RESUMO
Purpose: Recombinant human nerve growth factor (rhNGF; cenegermin-bkbj, OXERVATE) is the first and only U.S. Food and Drug Administration-approved treatment for moderate to severe neurotrophic keratopathy. The aim of this study was to determine the feasibility of incorporating a version of rhNGF in a mucoadhesive hydrogel capable of sustained drug release to the ocular surface. Methods: Hydrogels loaded with rhNGF were synthesized by conjugating chitosan with azidobenzoic acid (Az-Ch), adding rhNGF, and exposing the solution to ultraviolet (UV) radiation to induce photocrosslinking. Az-Ch hydrogels were evaluated for physical properties and rhNGF release profiles. Cytocompatbility of Az-Ch was assessed using immortalized human corneal limbal epithelial (HCLE) cells. TF1 erythroleukemic cell proliferation and HCLE cell proliferation and migration were used to assess the bioactivity of rhNGF released from Az-Ch hydrogels. Results: Az-Ch formed hydrogels in <10 seconds of UV exposure and demonstrated high optical transparency (75-85 T%). Az-Ch hydrogels exhibited good cytocompatibility with no demonstratable effect on HCLE cell morphology or viability. rhNGF was released gradually over 24 hours from Az-Ch hydrogels and retained its ability to induce TF1 cell proliferation. No significant difference was observed between rhNGF released from Az-Ch and freshly prepared rhNGF solutions on HCLE cell proliferation or percent wound closure after 12 hours; however, both were significantly better than control (P < 0.01). Conclusions: rhNGF-loaded Az-Ch hydrogels exhibited favorable physical, optical, and drug-release properties, as well as retained drug bioactivity. This drug delivery system has the potential to be further developed for in vivo and translational clinical applications. Translational Relevance: Az-Ch hydrogels may be used to enhance rhNGF therapy in patients with NK.
Assuntos
Proliferação de Células , Quitosana , Hidrogéis , Fator de Crescimento Neural , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/química , Fator de Crescimento Neural/administração & dosagem , Humanos , Quitosana/química , Quitosana/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Hidrogéis/síntese química , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Raios Ultravioleta , Reagentes de Ligações Cruzadas/química , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/citologia , Proteínas Recombinantes/química , Sistemas de Liberação de Medicamentos/métodosRESUMO
BACKGROUND: To assess the efficacy of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin [BDC]) and their analogs (tetrahydrocurcumin [THC], tetrahydrodemethoxycurcumin [THDC], tetrahydrobisdemethoxycurcumin) in reducing inflammatory cytokines and their toxicity to primary human corneal limbal epithelial cells, these cells were cultured and exposed to these compounds. METHODS: The PrestoBlue assay assessed cell viability after treatment. Anti-inflammatory effects on hyperosmotic cells were determined using real-time polymerase chain reaction and significance was gauged using one-way analysis of variance and Tukey's tests, considering p-values < 0.05 as significant. RESULTS: Curcuminoids and their analogs, at 1, 10, and 100 µM, exhibited no effect on cell viability compared to controls. However, cyclosporin A 1:500 significantly reduced cell viability more than most curcuminoid treatments, except 100 µM curcumin and BDC. All tested curcuminoids and analogs at these concentrations significantly decreased mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17 A, matrix metallopeptidase-9, and intercellular adhesion molecule-1 after 90 mM NaCl stimulation compared to untreated cells. Furthermore, proinflammatory cytokine levels from hyperosmotic cells treated with 1, 10, and 100 µM curcumin, 100 µM BDC, 100 µM THC, 1 and 100 µM THDC mirrored those treated with cyclosporin A 1:500. CONCLUSION: The anti-inflammatory efficiency of 1 and 10 µM curcumin, 100 µM THC, 1 and 100 µM THDC was comparable to that of cyclosporin A 1:500 while maintaining cell viability.
