elevation of Atg5, LC3 I, and II protein levels induced by 12.5 µmol/L H2O2. Exogenous H2O2 induces apoptosis and activation of autophagy in human adult retinal pigment epithelium cells through Akt-Bax signaling. ALA is effective in protecting RPE cells from H2O2-induced cell death.

[Research progress of anti-angiogenesis drug targets in diabetic retinopathy].

Diabetic retinopathy is one of the common ocular diseases. One of the pathological characteristic of proliferative diabetic retinopathy is retinal neovascularization which caused visual impairment in diabetic patients. With the anti-VEGF drugs applying in the clinic, it has become one of the main medical treatment of diabetic retinopathy. But it has its limitation. Researchers continue to explore new drug targeting for anti-angiogenesis and try combination therapy or customized treatment. There are many drug targets for anti-angiogenesis, such as promoting angiogenesis factor, integrins and matrix proteinases. Some drugs act on the typical tip cells which is much important in the process of angiogenesis differentiation and proliferation. Overall, these studies opened our view of anti-angiogenesis. The purpose of this article is to summarize the possible drug targets of angiogenesis and the key issues in the study of anti-angiogenic drugs.

[Research progress of vascular endothelial tip cells in diabetic retinopathy].

Tip cells are one kind of vascular endothelial cells. Migration and sprouting of tip cells is the first step of angiogenesis. In recent years, researchers found a large number of tip endothelial cells in new blood vessels of diabetic retinopathy patients and animal models. Vascular endothelial growth factor (VEGF) regulates the migration and budding of tip cells and dissolves the extracellular matrix. Pericyte recruitment guided by the VEGF-DLL-Notch signal pathway is necessary for neovascularization. The change of miRNA in tip cells is also involved in the regulation of angiogenesis. This paper focuses on the mechanism of endothelial tip cells in diabetic retinopathy and prospects for the future research of anti-angiogenesis drug targets.

Efficacy and Safety of Biosimilar QL1207 vs. the Reference Aflibercept for Patients with Neovascular Age-Related Macular Degeneration: A Randomized Phase 3 Trial.

INTRODUCTION: This trial aimed to compare the efficacy and safety between biosimilar QL1207 and the reference aflibercept for the treatment of neovascular age-related macular degeneration (nAMD). METHODS: This randomized, double-blind, phase 3 trial was conducted at 35 centers in China. Patients aged ≥ 50 years old with untreated subfoveal choroidal neovascularization secondary to nAMD and best-corrected visual acuity (BCVA) letter score of 73-34 were eligible. Patients were randomly assigned to receive intravitreous injections of QL1207 or aflibercept 2 mg (0.05 ml) in the study eye every 4 weeks for the first 3 months, followed by 2 mg every 8 weeks until week 48, stratified by baseline BCVA ≥ or < 45 letters. The primary endpoint was BCVA change from baseline at week 12. The equivalence margin was ± 5 letters. The safety, immunogenicity, pharmacokinetics (PK), and plasma vascular endothelial growth factor (VEGF) concentration were also evaluated. RESULTS: A total of 366 patients were enrolled (QL1207 group, n = 185; aflibercept group, n = 181) from Aug 2019 to Jan 2022 with comparable baseline characteristics. The least-squares mean difference in BCVA changes was - 1.1 letters (95% confidence interval - 3.0 to 0.7; P = 0.2275) between the two groups, within the equivalence margin. The incidences of treatment-emergent adverse events (TEAE; QL1207: 71.4% [132/185] vs. aflibercept: 71.8% [130/181]) and serious TEAE (QL1207: 14.1% [26] vs. aflibercept: 12.7% [23]) appeared comparable between treatment groups, and no new safety signal was found. Anti-drug antibody, PK profiles, and VEGF concentration were similar between the two groups. CONCLUSIONS: QL1207 has equivalent efficacy to aflibercept for nAMD with similar safety profiles. It could be used as an alternative anti-VEGF agent for clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05345236 (retrospectively registered on April 25, 2022); National Medical Products Administration of China: CTR20190937 (May 20, 2019).

