RESUMO
Glaucoma is a leading cause of irreversible blindness worldwide, caused by the gradual degeneration of retinal ganglion cells and their axons. While glaucoma is primarily considered a genetic and age-related disease, some inflammatory conditions, such as uveitis and viral-induced anterior segment inflammation, cause secondary or uveitic glaucoma. Viruses are predominant ocular pathogens and can impose both acute and chronic pathological insults to the human eye. Many viruses, including herpes simplex virus, varicella-zoster virus, cytomegalovirus, rubella virus, dengue virus, chikungunya virus, Ebola virus, and, more recently, Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), have been associated with sequela of either primary or secondary glaucoma. Epidemiological and clinical studies suggest the association between these viruses and subsequent glaucoma development. Despite this, the ocular manifestation and sequela of viral infections are not well understood. In fact, the association of viruses with glaucoma is considered relatively uncommon in part due to underreporting and/or lack of long-term follow-up studies. In recent years, literature on the pathological spectrum of emerging viral infections, such as ZIKV and SARS-CoV-2, has strengthened this proposition and renewed research activity in this area. Clinical studies from endemic regions as well as laboratory and preclinical investigations demonstrate a strong link between an infectious trigger and development of glaucomatous pathology. In this article, we review the current understanding of the field with a particular focus on viruses and their association with the pathogenesis of glaucoma.
Assuntos
Infecções Oculares Virais , Glaucoma , Uveíte Anterior , Infecção por Zika virus , Zika virus , Humanos , Uveíte Anterior/complicações , Infecções Oculares Virais/complicações , Infecção por Zika virus/complicações , Glaucoma/epidemiologia , Glaucoma/etiologia , Progressão da DoençaRESUMO
Visual disorders are common even after mild traumatic brain injury (mTBI) or blast exposure. The cost of blast-induced vision loss in civilians, military personnel, and veterans is significant. The visual consequences of blasts associated with TBI are elusive. Active military personnel and veterans report various ocular pathologies including corneal disorders post-combat blasts. The wars and conflicts in Afghanistan, Iraq, Syria, and Ukraine have significantly increased the number of corneal and other ocular disorders among military personnel and veterans. Binocular vision, visual fields, and other visual functions could be impaired following blast-mediated TBI. Blast-associated injuries can cause visual disturbances, binocular system problems, and visual loss. About 25% of veterans exposed to blasts report corneal injury. Blast exposure induces corneal edema, corneal opacity, increased corneal thickness, damage of corneal epithelium, corneal abrasions, and stromal and endothelial abnormality including altered endothelial density, immune cell infiltration, corneal neovascularization, Descemet membrane rupture, and increased pain mediators in animal models and the blast-exposed military personnel including veterans. Immune response exacerbates blast-induced ocular injury. TBI is associated with dry eyes and pain in veterans. Subjects exposed to blasts that cause TBI should undergo immediate clinical visual and ocular examinations. Delayed visual care may lead to progressive vision loss, lengthening/impairing rehabilitation and ultimately may lead to permanent vision problems and blindness. Open-field blast exposure could induce corneal injuries and immune responses in the cornea. Further studies are warranted to understand corneal pathology after blast exposure. A review of current advancements in blast-induced corneal injury will help elucidate novel targets for potential therapeutic options. This review discusses the impact of blast exposure-associated corneal disorders.
