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PURPOSE: To compare anterior capsule contraction (ACC) after cataract surgery with implantation of intraocular lens (IOLs) of different materials and designs. METHODS: We searched three electronic databases for relevant studies published up to January 1, 2023. Five randomized controlled trails (RCTs) and three cohort studies involving 1,221 eyes were included in quantitative synthesis. We extracted data, assessed their quality independently, and calculated standard mean difference (SMD) using a random-effects model. Six RCTs and one retrospective cohort were included in information summary. RESULTS: The contraction of the anterior capsule opening area in the hydrophilic group was larger than that of the hydrophobic group from one month to one year postoperatively (P < 0.001 and P < 0.001, respectively). Specifically, the hydrophilic group showed greater contraction of the anterior capsule opening area at one month postoperatively (Standardized mean difference [SMD] = -0.73, 95% confidence interval [CI] = -0.93 to -0.52), three months (SMD = -1.04, 95% CI = -1.32 to -0.75), six months (SMD = -0.99, 95% CI = -1.24 to -0.74) and one year (SMD = -1.33, 95% CI = -2.50 to -0.16). As of one year postoperatively, the anterior capsular opening area showed a trend of decreasing over time in both groups (P = 0.046 and P = 0.050, respectively). In information summary, three studies indicated no relationship between haptic design and ACC, while the other four studies reported that the number and shape of haptic would affect ACC. CONCLUSION: This meta-analysis suggested that the postoperative ACC after the implantation of hydrophobic IOLs was less than that induced by hydrophilic IOLs. Haptic design may also affect the degree of ACC.
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In this article, we examine the role of erythropoietin-producing hepatocellular receptor A2 (EphA2) in the apoptosis of lens epithelial cells (LECs) in H2O2 and UV radiation-induced cataracts. We treated SRA01/04 cells with H2O2 or ultraviolet (UV) radiation to create a cataract cell model. We constructed a cataract lens model by exposing mice to UV radiation. We used CCK8 assays, Annexin V-FITC analysis, and immunohistochemical staining to explore proliferation and apoptosis of the cataract model. Thereafter, we used quantitative real-time PCR (qPCR) analysis, Western blot assays, and immunofluorescence to determine gene and protein expression levels. We also employed Crispr/Cas9 gene editing to create an EphA2 knockout in SRA01/04 cells. Results: H2O2 or UV radiation induced SRA01/04 cells showed EphA2 gene upregulation. CCK8 and apoptosis assays showed that EphA2 over-expression (OE) reduced epithelial cell apoptosis, but knockout of EphA2 induced it in response to H2O2 and UV radiation, respectively. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on cell apoptosis. In vivo, the EphA2 protein level increased in the lenses of UV-treated mice. Our results showed that EphA2 was upregulated in SRA01/04 cells in response to H2O2 and UV radiation. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on H2O2 and UV radiation-induced cell apoptosis. We confirmed this change with an experiment on UV-treated mice. The present study established a novel association between EphA2 and LEC apoptosis.
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BACKGROUND: Limbal stem/progenitor cells (LSPCs) play a crucial role in maintaining corneal health by regulating epithelial homeostasis. Although PM2.5 is associated with the occurrence of several corneal diseases, its effects on LSPCs are not clearly understood. METHODS: In this study, we explored the correlation between PM2.5 exposure and human limbal epithelial thickness measured by Fourier-domain Optical Coherence Tomography in the ophthalmologic clinic. Long- and short-term PM2.5 exposed-rat models were established to investigate the changes in LSPCs and the associated mechanisms. RESULTS: We found that people living in regions with higher PM2.5 concentrations had thinner limbal epithelium, indicating the loss of LSPCs. In rat models, long-term PM2.5 exposure impairs LSPCs renewal and differentiation, manifesting as corneal epithelial defects and thinner epithelium in the cornea and limbus. However, LSPCs were activated in short-term PM2.5-exposed rat models. RNA sequencing implied that the circadian rhythm in LSPCs was perturbed during PM2.5 exposure. The mRNA level of circadian genes including Per1, Per2, Per3, and Rev-erbα was upregulated in both short- and long-term models, suggesting circadian rhythm was involved in the activation and dysregulation of LSPCs at different stages. PM2.5 also disturbed the limbal microenvironment as evidenced by changes in corneal subbasal nerve fiber density, vascular density and permeability, and immune cell infiltration, which further resulted in the circadian mismatches and dysfunction of LSPCs. CONCLUSION: This study systematically demonstrates that PM2.5 impairs LSPCs and their microenvironment. Moreover, we show that circadian misalignment of LSPCs may be a new mechanism by which PM2.5 induces corneal diseases. Therapeutic options that target circadian rhythm may be viable options for improving LSPC functions and alleviating various PM2.5-associated corneal diseases.