Assuntos
Anti-Inflamatórios , Sobrevivência Celular , Curcumina , Células Epiteliais , Humanos , Curcumina/farmacologia , Curcumina/análogos & derivados , Anti-Inflamatórios/farmacologia , Células Epiteliais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Limbo da Córnea/efeitos dos fármacos , Células Cultivadas , Diarileptanoides/farmacologia , Epitélio Corneano/efeitos dos fármacosRESUMO
BACKGROUND: Topical application of ß-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of ß-blocker eye drops on corneal epithelial wound healing. METHODS: Corneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL ß-blocker levobunolol, ß1-adrenoceptor (ß1AR)-specific antagonist atenolol or ß2-adrenoceptor (ß2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining. RESULTS: Levobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium. CONCLUSION: ß-blocker eye drops impaired corneal wound healing by inhibiting the ß2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective ß1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Epitélio Corneano , Limbo da Córnea , Soluções Oftálmicas/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Epitélio Corneano/efeitos dos fármacos , Limbo da Córnea/efeitos dos fármacos , Camundongos , Receptores AdrenérgicosRESUMO
Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of patients for the cornea transplantation all over the world and the list is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient's body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and has a potential role in mitigation of the adverse impact of inflammation induced by lipopolysaccharide.
Assuntos
Tecido Adiposo/citologia , Meios de Cultivo Condicionados/farmacologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Epitélio Corneano/citologia , Perfilação da Expressão Gênica , Humanos , Inflamação , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/crescimento & desenvolvimento , Limbo da Córnea/metabolismo , Lipopolissacarídeos/farmacologiaRESUMO
Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Soluções Oftálmicas/farmacologia , Fator de Transcrição PAX6/genética , Ritanserina/farmacologia , Antagonistas da Serotonina/farmacologia , Células-Tronco/efeitos dos fármacos , Aniridia/tratamento farmacológico , Aniridia/genética , Aniridia/metabolismo , Aniridia/patologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Regulação da Expressão Gênica , Células HEK293 , Haploinsuficiência , Humanos , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/metabolismo , Limbo da Córnea/patologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Fator de Transcrição PAX6/agonistas , Fator de Transcrição PAX6/metabolismo , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
Studies have reported that the incidence of ocular discomfort in people who often wear makeup is higher than that in the normal population. The incidence of ocular discomfort of these people may be also related to the daily ocular exposure to chemical surfactants during cleaning. The objectives of this study were to explore morphological and pathological changes in the murine ocular surface after low-dose repeated exposure to disodium cocoamphodiacetate (DC), a kind of chemical surfactant widely used in personal cleaning products, and to investigate the possible mechanisms. DC was administered in low dose (0.1%) to the ocular surface of C56BL/6 once daily for two weeks. We found that there were an increase of sodium fluorescein staining on the cornea, a significant thinning of corneal epithelial thickness, and increased TUNEL-positive cells in corneal epithelium in vivo. DC treatment also modulated the distribution of K14+ and P63+ epithelia from the limbal to the center on the cornea. In cultured murine corneal epithelial progenitor cell line (TKE2), DC treatment induced cell detachment and decreased the activation of Ak strain transforming protein (AKT), and extracellular signal-regulated kinase (ERK). And DC increased TUNEL-positive cells in vitro with increased expression of cleaved Caspase3 and B-cell lymphoma-2 associated X protein (Bax). Our results indicated that repeated low-dose DC exposure on ocular surface caused significant impairment on the structure and viability of the corneal epithelium by inhibiting epithelial proliferation and inducing apoptosis. It provides the foundations to understand the harmful effects of cleaning products daily exposure on the ocular surface.