Assuntos
Traumatismos por Explosões , Lesões da Córnea , Traumatismos por Explosões/complicações , Humanos , Lesões da Córnea/etiologia , Lesões da Córnea/patologia , Animais , Córnea/patologia , Transtornos da Visão/etiologia , Transtornos da Visão/fisiopatologiaRESUMO
Aquaporins (AQPs) are transmembrane water channel proteins that regulate the movement of water through the plasma membrane in various tissues including cornea. The cornea is avascular and has specialized microcirculatory mechanisms for homeostasis. AQPs regulate corneal hydration and transparency for normal vision. Currently, there are 13 known isoforms of AQPs that can be subclassified as orthodox AQPs, aquaglyceroporins (AQGPs), or supraquaporins (SAQPs)/unorthodox AQPs. AQPs are implicated in keratocyte function, inflammation, edema, angiogenesis, microvessel proliferation, and the wound-healing process in the cornea. AQPs play an important role in wound healing by facilitating the movement of corneal stromal keratocytes by squeezing through tight stromal matrix and narrow extracellular spaces to the wound site. Deficiency of AQPs can cause reduced concentration of hepatocyte growth factor (HGF) leading to reduced epithelial proliferation, reduced/impaired keratocyte migration, reduced number of keratocytes in the injury site, delayed and abnormal wound healing process. Dysregulated AQPs cause dysfunction in osmolar homeostasis as well as wound healing mechanisms. The cornea is a transparent avascular tissue that constitutes the anterior aspect of the outer covering of the eye and aids in two-thirds of visual light refraction. Being the outermost layer of the eye, the cornea is prone to injury. Of the 13 AQP isoforms, AQP1 is expressed in the stromal keratocytes and endothelial cells, and AQP3 and AQP5 are expressed in epithelial cells in the human cornea. AQPs can facilitate wound healing through aid in cellular migration, proliferation, migration, extracellular matrix (ECM) remodeling and autophagy mechanism. Corneal wound healing post-chemical injury requires an integrative and coordinated activity of the epithelium, stromal keratocytes, endothelium, ECM, and a battery of cytokines and growth factors to restore corneal transparency. If the chemical injury is mild, the cornea will heal with normal clarity, but severe injuries can lead to partial and/or permanent loss of corneal functions. Currently, the role of AQPs in corneal wound healing is poorly understood in the context of chemical injury. This review discusses the current literature and the role of AQPs in corneal homeostasis, wound repair, and potential therapeutic target for acute and chronic corneal injuries.
Assuntos
Aquaporinas , Lesões da Córnea , Humanos , Células Endoteliais/metabolismo , Microcirculação , Córnea/metabolismo , Lesões da Córnea/metabolismo , Cicatrização/fisiologia , Aquaporinas/metabolismoRESUMO
Pesticide exposure to eyes is a major source of ocular morbidities in adults and children all over the world. Carbofuran (CF), N-methyl carbamate, pesticide is most widely used as an insecticide, nematicide, and acaricide in agriculture, forestry, and gardening. Contact or ingestion of carbofuran causes high morbidity and mortality in humans and pets. Pesticides are absorbed in the eye faster than other organs of the body and damage ocular tissues very quickly. Carbofuran exposure to eye causes blurred vision, pain, loss of coordination, anti-cholinesterase activities, weakness, sweating, nausea and vomiting, abdominal pain, endocrine, reproductive, and cytotoxic effects in humans depending on amount and duration of exposure. Pesticide exposure to eye injures cornea, conjunctiva, lens, retina, and optic nerve and leads to abnormal ocular movement and vision impairment. Additionally, anticholinesterase pesticides like carbofuran are known to cause salivation, lacrimation, urination, and defecation (SLUD). Carbofuran and its two major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) are reversible inhibitors of acetylcholinesterase (AChE) which regulates acetylcholine (ACh), a neurohumoral chemical that plays an important role in corneal wound healing. The corneal epithelium contains high levels of ACh whose accumulation by AChE inhibition after CF exposure overstimulates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Hyper stimulation of mAChRs in the eye causes miosis (excessive constriction of the pupil), dacryorrhea (excessive flow of tears), or chromodacryorrhea (red tears). Recent studies reported alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure. This review describes carbofuran toxicity to the eye with special emphasis on corneal morbidities and blindness.