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Doenças da Córnea , Células-Tronco , Humanos , Ratos , Animais , Córnea , Homeostase , Material Particulado/toxicidade , Células EpiteliaisRESUMO
Lens itself has limited regeneration functionality, thus we aimed to create regenerated lens with biological function to treat cataracts rather than the intraocular lens used in cataract surgery. We induced exogenous human embryonic stem cells to directionally differentiate into lens fate like cells in vitro, mixed these cells with hyaluronate, and then implanted the mixture into lens capsule to regenerate in vivo. We successfully achieved near-complete lens regeneration, and the thickness of the regenerated lens reached 85% of the contralateral eye, showing the characteristics of biconvex shape, transparency, and a thickness and diopter close to that of natural lenses. Meanwhile, the participation of Wnt/PCP pathway in lens regeneration was verified. The regenerated lens in this study was the most transparent, thickest, and most similar to the original natural lens that has thus far been reported. Overall, these findings offer a new therapeutic strategy for cataracts and other lens diseases.
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Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. With aging and the accumulated effects of environmental stress, retinal pigment epithelial (RPE) cells are particularly susceptible to oxidative damage, which can lead to retinal degeneration. However, the underlying molecular mechanisms of how RPE responds and progresses under oxidative damage are still largely unknown. Here, we reveal that exogenous oxidative stress led to ferroptosis characterized by Fe2+ accumulation and lipid peroxidation in RPE cells. Glutathione specific gamma-glutamylcyclotransferase 1 (Chac1), as a component of the unfolded protein response (UPR) pathway, plays a pivotal role in oxidative-stress-induced cell ferroptosis via the regulation of glutathione depletion. These results indicate the biological significance of Chac1 as a novel contributor of oxidative-stress-induced ferroptosis in RPE, suggesting its potential role in AMD.
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Ferroptose , Degeneração Macular , Estresse Oxidativo , Epitélio Pigmentado da Retina , Idoso , Humanos , Células Epiteliais/metabolismo , Ferroptose/genética , Ferroptose/fisiologia , Glutationa/metabolismo , Degeneração Macular/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismoRESUMO
PURPOSE: The aim of this study was to investigate the expression profiles of long noncoding RNAs (lncRNAs) in human corneal epithelial cells (HCECs) exposed to fine particulate matter (PM2.5) and to identify potential biological pathways involved in PM2.5-induced toxicity in HCECs. METHODS: Using RNA sequencing (RNA-seq) and hierarchy clustering analysis, lncRNA expression profiles in PM2.5-treated and untreated HCECs were examined. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to predict the role of altered lncRNAs in biological processes and pathways. A quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was conducted to verify the RNA-seq results in HCECs and human corneal epithelial cell sheets. RESULTS: In total, 65 lncRNAs were altered in the PM2.5-treated HCECs, including 41 upregulated and 24 downregulated lncRNAs. The results of the qRT-PCR assay were consistent with those of the RNA-seq analysis. The expression of two significantly upregulated lncRNAs was confirmed in human corneal epithelial cell sheets. The GO analysis demonstrated that altered lncRNAs in the PM2.5-treated HCECs were significantly enriched in three domains: cellular component, molecular function, and biological process. The KEGG pathway analysis revealed enriched pathways of lncRNA co-expressed mRNAs, including cancer, RNA transport, and Rap1 signaling. CONCLUSIONS: Our results suggest that lncRNAs are involved in the pathogenesis of PM2.5-induced ocular diseases, exerting their effects through biological processes and pathogenic pathways. Among the altered lncRNAs, RP3-406P24.3 and RP11-285E9.5 may play significant roles in PM2.5-induced ocular surface injury.
