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1.
Invest Ophthalmol Vis Sci ; 62(12): 3, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34495288

RESUMO

Purpose: Age-related cataract is the leading cause of blindness worldwide. Variants in the EPHA2 gene increase the disease risk, and its knockout in mice causes cataract. We investigated whether age, sex, and genetic background, risk factors for age-related cataract, and Epha2 genotype influence Epha2-related cataract development in mice. Methods: Cataract development was monitored in Epha2+/+, Epha2+/-, and Epha2-/- mice (Epha2Gt(KST085)Byg) on C57BL/6J and FVB:C57BL/6J (50:50) backgrounds. Cellular architecture of lenses, endoplasmic reticulum (ER) stress, and redox state were determined using histological, molecular, and analytical techniques. Results: Epha2-/- and Epha2+/- mice on C57BL/6J background developed severe cortical cataracts by 18 and 38 weeks of age, respectively, compared to development of similar cataract significantly later in Epha2-/- mice and no cataract in Epha2+/- mice in this strain on FVB background, which was previously reported. On FVB:C57BL/6J background, Epha2-/- mice developed severe cortical cataract by 38 weeks and Epha2+/- mice exhibited mild cortical cataract up to 64 weeks of age. Progression of cataract in Epha2-/- and Epha2+/- female mice on C57BL/6J and mixed background, respectively, was slower than in matched male mice. N-cadherin and ß-catenin immunolabeling showed disorganized lens fiber cells and disruption of lens architecture in Epha2-/- and Epha2+/- lenses, coinciding with development of severe cataracts. EPHA2 immunolabeling showed intracellular accumulation of the mutant EPHA2-ß-galactosidase fusion protein that induced a cytoprotective ER stress response and in Epha2+/- lenses was also accompanied by glutathione redox imbalance. Conclusions: Both, Epha2-/- and Epha2+/- mice develop age-related cortical cataract; age as a function of Epha2 genotype, sex, and genetic background influence Epha2-related cataractogenesis in mice.


Assuntos
Catarata/genética , Regulação da Expressão Gênica , Cristalino/metabolismo , RNA/genética , Receptor EphA2/genética , Animais , Catarata/diagnóstico , Catarata/metabolismo , Modelos Animais de Doenças , Genótipo , Cristalino/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor EphA2/biossíntese
2.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502323

RESUMO

The aim of the study was the multi-elemental analysis of aqueous humor (AH) collected from patients undergoing cataract surgery. The study included: 16 patients with age-related macular degeneration AMD (99 controls), 10 patients with retinopathy (105 controls), 61 patients with hypertension (54 controls), and 33 patients with coexisting diabetes (82 controls). The control groups were recruited from patients with a lack of co-existing disease characterizing the specified studied group. The measurements were performed by the use of inductively coupled plasma optical emission spectrometry (ICP-OES). The statistical analysis was carried out using non-parametric testing (Mann-Whitney U). The level of significance was set at p = 0.05. The data obtained revealed substantial variations in elemental composition between the test groups in comparison to the controls. However, the significant variations concerned only a few elements. The phosphorous (P) level and the ratio of P/Ca were significant in retinopathy and diabetes, whereas cobalt (0.091 ± 0.107 mg/L vs. 0.031 ± 0.075 mg/L; p = 0.004) was significant in AMD. In co-existing hypertension, the levels of tin (0.293 ± 0.409 mg/L vs. 0.152 ± 0.3 mg/L; p = 0.031), titanium (0.096 ± 0.059 mg/L vs. 0.152 ± 0.192 mg/L; p = 0.045), and ruthenium (0.035 ± 0.109 mg/L vs. 0.002 ± 0.007 mg/L; p = 0.006) varied in comparison to the controls. The study revealed inter-elemental interactions. The correlation matrices demonstrated the domination of the positive correlations, whereas negative correlations mainly concerned sodium.