Assuntos
Acetatos/efeitos adversos , Doenças da Córnea/induzido quimicamente , Epitélio Corneano/efeitos dos fármacos , Glicina/análogos & derivados , Limbo da Córnea/efeitos dos fármacos , Tensoativos/efeitos adversos , Acetatos/administração & dosagem , Administração Oftálmica , Animais , Apoptose , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Feminino , Fluoresceína/metabolismo , Glicina/administração & dosagem , Glicina/efeitos adversos , Queratina-14/metabolismo , Limbo da Córnea/metabolismo , Limbo da Córnea/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Soluções Oftálmicas , Microscopia com Lâmpada de Fenda , Coloração e Rotulagem , Tensoativos/administração & dosagem , Transativadores/metabolismoRESUMO
Limbal stem cells (LSCs) are crucial for corneal transparency and vision. Any damages to LSCs might lead to limbal stem cell deficiency resulting in corneal opacification and even blindness. Here, we investigated the cytotoxicity of timolol and its underlying mechanisms in rabbit LSCs (rLSCs) in vitro. High concentrations of 0.5% and 0.25% timolol induced necroptosis in rLSCs to upregulate receptor interacting protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL) and phosphorylated MLKL along with downregulation of caspase-8 and caspase-2 within 4 h. While, median concentrations of 0.125% to 0.0625% timolol induced apoptosis in the rLSCs within 28 h. The apoptotic mechanism in the median-concentration timolol-treated rLSCs is probably via extrinsic apoptosis pathway by activating caspase-2, caspase-8 and caspase-3 and intrinsic apoptosis pathway triggered by excessive generation of ROS and subsequent DNA damage to upregulate Bax and Bad, downregulate Bcl-2 and Bcl-xL, subsequently disrupt mitochondrial membrane potential, cytosolically translocate cytochrome c and apoptosis-inducing factor, and activate caspase-9. In addition, low concentration of 0.03125% timolol induced senescence in the rLSCs by elevating ROS level and increasing number of senescence associated ß-galactosidase positive cells at 28 h. Our findings reveal that timolol induces necroptosis, apoptosis and senescence concentration-dependently in rLSCs in vitro.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Apoptose , Senescência Celular , Limbo da Córnea/patologia , Necroptose , Células-Tronco/patologia , Timolol/farmacologia , Animais , Técnicas In Vitro , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/metabolismo , Masculino , Fosforilação , Coelhos , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
AIM: To investigate the corneal epithelial and limbal epithelial alterations in patients under topical glaucoma treatment using anterior segment-OCT (AS-OCT) and to determine the changes of the limbal region due to the preservatives and glaucoma drugs, that can progress to limbal stem cell deficiency (LSCD). Limbal thickness was measured by AS-OCT to evaluate limbal cell deficiency. METHODS: Forty-seven patients using topical medication for glaucoma, and 48 control subjects were enrolled in this matched case-control study. The patients were divided into four groups according to the treatment regimens. Group 1: One-drug regimen, Group 2: Two-drug regimen, Group 3: Three-drug regimen, Group 4: Four-drug regimen For the ocular surface evaluation; tear break-up time with standard fluorescein sodium sterile strip application, Schirmer test-I, Ocular Surface Disease Index Questionnaire, and AS-OCT were performed. RESULTS: A total of 95 subjects were included: 47 eyes of 47 patients with glaucoma medication and 48 eyes of 48 healthy subjects. There was a statistically significant difference between patients and controls according to BUT, SCH, and OSDI (p < 0.001). The mean central corneal epithelium thickness was 48.5 ± 5.3 in patients and 54.5 ± 5.9 in controls (p < 0.001). The mean central total corneal thickness was 529.2 ± 41.2 in patients and 536 ± 35.3 in controls (p = 0.335). The mean limbal epithelium thickness was 64.1 ± 9.1 in patients and 76 ± 11.5 in controls (p < 0.001). CONCLUSION: Using at least one glaucoma drug caused limbal area injury, changed ocular surface measurements, and significantly reduced the limbal epithelial thickness where the stem cells reside. The limbal epithelial thickness measurement by AS-OCT seems to be an innovative, non-invasive, and promising technique for detecting and staging corneal damage in topical glaucoma therapy.