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RNA Longo não Codificante , Células Epiteliais , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Material Particulado/toxicidade , RNA Longo não Codificante/genética , RNA MensageiroRESUMO
Corneal damage forms scar tissue and manifests as permanent corneal opacity, which is the main cause of visual impairment caused by corneal diseases. To treat these diseases, herein, we developed a novel approach based on the exosome derived from induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) combined with a thermosensitive hydrogel, which reduces scar formation and accelerates the healing process. We found that a thermosensitive chitosan-based hydrogels (CHI hydrogel) sustained-release iPSC-MSC exosomes can effectively promote the repair of damaged corneal epithelium and stromal layer, downregulating mRNA expression coding for the three most enriched collagens (collagen type I alpha 1, collagen type V alpha 1 and collagen type V alpha 2) in corneal stroma and reducing scar formation in vivo. Furthermore, iPSC-MSCs secrete exosomes that contain miR-432-5p, which suppresses translocation-associated membrane protein 2 (TRAM2), a vital modulator of the collagen biosynthesis in the corneal stromal stem cells to avert the deposition of extracellular matrix (ECM). Our findings indicate that iPSC-MSCs secrete miRNA-containing exosomes to promote corneal epithelium and stroma regeneration, and that miR-432-5p can prevent ECM deposition via a mechanism most probably linked to direct repression of its target gene TRAM2. Overall, our exosomes-based thermosensitive CHI hydrogel, is a promising technology for clinical therapy of various corneal diseases.
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Epitélio Corneano , Exossomos , Células-Tronco Mesenquimais , Cicatriz/metabolismo , Substância Própria , Exossomos/metabolismo , Humanos , Hidrogéis/farmacologia , Glicoproteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , RegeneraçãoRESUMO
Congenital cataracts are the leading cause of childhood blindness. To date, surgical removal of cataracts is the only established treatment, but surgery is associated with multiple complications, which often lead to visual impairment. Therefore, mechanistic studies and drug-candidate screening have been intrigued by the aims of developing novel therapeutic strategies. However, these studies have been hampered by a lack of an appropriate human-disease model of congenital cataracts. Herein, we report the establishment of a human congenital cataract in vitro model through differentiation of patient-specific induced pluripotent stem cells (iPSCs) into regenerated lenses. The regenerated lenses derived from patient-specific iPSCs with known causative mutations of congenital cataracts (CRYBB2 [p. P24T] and CRYGD [p. Q155X]) showed obvious opacification that closely resembled that seen in patients' cataracts in terms of opacification severity and disease course accordingly, as compared with lentoid bodies (LBs) derived from healthy individuals. Increased protein aggregation and decreased protein solubility corresponding to the patients' cataract severity were observed in the patient-specific LBs and were attenuated by lanosterol treatment. Taken together, the in vitro model described herein, which recapitulates patient-specific clinical manifestations of congenital cataracts and protein aggregation in patient-specific LBs, provides a robust system for research on the pathological mechanisms of cataracts and screening of drug candidates for cataract treatment.
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BACKGROUND: To investigate the effects of small incision lenticule extraction (SMILE)-derived decellularized lenticules on intraocular pressure (IOP) and conjunctival scarring in a rabbit model of glaucoma filtration surgery. METHODS: Trabeculectomy was performed on both eyes of New Zealand rabbits. A decellularized lenticule was placed in the subconjunctival space in one eye of the rabbits (the decellularized lenticule group), and no adjunctive treatment was performed in the fellow eye (the control group). The filtering bleb features and IOP were evaluated 0, 3, 7, 14, 21, and 28 days after surgery, and histopathologic examination was performed 28 days after surgery. RESULTS: Decellularized lenticules significantly increased bleb survival and decreased IOP postoperatively in the rabbit model with no adverse side effects. The histopathologic results showed a larger subconjunctival space and less subconjunctival fibrosis in the decellularized lenticule group. CONCLUSIONS: Decellularized lenticules can prevent postoperative conjunctiva-sclera adhesion and fibrosis, and they may represent a novel antifibrotic agent for trabeculectomy.