Assuntos
Humor Aquoso/metabolismo , Catarata/metabolismo , Diabetes Mellitus/fisiopatologia , Retinopatia Diabética/fisiopatologia , Elementos Químicos , Hipertensão/fisiopatologia , Degeneração Macular/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Catarata/epidemiologia , Catarata/patologia , Catarata/terapia , Extração de Catarata , Feminino , Seguimentos , Humanos , Cristalino/cirurgia , Masculino , Pessoa de Meia-Idade , Polônia/epidemiologia , Prognóstico
3.
Sci Rep ; 11(1): 17401, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465795

RESUMO

Cataracts, named for pathological light scattering in the lens, are known to be associated with increased large protein aggregates, disrupted protein phase separation, and/or osmotic imbalances in lens cells. We have applied synchrotron phase contrast X-ray micro-computed tomography to directly examine an age-related nuclear cataract model in Cx46 knockout (Cx46KO) mice. High-resolution 3D X-ray tomographic images reveal amorphous spots and strip-like dense matter precipitates in lens cores of all examined Cx46KO mice at different ages. The precipitates are predominantly accumulated in the anterior suture regions of lens cores, and they become longer and dense as mice age. Alizarin red staining data confirms the presence of calcium precipitates in lens cores of all Cx46KO mice. This study indicates that the spatial and temporal calcium precipitation is an age-related event associated with age-related nuclear cataract formation in Cx46KO mice, and further suggests that the loss of Cx46 promotes calcium precipitates in the lens core, which is a new mechanism that likely contributes to the pathological light scattering in this age-related cataract model.


Assuntos
Cálcio/metabolismo , Catarata/metabolismo , Animais , Catarata/patologia , Cristalino/metabolismo , Camundongos , Camundongos Knockout , Microtomografia por Raio-X
4.
Exp Eye Res ; 210: 108709, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339681

RESUMO

Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death.


Assuntos
Envelhecimento/fisiologia , Biomarcadores/metabolismo , Catarata/metabolismo , Cristalino/metabolismo , Animais , Apoptose , Senescência Celular , Homeostase , Humanos , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
5.
Biomolecules ; 11(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34439816

RESUMO

Cataracts are a leading cause of blindness worldwide. Surgical removal of cataracts is a safe and effective procedure to restore vision. However, a large number of patients later develop vision loss due to regrowth of lens cells and subsequent degradation of the visual axis leading to visual disability. This postsurgical complication, known as posterior capsular opacification (PCO), occurs in up to 30% of cataract patients and has no clinically proven pharmacological means of prevention. Despite the availability of many compounds capable of preventing early steps in PCO development, there is currently no effective means to deliver such therapies into the eye for a suitable duration. To model a solution to this unmet medical need, we fabricated acrylic substrates as intraocular lens (IOL) mimics scaled to place into the capsular bag of the mouse lens following a mock-cataract surgery. Substrates were coated with a hydrophilic crosslinked acrylate nanogel designed to elute Sorbinil, an aldose reductase inhibitor previously shown to suppress PCO. Insertion of the Sorbinil-eluting device into the lens capsule at the time of cataract surgery resulted in substantial prevention of cellular changes associated with PCO development. This model demonstrates that a cataract inhibitor can be delivered into the postsurgical lens capsule at therapeutic levels.


Assuntos
Opacificação da Cápsula/prevenção & controle , Extração de Catarata/efeitos adversos , Portadores de Fármacos , Inibidores Enzimáticos/farmacologia , Imidazolidinas/farmacologia , Lentes Intraoculares , Actinas/genética , Actinas/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Opacificação da Cápsula/etiologia , Opacificação da Cápsula/genética , Opacificação da Cápsula/patologia , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Extração de Catarata/métodos , Modelos Animais de Doenças , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Cristalino/metabolismo , Cristalino/patologia , Cristalino/cirurgia , Camundongos , Nanogéis/administração & dosagem , Nanogéis/química , Transdução de Sinais , Vimentina/genética , Vimentina/metabolismo
6.
J Biol Chem ; 297(3): 101089, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34416235

RESUMO

Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-ß precursor protein (APP), whose mutations cause familial forms of Alzheimer's disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2bD rats). Given the BRI2/APP interaction and the widely accepted relevance of human amyloid ß (Aß), a proteolytic product of APP, to AD, Itm2bD rats were engineered to express two humanized App alleles and produce human Aß. Here, we studied young Itm2bD rats to investigate early pathogenic changes in these diseases. We found that periadolescent Itm2bD rats not only present subtle changes in human Aß levels along with decreased spontaneous glutamate release and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated responses but also had increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These alterations in excitatory interneuronal communication can impair learning and memory processes and were akin to those observed in adult mice producing rodent Aß and carrying either the Danish or British mutations in the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses across species and early in life. Future studies will determine whether this phenomenon represents an early pathogenic event in human dementia.