Assuntos
Carbofurano , Inseticidas , Praguicidas , Adulto , Criança , Humanos , Carbofurano/toxicidade , Carbofurano/metabolismo , Acetilcolinesterase/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Inibidores da Colinesterase , Praguicidas/toxicidade , Receptores ColinérgicosRESUMO
Sulfur mustard (SM) is a chemical warfare agent (CWA) that causes severe eye pain, photophobia, excessive lacrimation, corneal and ocular surface defects, and blindness. However, SM's effects on retinal cells are relatively meager. This study investigated the role of SM toxicity on Müller glial cells responsible for cellular architecture, inner blood-retinal barrier maintenance, neurotransmitter recycling, neuronal survival, and retinal homeostasis. Müller glial cells (MIO-M1) were exposed to SM analog, nitrogen mustard (NM), at varying concentrations (50-500 µM) for 3 h, 24 h, and 72 h. Müller cell gliosis was evaluated using morphological, cellular, and biochemical methods. Real-time cellular integrity and morphological evaluation were performed using the xCELLigence real-time monitoring system. Cellular viability and toxicity were measured using TUNEL and PrestoBlue assays. Müller glia hyperactivity was calculated based on glial fibrillary acidic protein (GFAP) and vimentin immunostaining. Intracellular oxidative stress was measured using DCFDA and DHE cell-based assays. Inflammatory markers and antioxidant enzyme levels were determined by quantitative real-time PCR (qRT-PCR). AO/Br and DAPI staining further evaluated DNA damage, apoptosis, necrosis, and cell death. Inflammasome-associated Caspase-1, ASC, and NLRP3 were studied to identify mechanistic insights into NM toxicity in Müller glial cells. The cellular and morphological evaluation revealed the Müller glia hyperactivity after NM exposure in a dose- and time-dependent manner. NM exposure caused significant oxidative stress and enhanced cell death at 72 h. A significant increase in antioxidant indices was observed at the lower concentrations of NM. Mechanistically, we found that NM-treated MIO-M1 cells increased caspase-1 levels that activated NLRP3 inflammasome-induced production of IL-1ß and IL-18, and elevated Gasdermin D (GSDMD) expression, a crucial component actuating pyroptosis. In conclusion, NM-induced Müller cell gliosis via increased oxidative stress results in caspase-1-dependent activation of the NLRP3 inflammasome and cell death driven primarily by pyroptosis.
Assuntos
Células Ependimogliais , Gás de Mostarda , Humanos , Células Ependimogliais/metabolismo , Gliose/etiologia , Gás de Mostarda/toxicidade , Antioxidantes/farmacologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspases/metabolismoRESUMO
Sulfur mustard (SM) ocular exposure severely damages the cornea and causes vision impairment. At present, no specific therapy exists to mitigate SM-induced corneal injury and vision loss. This study performed transcriptome profiling of naïve, SM-damaged, and SM-undamaged rabbit corneas using RNA-seq analysis and bioinformatic tools to gain a better mechanistic understanding and develop SM-specific medical countermeasures. The mRNA profiles of rabbit corneas 4 weeks post SM vapor exposure were generated using Illumina-NextSeq deep sequencing (Gene Expression Omnibus accession # GSE127708). The RNA sequences of naïve (n = 4), SM-damaged (n = 5), and SM-undamaged (n = 5) corneas were subjected to differential expression (DE) analysis after quality control profiling with FastQC. DE analysis was performed using HISAT2, StringTie, and DESeq2. The log2(FC)±2 and adjusted pË0.05 were chosen to identify the most relevant genes. A total of 5930 differentially expressed genes (DEGs) (upregulated: 3196, downregulated: 2734) were found in SM-damaged corneas compared to naïve corneas, whereas SM-undamaged corneas showed 1884 DEGs (upregulated: 1029, downregulated: 855) compared to naïve corneas. DE profiling of SM-damaged corneas to SM-undamaged corneas revealed 985 genes (upregulated: 308, downregulated: 677). The DE profiles were subsequently subjected to signaling pathway enrichment, and proteinâprotein interactions (PPIs) were analyzed. Pathway enrichment was performed for the genes associated with cellular apoptosis, death, adhesion, migration, differentiation, proliferation, extracellular matrix, and tumor necrosis factor production. To identify novel targets, we narrowed the pathway analysis to upregulated and downregulated genes associated with cell proliferation and differentiation, and PPI networks were developed. Furthermore, protein targets associated with cell differentiation and proliferation that may play vital roles in corneal fibrosis and wound healing post SM injury were identified.