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Cirurgia Filtrante , Glaucoma , Trabeculectomia , Animais , Túnica Conjuntiva/cirurgia , Modelos Animais de Doenças , Glaucoma/cirurgia , Pressão Intraocular , CoelhosRESUMO
Cataracts, the leading cause of vision impairment worldwide, arise from abnormal aggregation of lens proteins. According to the World Health Organization, cataracts cause more than 40% of blindness cases. As the population ages, the prevalence of cataracts will increase rapidly. Although cataract surgery is regarded as effective, it still suffers from complications and high cost, and could not meet the increasingly surgery demand. Therefore, pharmacological treatment for cataracts is a cheaper and more readily available option for patients, which is also a hot topic for years. Anti-cataract drug screening was previously mainly based on the specific pathogenic factors: oxidative stress, excess of quinoid substances, and aldose reductase (AR) activation. And several anti-cataract drugs have been applied in the clinic, while the effect is still unsatisfied. Makley and Zhao recently identified two kinds of novel pharmacological substances (25-hydroxycholesterol, lanosterol) that can reverse lens opacity by dissolving the aggregation of crystallin proteins, indicating that protein aggregation is not an endpoint and could be reversed with specific small-molecule drugs, significantly boosting the development of the cataract pharmacopeia and being regarded as a new dawn for cataract treatment. Our team built a novel optimized platform and had screened several potential therapeutic agents from a collection of lanosterol derivatives. In this review, we would mainly focus on the advancement of cataract pharmacotherapy based on the targets for anti-cataract drugs.
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AIM: Diabetic patients are reported to have a higher incidence of cataract surgery-induced retinal complications, possibly due to retinal inflammation. Our goal is to identify the key inflammatory cytokines, cells and regulatory pathways involved. MAIN METHODS: Diabetes mellitus (DM) induced by streptozotocin and control mice received extracapsular lens extraction (ECLE) in one eye. Neuroretinas were collected at postoperative day1(P1), day2(P2), and day7(P7). BV2 cells were harvested under the treatment of high glucose, lipopolysaccharide (LPS) and inhibitors. The method of qPCR, western blot and immunohistochemistry were used to identify the expression of cytokines and signaling pathways. KEY FINDINGS: ECLE induced increased inflammation in the neuroretina of surgery eye with a peak at P1. MCP-1 surge in long-term diabetes mellitus (LDM) mice at P1 is higher than short-term diabetes mellitus (SDM) mice and normal mice. Significant activation of c-jun and c-fos were found in LDM compared to normal and SDM. Advanced activation of stat1 and ERK was found at P1 in LDM instead of at P2 in SDM and Normal. Activation of microglia/macrophage was also detected in the LDM mice. Besides the inhibition of c-jun/JNK, MCP-1 expression can be attenuated by inhibiting stat1 and ERK under high glucose condition after LPS stimulation. SIGNIFICANCE: Enhancement of lens extraction-induced MCP-1 upregulation and microglia response in long-term diabetes might be due to the activation of cjun, stat1 and ERK, which provided potential therapeutic targets to attenuate retinal inflammation after surgery in diabetic individuals.
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Extração de Catarata , Quimiocina CCL2/metabolismo , Diabetes Mellitus Experimental/genética , Sistema de Sinalização das MAP Quinases , Microglia/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição STAT1/metabolismo , Regulação para Cima/genética , Animais , Glucose/toxicidade , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Retina/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
Indoor pollution with cooking oil fumes (COF) as one of the main components is closely related to ocular surface disorders. However, as the most abundant aldehyde in COF, the toxicity of trans, trans-2,4-decadienal (tt-DDE) on human cornea has not been explored before. In the present study, we observed a time- and dose-dependent cytotoxicity induced by tt-DDE in human corneal epithelial (HCE) cells, as evidenced by decreased cell viability, altered cell morphology, and increased proportion of apoptotic cells. Exposure to tt-DDE also led to an increase in reactive oxygen species (ROS) production, MMP loss, and a decrease in intracellular ATP levels. In addition, after exposure to tt-DDE, the expression of endoplasmic reticulum (ER) stress-related proteins (Bip, pIRE1, XBP1, pPERK, peIF2α, ATF4, and CHOP) increased, indicating that ER stress was activated. Moreover, pretreatment of HCE cells with two ER stress inhibitors (200 nM ISRIB or 1 mM 4-PBA) effectively attenuated oxidative stress induced by tt-DDE. These results suggested that tt-DDE could cause damage to HCE cells by triggering oxidative stress and ER stress. Furthermore, regulation of ER stress can be considered as a potential protective method for tt-DDE-induced ocular surface disorders.