Assuntos
Catarata/fisiopatologia , Ataxia Cerebelar/fisiopatologia , Surdez/fisiopatologia , Demência/fisiopatologia , Proteínas de Membrana/genética , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Catarata/metabolismo , Ataxia Cerebelar/metabolismo , Surdez/metabolismo , Demência/genética , Demência/metabolismo , Modelos Animais de Doenças , Fármacos Atuantes sobre Aminoácidos Excitatórios/metabolismo , Feminino , Masculino , Proteínas de Membrana/metabolismo , Memória , Terminações Pré-Sinápticas/metabolismo , Ratos , Receptores de Glutamato/metabolismo , Sinapses/metabolismo
7.
Exp Eye Res ; 210: 108705, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34297945

RESUMO

Crystallins, the most prevalent lens proteins, have no turnover throughout the entire human lifespan. These long-lived proteins are susceptible to post-synthetic modifications, including oxidation and glycation, which are believed to be some of the primary mechanisms for age-related cataractogenesis. Thanks to high glutathione (GSH) and ascorbic acid (ASA) levels as well as low oxygen content, the human lens is able to maintain its transparency for several decades. Aging accumulates substantial changes in the human lens, including a decreased glutathione concentration, increased reactive oxygen species (ROS) formation, impaired antioxidative defense capacity, and increased redox-active metal ions, which induce glucose and ascorbic acid degradation and protein glycation. The glycated lens crystallins are either prone to UVA mediated free radical production or they attract metal ion binding, which can trigger additional protein oxidation and modification. This vicious cycle is expected to be exacerbated with older age or diabetic conditions. ASA serves as an antioxidant in the human lens under reducing conditions to protect the human lens from damage, but ASA converts to the pro-oxidative role and causes lens protein damage by ascorbylation in high oxidation or enriched redox-active metal ion conditions. This review is dedicated in honor of Dr. Frank Giblin, a great friend and superb scientist, whose pioneering and relentless work over the past 45 years has provided critical insight into lens redox regulation and glutathione homeostasis during aging and cataractogenesis.


Assuntos
Envelhecimento/fisiologia , Catarata/metabolismo , Glicosilação , Cristalino/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Ácido Ascórbico/farmacologia , Catarata/fisiopatologia , Cristalinas/metabolismo , Glutationa/metabolismo , Humanos , Cristalino/efeitos dos fármacos , Oxirredução , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo
8.
Exp Eye Res ; 210: 108704, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34302851

RESUMO

Advanced glycation end products (AGEs) accumulate with age in human lens capsules. AGEs in lens capsules potentiate the transforming growth factor beta-2-mediated mesenchymal transition of lens epithelial cells, which suggests that they play a role in posterior capsule opacification after cataract surgery. We measured AGEs by liquid chromatography-mass spectrometry in capsulorhexis specimens obtained during cataract surgery from nondiabetic and diabetic patients with and without established retinopathy. Our data showed that the levels of most AGEs (12 out of 13 measured) were unaltered in diabetic patients and diabetic patients with retinopathy compared to nondiabetic patients. There was one exception: glucosepane, which was significantly higher in diabetic patients, both with (6.85 pmol/µmol OH-proline) and without retinopathy (8.32 pmol/µmol OH-proline), than in nondiabetic patients (4.01 pmol/µmol OH-proline). Our study provides an explanation for the similar incidence of posterior capsule opacification between nondiabetic and diabetic cataract patients observed in several studies.


Assuntos
Catarata/metabolismo , Retinopatia Diabética/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Cápsula do Cristalino/metabolismo , Idoso , Glicemia/metabolismo , Capsulorrexe , Catarata/patologia , Cromatografia Líquida , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retinopatia Diabética/patologia , Feminino , Hemoglobina A Glicada/metabolismo , Humanos , Cápsula do Cristalino/patologia , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem
9.
Exp Eye Res ; 210: 108697, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34233175

RESUMO

Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.