Assuntos
Gás de Mostarda , Animais , Coelhos , Gás de Mostarda/toxicidade , Mapas de Interação de Proteínas , RNA-Seq , Córnea , Perfilação da Expressão Gênica , Expressão Gênica , Biologia ComputacionalRESUMO
Acrolein is a highly reactive volatile toxic chemical that injures the eyes and many organs. It has been used in wars and terrorism for wounding masses on multiple occasions and is readily accessible commercially. Our earlier studies revealed acrolein's toxicity to the cornea and witnessed damage to other ocular tissues. Eyelids play a vital role in keeping eyes mobile, moist, lubricated, and functional utilizing a range of diverse lipids produced by the Meibomian glands located in the upper and lower eyelids. This study sought to investigate acrolein's toxicity to eyelid tissues by studying the expression of inflammatory and lipid markers in rabbit eyes in vivo utilizing our reported vapor-cap model. The study was approved by the institutional animal care and use committees and followed ARVO guidelines. Twelve New Zealand White Rabbits were divided into 3 groups: Naïve (group 1), 1-min acrolein exposure (group 2), or 3-min acrolein exposure (group 3). The toxicological effects of acrolein on ocular health in live animals were monitored with regular clinical eye exams and intraocular pressure measurements and eyelid tissues post-euthanasia were subjected to H&E and Masson's trichrome histology and qRT-PCR analysis. Clinical eye examinations witnessed severely swollen eyelids, abnormal ocular discharge, chemosis, and elevated intraocular pressure (p < 0.001) in acrolein-exposed eyes. Histological studies supported clinical findings and exhibited noticeable changes in eyelid tissue morphology. Gene expression studies exhibited significantly increased expression of inflammatory and lipid mediators (LOX, PAF, Cox-2, and LTB4; p < 0.001) in acrolein-exposed eyelid tissues compared to naïve eyelid tissues. The results suggest that acrolein exposure to the eyes causes acute damage to eyelids by altering inflammatory and lipid mediators in vivo.
Assuntos
Acroleína , Glândulas Tarsais , Coelhos , Animais , Acroleína/toxicidade , Acroleína/metabolismo , Córnea/metabolismo , LipídeosRESUMO
C-X-C chemokine receptor type 5 (CXCR5) regulates inflammatory responses in ocular and non-ocular tissues. However, its expression and role in the cornea are still unknown. Here, we report the expression of CXCR5 in human cornea in vitro and mouse corneas in vivo, and its functional role in corneal inflammation using C57BL/6J wild-type (CXCR5+/+) and CXCR5-deficient (CXCR5-/-) mice, topical alkali injury, clinical eye imaging, histology, immunofluorescence, PCR, qRT-PCR, and western blotting. Human corneal epithelial cells, stromal fibroblasts, and endothelial cells demonstrated CXCR5 mRNA and protein expression in PCR, and Western blot analyses, respectively. To study the functional role of CXCR5 in vivo, mice were divided into four groups: Group-1 (CXCR5+/+ alkali injured cornea; n = 30), Group-2 (CXCR5-/- alkali injured cornea; n = 30), Group-3 (CXCR5+/+ naïve cornea; n = 30), and Group-4 (CXCR5-/- naïve cornea; n = 30). Only one eye was wounded with alkali. Clinical corneal evaluation and imaging were performed before and after injury. Mice were euthanized 4 h, 3 days, or 7 days after injury, eyes were excised and used for histology, immunofluorescence, and qRT-PCR. In clinical eye examinations, CXCR5-/- mouse corneas showed ocular health akin to the naïve corneas. Alkali injured CXCR5+/+ mouse corneas showed significantly increased mRNA (p < 0.001) and protein (p < 0.01 or p < 0.0001) levels of the CXCR5 compared to the naïve corneas. Likewise, alkali injured CXCR5-/- mouse corneas showed remarkably amplified inflammation in clinical eye exams in live animals. The histological and molecular analyses of these corneas post euthanasia exhibited markedly augmented inflammatory cells in H&E staining and significant CD11b + cells in immunofluorescence (p < 0.01 or < 0.05); and tumor necrosis factor-alpha (TNFα; p < 0.05), cyclooxygenase 2 (COX-2; p < 0.0001), interleukin (IL)-1ß (p < 0.0001), and IL-6 (p < 0.0001 or < 0.01) mRNA expression compared to the CXCR5+/+ mouse corneas. Interestingly, CXCR5-/- alkali injured corneas also showed altered mRNA expression of fibrotic alpha smooth muscle actin (α-SMA; p > 0.05) and angiogenic vascular endothelial growth factor (VEGF; p < 0.01) compared to the CXCR5+/+ alkali injured corneas. In summary, the CXCR5 gene is expressed in all three major layers of the cornea and appears to influence corneal inflammatory and repair events post-injury in vivo. More studies are warranted to tease the mechanistic role of CXCR5 in corneal inflammation and wound healing.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Queimaduras Oculares , Humanos , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BL , Córnea/metabolismo , Lesões da Córnea/metabolismo , Fatores de Crescimento do Endotélio Vascular , Álcalis , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Inflamação/metabolismo , Receptores de Quimiocinas/metabolismo , Queimaduras Químicas/metabolismo , Queimaduras Oculares/metabolismoRESUMO
Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.