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Poluentes Atmosféricos/toxicidade , Aldeídos/toxicidade , Células Epiteliais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Culinária , Córnea/citologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Óleos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: To explore the molecular mechanisms of PM2.5-induced dysfunction in human corneal epithelial cells (HCECs) and the potential role of the plasminogen activator inhibitor type-2 (PAI-2) in PM2.5-induced autophagy in vitro and in vivo. METHODS: RNA-Seq was performed to identify the differentially expressed genes (DEGs) in PM2.5-exposed HCECs compared to unexposed condition, followed by validation via real-time PCR (qRT-PCR). Corneal fluorescein staining and tear secretion were assessed in the PM2.5-exposed rat model. The expression of PAI-2 and autophagy-related markers were examined via immunoblotting, immunofluorescence staining and/or qRT-PCR in PM2.5-exposed or unexposed HCECs and rat corneas. PAI-2-knockdown HCECs were generated to study PAI-2's role in the PM2.5-induced autophagy in HCECs. RESULTS: A total of 434 DEGs-240 up-regulated and 194 down-regulated-were identified in PM2.5-exposed HCECs rather than unexposed HCECs. The expression of a few genes related to proliferation, inflammation, and aryl hydrocarbon stimulation were significantly altered by PM2.5 exposure. PAI-2 expression was up-regulated in PM2.5-exposed HCECs, sharing a similar fluctuation trend with autophagy-related markers LC3B II and BECN1 according to various exposure periods. Moreover, PAI-2 knockdown significantly suppressed the expression of LC3B and BECN1 in PM2.5-exposed HCECs. The corneal fluorescein staining was enhanced and tear secretion was significantly reduced in PM2.5-exposed rat eyes. PAI-2 expression was also increased in PM2.5-exposed rat corneas, together with the up-regulation of several autophagy-related markers. CONCLUSION: The present study identified the altered expression of hundreds of genes in PM2.5-exposed HCECs, which suggests the importance of PM2.5 for cornea health. The involvement of PAI-2 was discovered in the PM2.5-induced autophagy in HCECs as well as likely in rat corneas, which implied that PAI-2 may become a potential target of clinical treatment of PM2.5-associated ocular surface diseases.
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Transcriptoma , Animais , Autofagia , Córnea , Células Epiteliais , Humanos , Material Particulado/toxicidade , RatosRESUMO
Despite the recent breakthrough in cataract drug development, further improvements have been limited by the lack of human in vitro cataract disease models. This study, therefore, aims to generate a qualified cataract disease model. Mature lentoid bodies (LBs) on Day 25 (D25), which were differentiated from human induced pluripotent stem cells (iPSCs) using the "fried egg" method, were continually culturing (control) or extra treated with either ultraviolet (UV) radiation or hydrogen peroxide (H2 O2 ). The LBs' shape alteration and opacity were examined using light microscopy and mean gray value evaluation. Their structure and crystallin expression were examined using immunofluorescence and transmission electron microscopy (TEM). Real-time polymerase chain reaction and western blot were used to investigate the potential role of autophagy in cloudy LBs. Mature LBs became cloudy with time which was accelerated by H2 O2 . Immunofluorescence examinations and TEM showed that the H2 O2 -treated and control LBs had similar shapes, lens capsule, and monolayer lens epithelial cell (LEC) structures. However, we were unable to do further assessment of the UV-treated LBs as the structures of LBs were easily damaged when treated with UV radiation. Cells containing aggregated protein (αA-crystallin and αB-crystallin) puncta were more abundant in the H2 O2 -treated LBs as compared with control LBs. Moreover, LC3B expression decreased with age in anterior lens capsules obtained from age-related cataracts (ARCs) patients as compared with LC3B levels in primary LECs, which is consistent with that LC3B expression in LBs was lower on D45 than on D25. Our study found that human iPSCs-derived LBs became cloudy with time which was accompanied by protein aggregation, and this phenomenon was accelerated by H2 O2 , suggesting that LBs with extending culture may serve as a human model for in vitro ARCs.