Assuntos
Envelhecimento/fisiologia , Catarata/etiologia , Cristalinas/metabolismo , Oxigenação Hiperbárica/efeitos adversos , Núcleo do Cristalino/metabolismo , Proteólise/efeitos dos fármacos , Animais , Aquaporinas/metabolismo , Catarata/metabolismo , Catarata/patologia , Cromatografia Líquida , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Cobaias , Núcleo do Cristalino/patologia , Espectrometria de Massas em Tandem , Cadeia A de alfa-Cristalina/metabolismo
10.
Invest Ophthalmol Vis Sci ; 62(7): 23, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156426

RESUMO

Purpose: The purpose of this study was to determine the importance of the xCT is a subunit. The cystine/glutamate antiporter is actually system xc-xCT subunit of the cystine/glutamate antiporter in maintaining redox balance by investigating the effects of the loss of xCT on lens transparency and cystine/cysteine balance in the aqueous humour. Methods: C57Bl/6 wild-type and xCT knockout mice at five age groups (6 weeks to 12 months) were used. Lens transparency was examined using a slit-lamp and morphological changes visualized by immunolabelling and confocal microscopy. Quantification of glutathione in lenses and cysteine and cystine levels in the aqueous was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Slit-lamp examinations revealed that 3-month-old wild-type mice and xCT knockout mice lenses exhibited an anterior localized cataract. The frequency of this cataract significantly increased in the knockout mice compared to the wild-type mice. Morphological studies revealed a localized swelling of the lens fiber cells at the anterior pole. Glutathione levels in whole lenses were similar between wild-type and knockout mice. However, glutathione levels were significantly decreased at 3 months in the knockout mice in the lens epithelium compared to the wild-type mice. Aqueous cysteine levels remained similar between wild-type and knockout mice at all age groups, whereas cystine levels were significantly increased in 3-, 9-, and 12-month-old knockout mice compared to wild-type mice. Conclusions: Loss of xCT resulted in the depletion of glutathione in the epithelium and an oxidative shift in the cysteine/cystine ratio of the aqueous. Together, these oxidative changes may contribute to the accelerated development of an anterior cataract in knockout mice, which appears to be a normal feature of aging in wild-type mice.


Assuntos
Envelhecimento/fisiologia , Humor Aquoso , Catarata , Cistina/metabolismo , Ácido Glutâmico/metabolismo , Cristalino , Animais , Antiporters/metabolismo , Humor Aquoso/diagnóstico por imagem , Humor Aquoso/fisiologia , Catarata/diagnóstico , Catarata/metabolismo , Catarata/fisiopatologia , Cristalino/diagnóstico por imagem , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal/métodos , Oxirredução , Estresse Oxidativo , Microscopia com Lâmpada de Fenda/métodos
11.
J Photochem Photobiol B ; 221: 112248, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34192628

RESUMO

Melatonin is mainly secreted by the pineal gland, and it is also produced by various ocular structures such as the lens. It has been recently demonstrated that melatonin ocular synthesis can be induced by blocking the blue component of white light by means of filters. Melatonin exhibits antioxidant properties that can be useful to face light-induced oxidative stress as well as oxidative events associated to ocular pathologies like cataracts. Moreover, as oxidative stress is a main event in cataract development, changes in melatonin levels could happen and be relevant in the progression of this pathology, a subject that remains uncertain. The goal of this work was to analyze the ability of a short wavelength light blocking (yellow) filter to modulate endogenous melatonin concentration and the antioxidant and cytoprotective actions induced by yellow filter's use in lens. Furthermore, we evaluated the potential changes in aqueous humor melatonin concentration from patients with cataracts. In human lens epithelial cells, white light-emitting diode (LED) light challenge reduced melatonin secretion, protein levels of the enzymes involved in melatonin synthesis (hydroxyindole-O-methyltransferase and unphosphorylated and phosphorylated forms of arylalkylamine N-acetyltransferase) and cell viability whereas increased reactive oxygen species production. Yellow filter exposure precluded melatonin secretion reduction and protected cells from oxidative damage. Consistent with cataract patient's results, significantly lower levels of melatonin were observed in aqueous humor of alloxan-induced diabetic cataract rabbits as compared to those of control rabbits. In contrast, aqueous humor melatonin levels of diabetic cataract animals maintaining in cages covered with a yellow filter resembled control values. This recovery seems to be mediated by the induction of melatonin biosynthetic enzymes protein expression. Yellow filter also preserved Nrf2 lens protein expression and superoxide dismutase protein levels and activity in diabetic animals. Modulation of endogenous ocular melatonin concentration using blocking filters might be a promising approach to prevent premature lens opacification.