Assuntos
Doenças da Córnea , Lesões da Córnea , Opacidade da Córnea , Actinas/genética , Álcalis , Animais , Cicatriz/patologia , Cicatriz/terapia , Córnea , Doenças da Córnea/genética , Doenças da Córnea/terapia , Lesões da Córnea/patologia , Lesões da Córnea/terapia , Opacidade da Córnea/patologia , Opacidade da Córnea/terapia , Dependovirus , Fibronectinas/genética , Fibrose , Terapia Genética/métodos , RNA Mensageiro , Coelhos , Fatores de Crescimento Transformadores/genéticaRESUMO
A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.
Assuntos
Lesões da Córnea/metabolismo , Decorina/fisiologia , Colágenos Fibrilares/metabolismo , Organogênese/fisiologia , Cicatrização/fisiologia , Animais , Queimaduras Químicas/metabolismo , Lesões da Córnea/patologia , Proteínas da Matriz Extracelular/metabolismo , Queimaduras Oculares/induzido quimicamente , Colágenos Fibrilares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microscopia com Lâmpada de Fenda , Hidróxido de SódioRESUMO
Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5'TG3'-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-ß1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.
Assuntos
Doenças da Córnea , Edição de Genes , Actinas/genética , Actinas/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular , Células Cultivadas , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Colágeno/metabolismo , Doenças da Córnea/patologia , Fibroblastos/metabolismo , Fibrose , Proteínas de Homeodomínio , Humanos , Miofibroblastos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
An array of corneal pathologies collectively called mustard gas keratopathy (MGK) resulting from ocular exposure to sulfur mustard (SM) gas are the most prevalent chemical warfare injury. MGK involves chronic ocular discomfort that results in vision impairment. The etiology of MGK remains unclear and poorly understood primarily due to a lack of scientific data regarding structural and cellular changes in different layers of the cornea altered by mustard vapor exposure in vivo. The goals of this study were to (a) characterize time-dependent changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ by employing state-of-the-art multimodal clinical ophthalmic imaging techniques and (b) determine if SM-induced acute changes in corneal cells could be rescued by a topical eye drop (TED) treatment using in an established rabbit in vivo model. Forty-five New Zealand White Rabbit eyes were divided into four groups (Naïve, TED, SM, and SM + TED). Only one eye was exposed to SM (200 mg-min/m3 for 8 min), and each group had three time points with six eyes each (Table-1). TED was topically applied twice a day for seven days. Clinical eye examinations and imaging were performed in live rabbits with stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, and applanation tonometry were conducted to measure corneal haze, ocular surface aberrations, tears, and intraocular pressure respectively. H&E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of corneal epithelium, stroma, and endothelium in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naïve cornea. Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Neither naive nor TED-alone treated eyes showed any structural, cellular, and functional abnormalities. Topical TED treatment significantly reduced SM-induced abnormalities in primary corneal layers. We conclude that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo, and efficient targeting of them with suitable agents has the potential to mitigate SM ocular injury.
Assuntos
Queimaduras Químicas , Substâncias para a Guerra Química , Doenças da Córnea , Gás de Mostarda , Coelhos , Animais , Gás de Mostarda/toxicidade , Substâncias para a Guerra Química/toxicidade , Córnea/patologia , Doenças da Córnea/patologia , Queimaduras Químicas/patologia , Soluções Oftálmicas/farmacologia , FluoresceínasRESUMO
The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-ß. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGß1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.