Assuntos
Epitélio Corneano/efeitos dos fármacos , Glaucoma/tratamento farmacológico , Limbo da Córnea/efeitos dos fármacos , Idoso , Anti-Hipertensivos/uso terapêutico , Compostos de Benzalcônio/uso terapêutico , Tartarato de Brimonidina/uso terapêutico , Estudos de Casos e Controles , Epitélio Corneano/patologia , Feminino , Humanos , Latanoprosta/uso terapêutico , Limbo da Córnea/patologia , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas/uso terapêutico , Conservantes Farmacêuticos/uso terapêutico , Método Simples-Cego , Sulfonamidas/uso terapêutico , Tiofenos/uso terapêutico , Timolol/uso terapêutico , Tomografia de Coerência ÓpticaRESUMO
Purpose: NCX 667, a novel nitric oxide (NO) donor with an isomannide core, was characterized for its IOP-lowering ability in animal models of ocular hypertension and glaucoma. Bioengineered human trabecular meshwork/Schlemm's canal (HTM/HSC) constructs were used to explore the mode of action. Methods: Ocular normotensive New Zealand white (NZW) rabbits (ONT-rabbits), spontaneously ocular hypertensive pigmented Dutch-belted rabbits (sOHT-rabbits), hypertonic saline (5%)-induced transient ocular hypertensive NZW rabbits (tOHT-rabbits), ocular normotensive Beagle dogs (ONT-dogs), and laser-induced ocular hypertensive cynomolgus monkeys (OHT-monkeys) were used. NCX 667 or vehicle (30 µL) was instilled in a crossover, masked fashion and intraocular pressure (IOP) measured before dosing (baseline) and for several hours thereafter. The ONT-rabbits were used for cyclic guanosine monophosphate (cGMP) determination in ocular tissues after ocular dosing with NCX 667. Transforming growth factor-beta2 (TGFß2) (2.5 ng/mL, six days)-treated HTM/HSC constructs were used to address changes in outflow facility. Results: NCX 667 resulted in robust and dose-dependent IOP decrease in all models used. Maximal IOP-lowering efficacy at 1% was -4.1 ± 0.6, -12.2 ± 2.7, -10.5 ± 2.0, -5.3 ± 0.8, and -6.6 ± 1.9 mmHg, respectively, in ONT-dogs, sOHT-rabbits, tOHT-rabbits, ONT-rabbits, and OHT-monkeys. In ONT-rabbits NCX 667 (1%) increased cGMP in aqueous humor (AH) but not in retina and iris/ciliary body. NCX 667 concentration-dependently increased outflow facility in TGFß2-treated HTM/HSC constructs (outflow facility, 0.10 ± 0.06 and 0.30 ± 0.10 µL/min/mmHg/mm2, respectively, in vehicle- and NCX 667-treated constructs). Conclusions: NCX 667 leads to robust IOP lowering in several animal models. Evidence in HTM/HSC constructs indicate that the IOP reduction likely results from NO-mediated increase of the conventional outflow pathway. Other mechanisms including changes in AH production and episcleral vein pressure may not be excluded at this time.
Assuntos
Pressão Intraocular/efeitos dos fármacos , Limbo da Córnea/efeitos dos fármacos , Doadores de Óxido Nítrico/uso terapêutico , Hipertensão Ocular/tratamento farmacológico , Malha Trabecular/efeitos dos fármacos , Animais , Humor Aquoso/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Cães , Feminino , Limbo da Córnea/metabolismo , Macaca fascicularis , Coelhos , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
PURPOSE: To study the efficacy of interferon alfa-2b (IFN-a2b) on pigmented ocular surface squamous neoplasia (p-OSSN) and assess the resolution of the pigment to the treatment. METHODS: A retrospective case series of 8 tumors in 7 patients. RESULTS: The mean age at diagnosis of p-OSSN was 65 years (median, 61 years; range, 51-84 years), and all patients were men. The mean duration of symptoms was 2 months (median, 1 month; range, 1-4 months). One patient had 2 distinct lesions in the same eye. Tumor epicenter was located at the limbus (n = 5) or bulbar conjunctiva (n = 2). Complexion-associated melanosis was noted in all eyes. The mean basal dimension of the tumor was 8 mm (median, 7 mm; range, 5-12 mm). The mean % of tumor pigmentation was 47% (median, 30%; range, 10%-100%). The treatment details included topical IFN-a2b (n = 1) or a combination of topical and subconjunctival injection of IFN-a2b (n = 7). All patients with p-OSSN showed excellent response to IFN-a2b with complete tumor regression and resolution of tumor-associated pigment with a mean number of 2 subconjunctival IFN-a2b injections (median, 2; range, 0-3) and topical IFN-a2b for an average of 2 months (median, 2 months; range, 1-3 months). There was no change in the complexion-associated melanosis with IFN-a2b. CONCLUSIONS: IFN-a2b is very effective in the management of p-OSSN. There is a complete resolution of the pigment along with the tumor.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias da Túnica Conjuntiva/tratamento farmacológico , Doenças da Córnea/tratamento farmacológico , Neoplasias Oculares/tratamento farmacológico , Interferon alfa-2/uso terapêutico , Limbo da Córnea/efeitos dos fármacos , Administração Oftálmica , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Neoplasias da Túnica Conjuntiva/patologia , Doenças da Córnea/patologia , Neoplasias Oculares/patologia , Feminino , Seguimentos , Humanos , Injeções Intraoculares , Limbo da Córnea/patologia , Masculino , Melanose/patologia , Pessoa de Meia-Idade , Soluções Oftálmicas , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) ß2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFß2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFß2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.