Assuntos
Humor Aquoso/metabolismo , Melatonina/metabolismo , Substâncias Protetoras/metabolismo , Idoso , Animais , Catarata/metabolismo , Catarata/patologia , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Humanos , Cristalino/citologia , Luz , Masculino , Melatonina/farmacologia , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para Cima/efeitos dos fármacos
12.
Turk J Ophthalmol ; 51(2): 107-113, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33951899

RESUMO

Congenital cataract is a challenging ophthalmological disorder which can cause severe visual loss. It can be diagnosed at birth or during the first year of life. Early diagnosis and treatment are crucial for the visual prognosis. It can be associated with various ocular and systemic abnormalities. Determining whether congenital cataract is isolated or associated with other pathology is an indispensable step for the prediction of potential vision as well as early diagnosis and treatment of conditions that can cause morbidity or mortality. Many genes have been identified in the molecular etiology of congenital cataract. Most mutations have been reported in the crystallin genes. Determination of the genetic cause may not only enable individualized genetic counseling but also help to identify concomitant ocular and/or systemic disorders depending on the characteristics of the genetic test used. Recently, next-generation sequencing in particular has become an evolving technology for determining the molecular etiology of congenital cataract and furthering our knowledge of the disease.


Assuntos
Catarata/congênito , Cristalinas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Catarata/genética , Catarata/metabolismo , Cristalinas/metabolismo , Testes Genéticos , Humanos , Linhagem
13.
PLoS One ; 16(4): e0250277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857260

RESUMO

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66-80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66-80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact ß-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.


Assuntos
Amiloide/química , Ácido Aspártico/metabolismo , Ácido Isoaspártico/metabolismo , Processamento de Proteína Pós-Traducional , Cadeia A de alfa-Cristalina/metabolismo , Envelhecimento/genética , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Ácido Aspártico/química , Benzotiazóis/química , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ácido Isoaspártico/química , Isomerismo , Cristalino/metabolismo , Cristalino/patologia , Microscopia Eletrônica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/química , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Eletricidade Estática , Cadeia A de alfa-Cristalina/genética
14.
Biomolecules ; 11(4)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807297

RESUMO

Cells encounter a myriad of endogenous and exogenous stresses that could perturb cellular physiological processes. Therefore, cells are equipped with several adaptive and stress-response machinery to overcome and survive these insults. One such machinery is the heat shock response (HSR) program that is governed by the heat shock factors (HSFs) family in response towards elevated temperature, free radicals, oxidants, and heavy metals. HSF4 is a member of this HSFs family that could exist in two predominant isoforms, either the transcriptional repressor HSFa or transcriptional activator HSF4b. HSF4 is constitutively active due to the lack of oligomerization negative regulator domain. HSF4 has been demonstrated to play roles in several physiological processes and not only limited to regulating the classical heat shock- or stress-responsive transcriptional programs. In this review, we will revisit and delineate the recent updates on HSF4 molecular properties. We also comprehensively discuss the roles of HSF4 in health and diseases, particularly in lens cell development, cataract formation, and cancer pathogenesis. Finally, we will posit the potential direction of HSF4 future research that could enhance our knowledge on HSF4 molecular networks as well as physiological and pathophysiological functions.