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Catarata/patologia , Células Epiteliais/patologia , Peróxido de Hidrogênio/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cápsula do Cristalino/citologia , Agregados Proteicos/fisiologia , Idoso , Envelhecimento , Autofagia/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Cristalinas/metabolismo , Imunofluorescência , Humanos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/biossíntese , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown antifibrotic effects on several diseases. The aims of the present in vitro study were to investigate the antifibrotic effects of bromfenac (a kind of NSAID) on primary human pterygium fibroblasts (HPFs) and primary human conjunctival fibroblasts (HConFs), as well as to explore the possible mechanisms of these effects. Methods: The cells used in this study were primary HPFs and HConFs, and profibrotic activation was induced by transforming growth factor-beta1 (TGF-ß1). Western blot, quantitative real-time PCR, and immunofluorescence (IF) assays were used to detect the effects of TGF-ß1 and bromfenac on the synthesis of fibronectin (FN), type III collagen (COL3), and alpha-smooth muscle actin (α-SMA) in HPFs and HConFs; the changes of signaling pathways were detected by Western blot; cell migration ability was detected by wound healing assay; cell proliferation ability was detected by CCK-8 assay; and pharmaceutical inhibitions of the downstream signaling pathways of TGF-ß1 were used to assess their possible associations with the effects of bromfenac. Results: Bromfenac suppressed the TGF-ß1-induced protein expression of FN (0.59 ± 0.07 folds, P = 0.008), COL3 (0.48 ± 0.08 folds, P = 0.001), and α-SMA (0.61 ± 0.03 folds, P = 0.008) in HPFs. Bromfenac also attenuated TGF-ß1-induced cell migration (0.30 ± 0.07 folds, P < 0.001), cell proliferation (0.64 ± 0.03 folds, P = 0.002) and the expression levels of p-AKT (0.66 ± 0.08 folds, P = 0.032), p-ERK1/2 (0.69 ± 0.11 folds, P = 0.003), and p-GSK-3ß-S9 (0.65 ± 0.10 folds, P = 0.002) in HPFs. PI3K/AKT inhibitor (wortmannin) and MEK/ERK inhibitor (U0126) reduced the TGF-ß1-induced synthesis of FN, COL3, and α-SMA in HPFs. All the results were similar in HConFs. Conclusions: Bromfenac protects against TGF-ß1-induced synthesis of FN, α-SMA, and COL3 in HPFs and HConFs at least in part by inactivating the AKT and ERK pathways.
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Anti-Inflamatórios não Esteroides/farmacologia , Benzofenonas/farmacologia , Bromobenzenos/farmacologia , Túnica Conjuntiva/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pterígio/patologia , Pterígio/prevenção & controle , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Idoso , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose/patologia , Fibrose/prevenção & controle , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt , Pterígio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Corneal epithelium integrity depends on continuous self-renewing of epithelium and connections between adjacent cells or between the cells and the basement membrane. Self-renewing epithelium cells mainly arise from the continuous proliferation and differentiation of the basal layer and limbal stem cells. The aim of the present study was to generate a bioactive, thermosensitive chitosan-gelatin hydrogel (CHI hydrogel) by incorporating exogenous recombinant human stromal cell-derived factor-1 alpha (SDF-1 alpha) for corneal epithelium regeneration. The exogenous SDF-1 alpha could enhance the stem cells proliferation, chemotaxis and migration, and the expression levels of related genes were significantly elevated in LESCs and mesenchymal stem cells (MSCs) in vitro. Moreover, the MSCs promoted the proliferation and maintained the corneal fate of the LESCs. The rat alkali injury model was used for in vivo study. The injured eyes were covered with CHI hydrogel alone or rhSDF-1 alpha-loaded CHI hydrogel. All rats were followed for 13days. Histological examination showed that the SDF-1 alpha/CHI hydrogel complex group had a nearly normal thickness; moreover, it was also found that this group could upregulate the expression of some genes and had more ΔNp63-positive cells. The SDF-1 alpha/CHI hydrogel complex group had a more tightly arranged epithelium compared with the control group using transmission electron microscopy (TEM). The mechanism for this may have involved the activation of stem cell homing and the secretion of growth factors via the SDF-1/CXCR4 chemokine axis. Therefore, SDF-1 alpha/CHI hydrogel complexes could provide a new idea for the clinical application. STATEMENT OF SIGNIFICANCE: The clarity of cornea is important for normal vision. The loss or dysfunction of LESCs leads to the impairment of corneal epithelium. The complete regeneration of corneal epithelium has not been achieved. Our study demonstrated that the incorporation of rhSDF-1 alpha with CHI hydrogel accelerated corneal epithelium reconstruction with more native structural and functional properties. The mechanism may involve in inducing proliferation and migration of the LESCs and MSCs to the injury site via the SDF-1/CXCR4 chemokine axis. Therefore, SDF-1 alpha/CHI hydrogel complexes could be a practical application for clinical therapy.