Assuntos
Dexametasona/farmacologia , Limbo da Córnea/metabolismo , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/patologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
Purpose: In contact with the external environment, the cornea can easily be injured. Although corneal wounds generally heal rapidly, the pain and increased risk of infection associated with a damaged cornea, as well as the impaired healing observed in some individuals, emphasize the need for novel treatments to accelerate corneal healing. We previously demonstrated in epidermal keratinocytes that the glycerol channel aquaporin-3 (AQP3) interacts with phospholipase D2 (PLD2) to produce the signaling phospholipid phosphatidylglycerol (PG), which has been shown to accelerate skin wound healing in vivo. We hypothesized that the same signaling pathway might be operational in corneal epithelial cells. Methods: We used co-immunoprecipitation, immunohistochemistry, scratch wound healing assays in vitro, and corneal epithelial wound healing assays in vivo to determine the role of the AQP3/PLD2/PG signaling pathway in corneal epithelium. Results: AQP3 was present in human corneas in situ, and AQP3 and PLD2 were co-immunoprecipitated from corneal epithelial cell lysates. The two proteins could also be co-immunoprecipitated from insect cells simultaneously infected with AQP3- and PLD2-expressing baculoviruses, suggesting a likely direct interaction. A particular PG, dioleoylphosphatidylglycerol (DOPG), enhanced scratch wound healing of a corneal epithelial monolayer in vitro. DOPG also accelerated corneal epithelial wound healing in vivo, both in wild-type mice and in a mouse model exhibiting impaired corneal wound healing (AQP3 knockout mice). Conclusions: These results indicate the importance of the AQP3/PLD2/PG signaling pathway in corneal epithelial cells and suggest the possibility of developing DOPG as a pharmacologic therapy to enhance corneal wound healing in patients.
Assuntos
Epitélio Corneano/efeitos dos fármacos , Limbo da Córnea/efeitos dos fármacos , Fosfatidilgliceróis/farmacologia , Cicatrização/fisiologia , Animais , Aquaporina 3/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Epitélio Corneano/metabolismo , Humanos , Imunoprecipitação , Limbo da Córnea/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosfolipase D/metabolismo , Células Sf9/metabolismo , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30â¯min and then irradiated by a custom-made green LED light source (525â¯nm, 6â¯mW/cm2) for 15â¯min (5.4â¯J/cm2). Three rabbits were sacrificed and enucleated after 24â¯h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.
Assuntos
Anti-Infecciosos/farmacologia , Ceratócitos da Córnea/efeitos dos fármacos , Endotélio Corneano/efeitos dos fármacos , Corantes Fluorescentes/farmacologia , Fotoquimioterapia , Rosa Bengala/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Animais , Apoptose , Biomarcadores/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Ceratócitos da Córnea/metabolismo , Ceratócitos da Córnea/patologia , Substância Própria/efeitos dos fármacos , Substância Própria/metabolismo , Substância Própria/patologia , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/metabolismo , Limbo da Córnea/patologia , CoelhosRESUMO
Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue's mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration.