Assuntos
Catarata/patologia , Fatores de Transcrição de Choque Térmico/metabolismo , Neoplasias/patologia , Catarata/metabolismo , Diferenciação Celular , Fatores de Transcrição de Choque Térmico/classificação , Fatores de Transcrição de Choque Térmico/genética , Humanos , Cristalino/citologia , Cristalino/metabolismo , Neoplasias/metabolismo , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína
15.
J Biochem Mol Toxicol ; 35(7): e22789, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33847027

RESUMO

Previously, we established several facts regarding hypertension-associated cataractogenesis. As a follow-on study, we evaluated the role of the renin-angiotensin system (RAS) in angiotensin-II (Ang-II)-induced cataract formation in experimental hypertensive rats. Sprague-Dawley male albino rats (150-180 g) were used for the present experiment. The animals were divided into four groups, with six animals in each group. During the 12 weeks of the experimental protocol, the normal group received sterile water (1 ml/kg/day, subcutaneously (sc), and the Ang-II control group received angiotensin (1 mg/kg/day) subcutaneously. The ARB (O) group received olmesartan (2 mg/kg/day) orally, and the ARB (T) group received two drops of olmesartan (5 mM) topically on the cornea; concurrently, both groups were treated with Ang-II (1 mg/kg/day, sc) to induce hypertension. Biweekly, the systolic and the diastolic blood pressures were recorded, and the eyes were examined; moreover, cataractogenic parameters, such as oxidative stress markers and protein contents in the lenses, were evaluated after completion of the experimental protocol. Twelve weeks of olmesartan administered, orally or topically, significantly reduced the progression of cataract formation and restored antioxidants, lipid peroxidation, nitrite content, and protein contents in the lenses of the mice in groups O and T, respectively, as compared with those in the Ang-II control group. On the basis of our results, we conclude that the ocular RAS exacerbates the lenticular oxidative stress that may lead to cataract formation. The results showed that the RAS has an independent and important role in cataract formation under hypertensive conditions.


Assuntos
Angiotensina II/efeitos adversos , Catarata , Sistema Renina-Angiotensina/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Catarata/induzido quimicamente , Catarata/metabolismo , Catarata/patologia , Imidazóis/farmacologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Tetrazóis/farmacologia
16.
Gene ; 786: 145621, 2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-33798680

RESUMO

KPNA4 (also called importin-α3) belongs to the importin α adaptor proteins family, which orchestrates classical nuclear transport processes, importin-α/importin-ß1 pathway, and involves in cellular homeostasis. Disruption of balanced transport pathways may result in ectopic nuclear proteins and eventually cause diseases, mainly under the situation of cellular stress, such as oxidative stress. Little evidence is available on its cellular functions for high specific expression in lens. We firstly studied the role of KPNA4 in cataract formation. Lens defects were observed at an early age in kpna4 gene knockout zebrafish, generated by the CRISPR/Cas9 system. Those phenotype, including cloudy center part of the lens, via bright field microscopy, and the thinning of the LE layer, wider space between the adjacent LE and LF cells, irregular cells morphology and the increased number of holes inside the LE cells, which were detected by transmission electron microscopy, recapitulate the clinical features of cataract patients. As the p53-specific adaptor of the nuclear import, KPNA4 upregulated with the same pattern of p53 in hydrogen peroxide-induced apoptosis in human lens epithelia cells. Furthermore, the loss of Kpna4 resulted in the accumulation of p53 in the center of lens. Taken together, we showed that KPNA4 was involved in the formation of cataract, likely by mediating p53 nuclear transport.


Assuntos
Catarata/diagnóstico por imagem , Proteína Supressora de Tumor p53/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Catarata/genética , Catarata/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Inativação de Genes , Humanos , Peróxido de Hidrogênio/efeitos adversos , Cristalino/citologia , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Microscopia Eletrônica de Transmissão , Peixe-Zebra
17.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917258

RESUMO

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.