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Quimiocina CXCL12/farmacologia , Quitosana/química , Epitélio Corneano/fisiologia , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Regeneração , Temperatura , Animais , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Epitélio Corneano/ultraestrutura , Gelatina/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Regeneração/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: To investigate particulate matter (PM2.5)-induced damage to human corneal epithelial cells (HCECs) and to determine the underlying mechanisms. METHODS: HCECs were exposed to PM2.5 at a series of concentrations for various periods. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated via 5-ethynyl-2'-deoxyuridine (EdU) analysis, while autophagy was determined by immunofluorescence and Western blot. RESULTS: PM2.5-induced cell damage of HCECs occurred in a time- and dose-dependent manner. Decreased cell viability and proliferation as well as increased apoptosis were observed in HCECs after PM2.5 exposure for 24 h. Autophagy in HCECs was slightly inhibited in the early stage (before 4 h) of exposure but significantly activated in the late stage (after 24 h), as evidenced by a decrease in the former and increase in the latter of the expression of the autophagy-associated markers LC3B, ATG5, and BECN1. Interestingly, rapamycin, an autophagy activator, attenuated early-stage but aggravated late-stage PM2.5-induced cell damage, suggesting that the role of autophagy in HCECs may change over time during PM2.5 exposure. In addition, in the early stage, the expression of LC3B and ATG5 increased in cells co-treated with rapamycin and PM2.5 compared to rapamycin-only or PM2.5-only treated cells, suggesting that autophagy may benefit cell viability after PM2.5 exposure. CONCLUSIONS: The results indicate the potential role of autophagy in the treatment of PM2.5-induced ocular corneal diseases and provide direct evidence for the cytotoxicity, possibly involving an autophagic process, of PM2.5 in HCECs.
Assuntos
Poluentes Atmosféricos/toxicidade , Material Particulado/toxicidade , Apoptose , Autofagia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Desoxiuridina/análogos & derivados , Células Epiteliais/metabolismo , Humanos , Sais de Tetrazólio , Tiazóis , Testes de ToxicidadeRESUMO
Purpose: The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells. Methods: Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter "X," respectively. Results: We developed a "fried egg" differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers αA-, αB-, ß-, and γ-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.7×) of the LBs generated from UiPSCs. Conclusions: Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.
Assuntos
Catarata/metabolismo , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Cristalino/metabolismo , RNA Mensageiro/genética , Western Blotting , Catarata/genética , Catarata/patologia , Diferenciação Celular , Células Cultivadas , Células Epiteliais/patologia , Proteínas do Olho/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/patologia , Cristalino/patologiaRESUMO
Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 µmol/L) on HPFs was lower than on HCFs (100 µmol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.
Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histamina/farmacologia , Pterígio/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Túnica Conjuntiva/citologia , Difenidramina/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Nizatidina/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Soro/fisiologiaRESUMO
The scarcity of corneal tissue to treat deep corneal defects and corneal perforations remains a challenge. Currently, small incision lenticule extraction (SMILE)-derived lenticules appear to be a promising alternative for the treatment of these conditions. However, the thickness and toughness of a single piece of lenticule are limited. To overcome these limitations, we constructed a corneal stromal equivalent with SMILE-derived lenticules and fibrin glue. In vitro cell culture revealed that the corneal stromal equivalent could provide a suitable scaffold for the survival and proliferation of corneal epithelial cells, which formed a continuous pluristratified epithelium with the expression of characteristic markers. Finally, anterior lamellar keratoplasty in rabbits demonstrated that the corneal stromal equivalent with decellularized lenticules and fibrin glue could repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization. Corneal neovascularization, graft degradation, and corneal rejection were not observed within 3 months. Taken together, the corneal stromal equivalent with SMILE-derived lenticules and fibrin glue appears to be a safe and effective alternative for the repair of damage to the anterior cornea, which may provide new avenues in the treatment of deep corneal defects or corneal perforations.