Assuntos
Córnea/citologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Adulto , Idoso , Animais , Fenômenos Biomecânicos , Diferenciação Celular/fisiologia , Colagenases/farmacologia , Córnea/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/transplante , Humanos , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/ultraestrutura , Mecanotransdução Celular , Microscopia de Fluorescência , Pessoa de Meia-Idade , Fenótipo , Coelhos , Nicho de Células-Tronco/efeitos dos fármacos , Nicho de Células-Tronco/fisiologia , Células-Tronco/efeitos dos fármacos , Engenharia Tecidual/métodos , Cicatrização/fisiologiaRESUMO
We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned silk films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned silk films. Cells cultured on nanoscale patterned silk films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/ß-catenin signaling. We conclude that patterned silk film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.
Assuntos
Diferenciação Celular , Epitélio Corneano/citologia , Regulação da Expressão Gênica , Limbo da Córnea/citologia , Seda/química , Seda/farmacologia , Células-Tronco/citologia , Proliferação de Células , Células Cultivadas , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Engenharia TecidualAssuntos
Doenças da Córnea/diagnóstico , Limbo da Córnea/patologia , Miosite/diagnóstico , Administração Oftálmica , Doenças da Córnea/tratamento farmacológico , Dexametasona/uso terapêutico , Feminino , Glucocorticoides/uso terapêutico , Humanos , Limbo da Córnea/efeitos dos fármacos , Pessoa de Meia-Idade , Miosite/tratamento farmacológico , Soluções OftálmicasRESUMO
PURPOSE: The effects of lithium (Li) and Valproic Acid (VA) drugs have been recently revealed to improve the Mesenchymal stem cells')MSCs (migration and proliferation processes. The aim of this study is to determine the expression of the genes involved in the proliferation and migration of limbal epithelial stem/progenitor cells (LESPCs) after treatment with Li and VA. METHODS: After extraction of LSCs from human Corneoscleral tissue, cells were subcultured three times. The cell culture media were divided into four separate groups including groups treated with VA, Li, combination, and control groups after determining the non-toxic concentration of drugs (64mml) Li and (28mml) VA based on MTT assay, and then cells cultures were treated for 3 hours. A real-time polymerase chain reaction was performed to detect the expression levels of CD44, Ki67, CXCR4, CXCR7, MMP-2, MMP-9, and SDF-1 genes. Changes in the expression of each gene in different treatments were calculated. Finally, the graphs were analyzed by SPSS (Version 18) software. RESULTS: The highest expression of CXCR4 and CXCR7 was in the Li-treated group. Additionally, the highest expression levels of MMP-9 and CD44 genes were observed in the VA-treated group. In contrast, the expression level of SDF-1a, MMP2, and Ki67 genes in all three treatment groups reduced compared to the control group. CONCLUSION: Increasing the LSCs migration genes (CXCR4 and MMP9) was more evident than cell proliferation genes (Ki67). In sum, Li and VA can affect the process of proliferation and migration of LSCs in vitro.
Assuntos
Limbo da Córnea/citologia , Lítio/farmacologia , Células-Tronco Mesenquimais/citologia , Ácido Valproico/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Limbo da Córnea/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Transdução de SinaisRESUMO
Recently, compressed collagen has attracted much attention as a potential alternative for a limbal epithelial stem cell (LESC) carrier to treat limbal stem cell deficiency (LSCD), in that it can provide mechanically improved collagen fibrillar structures compared to conventional collagen hydrogel. However, its clinical efficacy as an LESC carrier has not yet been studied through in vivo transplantation due to limited mechanical strength that cannot withstand a force induced by surgical suturing and low resistance to enzymatic degradation. This study firstly presents a suturable LESC carrier based on compressed collagen in the form of a biocomposite. The biocomposite was achieved by integrating a decellularized corneal lenticule, which is a decellularized stromal tissue obtained from corneal refractive surgery, inside a compressed collagen to form a sandwich structure. A suture retention test verified that the biocomposite has a much higher suture retention strength (0.56 ± 0.12 N) compared to the compressed collagen (0.02 ± 0.01 N). The biocomposite also exhibited more than 3 times higher resistance to enzymatic degradation, indicating long-term stability after transplantation. In vitro cell culture results revealed that the biocomposite effectively supported the expansion and stratification of the LESCs with expressions of putative stem cell and differentiated corneal epithelial cell markers. Finally, the biocomposite verified its clinical efficacy by stably delivering the LESCs onto an eye of a rabbit model of LSCD and effectively reconstructing the ocular surface.