Assuntos
Lesões da Córnea , Proteômica , Diferenciação Celular/fisiologia , Células Cultivadas , Córnea/metabolismo , Lesões da Córnea/metabolismo , Fibroblastos/metabolismo , Humanos , Profilinas/metabolismo , Células Estromais/metabolismo , Cicatrização/fisiologiaRESUMO
Purpose: Diabetes mellitus (DM) is a metabolic disorder that affects over 450 million people worldwide. DM is characterized by hyperglycemia, causing severe systemic damage to the heart, kidneys, skin, vasculature, nerves, and eye. Type 2 diabetes (T2DM) constitutes 90% of clinical cases and is the most common cause of blindness in working adults. Also, about 70% of T2DM patients show corneal complications including delayed wound healing, often described as diabetic keratopathy (DK). Despite the increasing severity of DM, the research on DK is bleak. This study investigated cellular morphology and collagen matrix alterations of the diabetic and non-diabetic corneas collected from Ossabaw mini pigs, a T2DM animal model with a "thrifty genotype." Methods: Pig corneas were collected from six-month-old Ossabaw miniature pigs fed on a western diet (WD) for ten weeks. The tissues were processed for immunohistochemistry and analyzed using hematoxylin and eosin staining, Mason Trichrome staining, Picrosirus Red staining, Collage I staining, and TUNEL assay. mRNA was prepared to quantify fibrotic gene expression using quantitative reverse-transcriptase PCR (qRT-PCR). Transmission electron microscopy (TEM) was performed to evaluate stromal fibril arrangements to compare collagen dynamics in WD vs. standard diet (SD) fed Ossabaw pig corneas. Results: Ossabaw mini pigs fed on a WD for 10 weeks exhibit classic symptoms of metabolic syndrome and hyperglycemia seen in T2DM patients. We observed significant disarray in cornea stromal collagen matrix in Ossabaw mini pigs fed on WD compared to the age-matched mini pigs fed on a standard chow diet using Masson Trichome and Picrosirius Red staining. Furthermore, ultrastructure evaluation using TEM showed alterations in stromal collagen fibril size and organization in diabetic corneas compared to healthy age-matched corneas. These changes were accompanied by significantly decreased levels of Collagen IV and increased expression of matrix metallopeptidase 9 in WD-fed pigs. Conclusions: This pilot study indicates that Ossabaw mini pigs fed on WD showed collagen disarray and altered gene expression involved in wound healing, suggesting that corneal stromal collagens are vulnerable to diabetic conditions.
Assuntos
Substância Própria , Diabetes Mellitus Tipo 2 , Animais , Colágeno Tipo IV , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Projetos Piloto , Suínos , Porco MiniaturaRESUMO
Corneal disease remains a leading cause of impaired vision world-wide, and advancements in gene therapy continue to develop with promising success to prevent, treat and cure blindness. Ideally, gene therapy requires a vector and gene delivery method that targets treatment of specific cells or tissues and results in a safe and non-immunogenic response. The cornea is a model tissue for gene therapy due to its ease of clinician access and immune-privileged state. Improvements in the past 5-10 years have begun to revolutionize the approach to gene therapy in the cornea with a focus on adeno-associated virus and nanoparticle delivery of single and combination gene therapies. In addition, the potential applications of gene editing (zinc finger nucleases [ZNFs], transcription activator-like effector nucleases [TALENs], Clustered Regularly Interspaced Short Palindromic Repeats/Associated Systems [CRISPR/Cas9]) are rapidly expanding. This review focuses on recent developments in gene therapy for corneal diseases, including promising multiple gene therapy, while outlining a practical approach to the development of such therapies and potential impediments to successful delivery of genes to the cornea.
Assuntos
Córnea/patologia , Doenças da Córnea/terapia , Terapia Genética/métodos , Doenças da Córnea/genética , HumanosRESUMO
Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn-/-) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn-/- and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn-/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn-/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn-/- mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn-/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn-/- mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn-/- mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn-/- mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.