A case of uveitis in adult-onset Still's disease with ophthalmologic symptoms.

Adult-onset Still's disease (AOSD) is a rare and systemic inflammatory disorder of unknown etiology and pathogenesis. AOSD is characterized by high fever accompanied by a range of systemic symptoms. However, there are rare cases of AOSD with ophthalmologic symptoms as well as with an obvious causation of corticosteroid withdrawal. In this case, a 43-year-old male patient diagnosed with AOSD showed ocular inflammation after withdrawing from corticosteroid treatment. This patient was treated with prednisolone for AOSD and discharged after achieving complete remission of breathlessness, backache, thoracalgia, joint pain, and spiking fever. The patient unauthorizedly stopped taking prednisolone after he was discharged from the hospital and returned to the Department of Ophthalmology with the complaint of decreased visual acuity in both eyes for half a month and sudden vision loss in the left eye for 3 days. After regular ophthalmologic examinations and fluorescence angiography examination, he was diagnosed with acute panuveitis as the manifestation of AOSD. Uveitis was effectively treated with corticosteroid drugs. This case reported a rare manifestation of AOSD in an ophthalmological system that was associated with the withdrawal of corticosteroid treatment. This report highlighted the therapeutic effect of local and systemic corticosteroid use for AOSD manifested with uveitis. This case is interesting for both rheumatologists and ophthalmologists.

Attenuation of Microglial Activation and Pyroptosis by Inhibition of P2X7 Pathway Promotes Photoreceptor Survival in Experimental Retinal Detachment.

Purpose: Photoreceptor (PR) death is the ultimate cause of irreversible vision loss in retinal detachment (RD). Although microglial infiltration in the subretinal space (SRS) was observed after RD, the molecular mechanism underlying microglial activation and the outcomes of infiltrating microglia remain unclear. We aimed to uncover the mechanism of initiation of microglial activation to help explore potential therapy to promote PR survival. Methods: An RD model was conducted by injecting sodium hyaluronate into SRS of C57BL/6J wild type mice. Adenosine triphosphate (ATP) was measured by a ATP Microplate Assay Kit. Bioinformatics analysis was used to evaluate the upregulated receptor relating to ATP binding in human datasets and mouse transcriptomes of RD. Expression of P2X7, its downstream signaling pathways, and microglial pyroptosis were confirmed by qPCR, WB, and immunofluorescence in vivo and in vitro. The cell viability of PR was measured by cell counting kit-8. Brilliant Blue G, a P2X7 antagonist, was subretinally or intraperitoneally injected to inhibit microglial activation in vivo and was applied for microglia cell line treatment in vitro. The decrease in microglial activation and pyroptosis was detected by immunofluorescence and WB. The protective effect on PR was measured by hematoxylin and eosin staining, TUNEL assay, and electroretinogram analysis. Results: The results showed that extracellular ATP released in the SRS after RD triggered P2X7 activation and attracted microglia. The downstream cascade of inflammasome activation induced by P2X7 activation contributed to microglial pyroptosis and then to PR death. ATP-activated microglia led to PR death in vitro. P2X7 blockade rescued PR morphologically and functionally by inhibiting microglial activation and pyroptosis. Conclusions: These results elucidate that ATP-induced P2X7-mediated microglial activation leads to microglial pyroptosis, contributing to PR death. Appropriate inhibition of microglial pyroptosis might serve as a pharmacotherapeutic strategy for decreasing PR death in RD.

[Comparison of growth of human fetal RPE cells on electrospun nanofibers and etched pore polyester membranes].