Assuntos
Catarata/tratamento farmacológico , Catarata/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/farmacologia , Animais , Catarata/diagnóstico , Catarata/etiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Olho/efeitos dos fármacos , Olho/metabolismo , Produtos Finais de Glicação Avançada/administração & dosagem , Cavalos , Humanos , Imuno-Histoquímica , Injeções Intravítreas , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/administração & dosagem , Fosfotransferases (Aceptor do Grupo Álcool)/uso terapêutico
18.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760112

RESUMO

Age-related cataract (ARC) is the primary cause of blindness worldwide. Abnormal expression of microRNAs (miRNAs/miRs) has been reported to be associated with multiple diseases, including ARC. However, the potential role of miR-124 in ARC remains unclear. The present study used the human lens epithelial cell line, SRA01/04, to investigate the potential role of miR-124 in ARC. Reverse transcription-quantitative PCR analysis was performed to detect the expression levels of miR-124, protein sprouty homolog 2 (SPRY2) and matrix metalloproteinase-2 (MMP-2) in ARC tissues, while western blotting was performed to detect the protein levels of SPRY2 and MMP-2. Cell viability and apoptosis of SRA01/04 cells were assessed via Cell Counting Kit-8 and TUNEL assays, respectively. The interaction between miR-124 and SPRY2 or MMP-2 was confirmed via the dual-luciferase reporter and RNA immunoprecipitation assays. The results of the present study demonstrated that miR-124 expression was significantly upregulated in ARC tissues, and knockdown of miR-124 increased SRA01/04 cell viability and suppressed apoptosis. In addition, SPRY2 and MMP-2 expression was decreased in ARC tissues, and were demonstrated to directly bind to miR-124. Overexpression of SPRY2 or MMP-2 increased SRA01/04 cell viability and repressed apoptosis, the effects of which were reversed following overexpression of miR-124. Taken together, these results suggested that miR-124 facilitates lens epithelial cell apoptosis by modulating SPRY2 or MMP-2 expression, providing a novel treatment approach for ARC.


Assuntos
Catarata/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cristalino/metabolismo , Metaloproteinase 2 da Matriz/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Idoso , Apoptose/genética , Catarata/metabolismo , Catarata/patologia , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Cristalino/patologia , Masculino , Pessoa de Meia-Idade
19.
Exp Eye Res ; 206: 108543, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33744257

RESUMO

Many long non-coding RNAs (lncRNAs) can exert crucial roles in the pathogenesis of cataract, including lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1). We aimed to further elucidate the biological role and regulatory molecular mechanism of KCNQ1OT1 in cataract. The expression of KCNQ1OT1 and miR-223-3p and BCL2 like 2 (BCL2L2) was examined by qRT-PCR. Cataract cell model was constructed by treatment with hydrogen peroxide (H2O2) in lens epithelial cells (SRA01/04). SRA01/04 cell viability and cell apoptosis were tested using CCK-8 assay and flow cytometry, respectively. Western blot (WB) was performed to measure the levels of apoptosis-related proteins and BCL2L2 protein. The oxidative stress factors were analyzed by corresponding kits. The interaction between miR-223-3p and KCNQ1OT1 or BCL2L2 was validated by dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. We found that KCNQ1OT1 was upregulated in cataract anterior lens capsule samples and H2O2-induced SRA01/04 cells. Knockdown of KCNQ1OT1 suppressed H2O2-induced SRA01/04 cell apoptosis and oxidative stress. KCNQ1OT1 acted as a sponge of miR-223-3p. Inhibition of miR-223-3p could abate the function of KCNQ1OT1 silence in H2O2-treated SRA01/04 cells. Additionally, BCL2L2 was a direct target of miR-223-3p, and miR-223-3p weakened H2O2-induced SRA01/04 cell apoptosis and oxidative stress by targeting BCL2L2. Collectively, the data suggest a role for the KCNQ1OT1/miR-223-3p/BCL2L2 axis in cataract formation but the data was generated using an epithelial cell line.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Catarata/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Estresse Oxidativo/genética , Idoso , Proteínas Reguladoras de Apoptose/biossíntese , Catarata/metabolismo , Catarata/patologia , Células Cultivadas , DNA/genética , Feminino , Humanos , Peróxido de Hidrogênio/toxicidade , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/biossíntese , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética
20.
Exp Eye Res ; 206: 108544, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33744256

RESUMO

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.


Assuntos
Catarata/metabolismo , Colesterol/metabolismo , Cristalino/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipídeos/metabolismo , alfa-Cristalinas/metabolismo , Catarata/patologia , Membrana Celular/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cristalino/patologia , Marcadores de Spin
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