Assuntos
Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/fisiopatologia , Decorina/fisiologia , Actinas/metabolismo , Animais , Neovascularização da Córnea/genética , Endoglina/genética , Endoglina/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGFß)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGFß-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation. Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGFß1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGFß-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (α-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis. Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGFß1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in α-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGFß1 showed the expected low expression of α-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGFß-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGFß-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein. Conclusions: Id3 regulates TGFß-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.
Assuntos
Diferenciação Celular/genética , Substância Própria/citologia , Fibroblastos/citologia , Proteínas Inibidoras de Diferenciação/genética , Proteínas de Neoplasias/genética , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Forma Celular/efeitos dos fármacos , Forma Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Diferenciação/metabolismo , Modelos Biológicos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM). International Diabetic Federations (IDF) estimates up to 629 million people with DM by the year 2045 worldwide. Nearly 50% of DM patients will show evidence of diabetic-related eye problems. Therapeutic interventions for DR are limited and mostly involve surgical intervention at the late-stages of the disease. The lack of early-stage diagnostic tools and therapies, especially in DR, demands a better understanding of the biological processes involved in the etiology of disease progression. The recent surge in literature associated with NOD-like receptors (NLRs) has gained massive attraction due to their involvement in mediating the innate immune response and perpetuating inflammatory pathways, a central phenomenon found in the pathogenesis of ocular diseases including DR. The NLR family of receptors are expressed in different eye tissues during pathological conditions suggesting their potential roles in dry eye, ocular infection, retinal ischemia, cataract, glaucoma, age-related macular degeneration (AMD), diabetic macular edema (DME) and DR. Our group is interested in studying the critical early components involved in the immune cell infiltration and inflammatory pathways involved in the progression of DR. Recently, we reported that NLRP3 inflammasome might play a pivotal role in the pathogenesis of DR. This comprehensive review summarizes the findings of NLRs expression in the ocular tissues with special emphasis on its presence in the retinal microglia and DR pathogenesis.
Assuntos
Retinopatia Diabética/imunologia , Glaucoma/imunologia , Inflamassomos/imunologia , Degeneração Macular/imunologia , Edema Macular/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas NLR/imunologia , Olho/imunologia , Humanos , Imunidade InataRESUMO
Retinal inflammation is an integral component of many retinal diseases including diabetic retinopathy (DR), age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). Inflammation is commonly initiated and perpetuated by myeloid-derived immune cells. In the retina, microglial cells are resident macrophages with myeloid origins, which acts as the first responders involved in the innate immune system. To understand the disease pathogenesis, the use of isolated retinal cell culture model is vital for the examination of multiple cellular responses to injury or trauma. The pig retina resembles human retina in terms of tissue architecture, vasculature, and topography. Additionally, it is a better model than the rodent retina because of the presence of the pseudomacula. In the present study, we sought to establish and characterize pig retinal primary microglial cell (pMicroglia) culture. We used pig eyes from the local abattoir and optimized pMicroglia cultures using multiple cell culture conditions and methods. The best results were obtained by seeding cells in DMEM-high glucose media for 18 days followed by shaking of the culture plate. The resulting pMicroglia were characterized by cellular morphology, phenotype, and immunostaining with Iba-1, CD68, P2Y12, CD163, CD14, and Isolectin GS-IB4. Generated pMicroglia were found functionally active in phagocytosis assay and responsive to lipopolysaccharides (LPS) in dose-dependent production of IL-1ß. Furthermore, they showed increased secretion of pro-inflammatory cytokines with LPS treatment. Thus, we report a novel and reproducible method for the isolation of primary microglial cells from pig eyes, which may be useful for studying retinal diseases.
Assuntos
Microglia/citologia , Retina/citologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células , Meios de Cultura , Relação Dose-Resposta a Droga , Interleucina-1beta/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Microglia/metabolismo , Fagocitose/fisiologia , Cultura Primária de Células , Receptores de Superfície Celular/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Sus scrofaRESUMO
Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48â¯h and then treated with decorin (250â¯nM) in the presence or absence of EGF (100â¯ng/ml) for various time points (10-60â¯min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10â¯min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-ß-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.