Assuntos
Colágeno/farmacologia , Limbo da Córnea/fisiologia , Regeneração , Animais , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Limbo da Córnea/efeitos dos fármacos , Coelhos , Ratos , Regeneração/efeitos dos fármacos , Transplante de Células-Tronco , Células-Tronco/citologia , SuturasRESUMO
Purpose: To investigate the roles of vasoactive intestinal peptides (VIPs) in regulating the morphology and F-actin distribution of Schlemm's canal (SC) of rat eyes. Methods: Chronic intraocular pressure (IOP) hypertension models with episcleral venous cauterization (EVC) were treated with topical VIP or PG99-465 (vasoactive intestinal peptide receptors 2 [VPAC2] antagonist). IOPs were measured with Tono-Pen, and the SC parameters, including the cross-section area, circumference, and length, were statistically evaluated by hematoxylin-eosin and CD31 immunohistochemical staining. Immunofluorescence was performed to detect the distribution of F-actin in the SC. Moreover, the distribution of filamentous actin (F-actin) and globular actin (G-actin) in human umbilical vein endothelial cells (HUVECs) was studied under a pressure system by immunofluorescence and Western blotting. Results: Increased expressions of VIP and VPAC2 receptors, as well as a disordered distribution of F-actin were found in SC endothelial cells (SCEs) in the EVC model. Moreover, topical VIP maintained the normal distribution of F-actin in SCEs, expanded the collapsed SC, and induced a significant decrease in IOP in the EVC model. In in vitro HUVECs, the F-actin/G-actin ratio increased significantly under stress stimulation for 30 minutes. A total of 50 µM VIP helped maintain the normal F-actin/G-actin ratio of HUVECs against stress stimulation. Conclusions: VIP regulates the distribution of F-actin in SCEs via the VPAC2 receptor in order to induce a decrease in IOP. VIP may represent a new target for antiglaucoma drugs.
Assuntos
Actinas/metabolismo , Modelos Animais de Doenças , Pressão Intraocular/efeitos dos fármacos , Limbo da Córnea/efeitos dos fármacos , Hipertensão Ocular/tratamento farmacológico , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Western Blotting , Doença Crônica , Técnica Indireta de Fluorescência para Anticorpo , Células Endoteliais da Veia Umbilical Humana , Humanos , Pressão Intraocular/fisiologia , Limbo da Córnea/metabolismo , Masculino , Hipertensão Ocular/metabolismo , Ratos , Ratos Sprague-Dawley , Tonometria Ocular , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismoRESUMO
PURPOSE: To report the safety and efficacy of single-port pars plana anterior vitrectomy. SETTING: Cincinnati Eye Institute, Cincinnati, Ohio, USA. DESIGN: Retrospective case series. METHODS: Eyes that had anterior vitrectomy from September 2010 to June 2016 were electronically identified. Charts were reviewed for demographics, history of ocular trauma, underlying ocular or systemic comorbidity, surgical indications, outcomes, and postoperative complications. RESULTS: The mean postoperative follow-up was 10.9 months with a mean patient age of 62.4 years. Three hundred thirty-five eyes (97.7%) were scheduled as planned anterior vitrectomies, whereas 8 eyes (2.3%) were operated on unexpectedly after posterior capsule ruptures. Eighty-two eyes (23.9%) had a history of trauma. Twenty-five eyes (7.3%) had documented postoperative cystoid macular edema (CME), whereas 7 (2.0%) of these eyes had known preoperative CME. There were 3 eyes (0.9%) with retinal detachments and 1 eye (0.3%) with a retinal tear without detachment. There were no cases of endophthalmitis and no evidence of residual vitreous prolapse in the anterior chamber in any eye postoperatively. CONCLUSIONS: The safety and efficacy profile of a pars plana technique compared favorably against historical data for both coaxial and bimanual limbal clear corneal infusion and cutting. Sutureless pars plana anterior vitrectomy might be considered a safe and reliable solution for the anterior segment surgeon in managing vitreous prolapse during anterior segment surgeries.