OBJECTIVE: To investigate and compare the growth of human fetal retinal pigment epithelial (RPE) cells seeded onto electrospun polyamide nanofibers (EPN) or etched pore polyester (EPP), and, further, to explore their possible use as prosthetic Bruch's membrane. METHODS: Human fetal RPE cells were planted onto the EPN, EPP and plastic (control) substrates in Transwells. The cultures were assessed with respect to cell attachment at 2, 4, 8 hours and proliferation at 1, 4, 8 days after seeding. Growth and morphology of the cells were monitored under the phase contrast microscope, and the phenotype was identified by immunofluorescence staining with antibodies against tight junction protein ZO-1. Strips of single EPP coated with nothing or EPP coated with EPN was differently implanted into the subretinal space of two P21 RCS rats for two weeks and the histologic slides of the retina were assessed. RESULTS: Cultured human fetal RPE cells were attached to either EPN or EPP substrates (with seeding on plastic substrate as control). After 8 h, the numbers of adherent cells in the EPN, EPP and control groups were 1.23*10(5)/cm(2), 1.70*10(5)/cm(2), and 1.64*10(5)/cm(2), respectively. The number of RPE cells attached to EPN was obviously less than that to both EPP and control (P<0.05). On the first day, the proliferation of cells on EPN was less than that of EPP and control (P<0.05); but by the 8th day in culture, the proliferation of cells on EPN had increased and was higher than proliferation on both EPP and control (P<0.05). All of the RPE cells cultured on EPN and EPP substrates were in monolayer, and the EPN-attached cells resembled the inner collagenous layer of Bruch's membrane. Immunofluorescence staining showed that the RPE cells cultured on EPN and EPP substrates adopted a higher expression of ZO-1 than that on the plastic control substrate. Subretinal implantation of either EPP alone or EPP as a carrier for free EPN for 2 weeks in P21RCS rats resulted in an expected encapsulation and loss of photoreceptor layer. No toxicity or other adverse reaction was observed in the vicinity of the transplant. CONCLUSION: EPN and EPP could maintain human fetal RPE cell attachment and proliferation. Both EPN and EPP appeared to be grossly tolerance and biocompatible with subretinal implantation. EPN represents an intriguing prospect for prosthetic Bruch's membrane replacement because of its similarity in structure to native Bruch's membrane.

[Ultraviolet radiation-induced apoptosis in human lens epithelial cells and its effect on Bcl-2 and Bax].

OBJECTIVE: To explore the apoptosis-inducing effect of ultraviolet(UV) radiation on human lens epithelial cells (HLEC), with particular focus on changes in Bcl-2 or Bax expression as possible mechanisms. METHODS: All experimental groups were exposed to the same UV light source. HLEC were divided into 6 groups according to duration of UV radiation : 0 min group (control group), 5 min group, 10 min group,15 min group, and 30 min group. Analysis on apoptosis of HLEC was performed by flow cytometry analysis (FCA, Annexin V + PI staining). Changes of Bax and Bcl-2 expression in HLEC were detected by hybridization in situ. RESULTS: Apoptosis in HLEC increased with UV exposure time. The expression level of Bax mRNA was increased with the increase of UV exposure time, whereas the expression level of Bcl-2 mRNA decreased with the increase of UV exposure time. The proportion of apoptotic cells was negatively correlated with ratio of Bcl-2/Bax (r=-0.874, P<0.05). CONCLUSION: UA radiation can induce apoptosis of HLEC in vitro. Bcl-2 and Bax genes may play an important role in regulating this apoptotic process.

[Effect of ultraviolet radiation on ALDH1 expression in human lens epithelial cells].

OBJECTIVE: To determine the apoptosis-inducing effect of ultraviolet light (UV) on human lens epithelial cell (HLEC) and to explore the involvement of changes in ALDH1 folowing UV radiation. METHODS: HLEC was exposed to the same UV light source and was subsequently divided into 6 groups according to UV radiation time of 0 (control group), 5, 10, 15, and 30 min. Apoptosis was detected by AO/EB staining. Changes of ALDH1 in HLEC were detected by immunohistochemical staining and Western blot. RESULTS: The intensity of immunohistochemical staining and the rate of positive cells decreased with increase of UV time (P<0.05). The rate of positive ALDH1 cells was negatively correlated with the rate of apoptosis (r= -0.92, P<0.05). Western blot showed the integrated absorbance values significantly decreased with the increase of UV time (P<0.05). CONCLUSION: ALDH1 in HLEC decreases with an increase of UV exposure, which may be related to UV induced apoptosis of HLEC.