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1.
Cell ; 155(5): 1104-18, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24238962

RESUMO

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-ß/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.


Assuntos
Senescência Celular , Desenvolvimento Embrionário , Saco Endolinfático/embriologia , Mesonefro/embriologia , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Saco Endolinfático/citologia , Feminino , Humanos , Rim/embriologia , Masculino , Mesonefro/citologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo
2.
Mamm Genome ; 34(3): 389-407, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37421464

RESUMO

The laboratory mouse is the foremost mammalian model used for studying human diseases and is closely anatomically related to humans. Whilst knowledge about human anatomy has been collected throughout the history of mankind, the first comprehensive study of the mouse anatomy was published less than 60 years ago. This has been followed by the more recent publication of several books and resources on mouse anatomy. Nevertheless, to date, our understanding and knowledge of mouse anatomy is far from being at the same level as that of humans. In addition, the alignment between current mouse and human anatomy nomenclatures is far from being as developed as those existing between other species, such as domestic animals and humans. To close this gap, more in depth mouse anatomical research is needed and it will be necessary to extent and refine the current vocabulary of mouse anatomical terms.


Assuntos
Animais Domésticos , Mamíferos , Humanos , Camundongos , Animais , Anatomia Comparada
3.
Exp Eye Res ; 212: 108801, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34688624

RESUMO

Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels.


Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética/metabolismo , Endostatinas/metabolismo , Disco Óptico/metabolismo , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Corpo Vítreo/irrigação sanguínea , Animais , Retinopatia Diabética/diagnóstico , Humanos , Masculino , Camundongos , Disco Óptico/patologia , Neovascularização Retiniana/patologia , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/diagnóstico por imagem , Corpo Vítreo/diagnóstico por imagem
4.
Exp Eye Res ; 202: 108292, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065090

RESUMO

Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and the progression of several retinopathies. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter protein, through its binding to Scara5 and TIM2 membrane receptors. In this study, the presence and iron-related functions of TIM2 in the mouse retina were investigated. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly in Müller cells. Experimental TIM2 downregulation in the mouse retina promoted, probably due to a compensatory mechanism, Scara5 overexpression that increased retinal ferritin uptake and induced iron overload. Consecutive reactive oxygen species (ROS) overproduction and vascular endothelial growth factor (VEGF) overexpression led to impaired paracellular and transcellular endothelial transport characterized by tight junction degradation and increased caveolae number. In consequence, blood-retinal barrier (BRB) breakdown and retinal edema were observed. Altogether, these results point to TIM2 as a new modulator of retinal iron homeostasis and as a potential target to counteract retinopathy.


Assuntos
Barreira Hematorretiniana/fisiologia , Células Ependimogliais/metabolismo , Ferritinas/metabolismo , Proteínas de Membrana/fisiologia , Animais , Transporte Biológico , Western Blotting , Homeostase/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Oftalmoscopia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Depuradores Classe A/metabolismo , Espectrometria por Raios X , Espectrometria de Massas em Tandem , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Mamm Genome ; 31(1-2): 49-53, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32088735

RESUMO

Design and production of genetically engineered mouse strains by individual research laboratories, research teams, large-scale consortia, and the biopharmaceutical industry have magnified the need for qualified personnel to identify, annotate, and validate (phenotype) these potentially new mouse models of human disease. The PATHBIO project has been recently established and funded by the European Union's ERASMUS+ Knowledge Alliance program to address the current shortfall in formally trained personnel. A series of teaching workshops will be given by experts on anatomy, histology, embryology, imaging, and comparative pathology to increase the availability of individuals with formal training to contribute to this important niche of Europe's biomedical research enterprise. These didactic and hands-on workshops are organized into three modules: (1) embryology, anatomy, histology, and the anatomical basis of imaging, (2) image-based phenotyping, and (3) pathology. The workshops are open to all levels of participants from recent graduates to Ph.D., M.D., and veterinary scientists. Participation is available on a competitive basis at no cost for attending. The first series of Workshop Modules was held in 2019 and these will continue for the next 2 years.


Assuntos
Pesquisa Biomédica/educação , Fenótipo , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica/organização & administração , Currículo , Modelos Animais de Doenças , Humanos , Camundongos , Pesquisadores/educação
6.
Hum Mol Genet ; 26(8): 1535-1551, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28334745

RESUMO

Gene therapy is a promising therapeutic alternative for Lysosomal Storage Disorders (LSD), as it is not necessary to correct the genetic defect in all cells of an organ to achieve therapeutically significant levels of enzyme in body fluids, from which non-transduced cells can uptake the protein correcting their enzymatic deficiency. Animal models are instrumental in the development of new treatments for LSD. Here we report the generation of the first mouse model of the LSD Muccopolysaccharidosis Type IIID (MPSIIID), also known as Sanfilippo syndrome type D. This autosomic recessive, heparan sulphate storage disease is caused by deficiency in N-acetylglucosamine 6-sulfatase (GNS). Mice deficient in GNS showed lysosomal storage pathology and loss of lysosomal homeostasis in the CNS and peripheral tissues, chronic widespread neuroinflammation, reduced locomotor and exploratory activity and shortened lifespan, a phenotype that closely resembled human MPSIIID. Moreover, treatment of the GNS-deficient animals with GNS-encoding adeno-associated viral (AAV) vectors of serotype 9 delivered to the cerebrospinal fluid completely corrected pathological storage, improved lysosomal functionality in the CNS and somatic tissues, resolved neuroinflammation, restored normal behaviour and extended lifespan of treated mice. Hence, this work represents the first step towards the development of a treatment for MPSIIID.


Assuntos
Terapia Genética , Doenças por Armazenamento dos Lisossomos/terapia , Mucopolissacaridose III/terapia , Sulfatases/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Camundongos , Mucopolissacaridose III/genética , Mucopolissacaridose III/patologia , Fenótipo , Sulfatases/administração & dosagem
7.
Hum Mol Genet ; 24(7): 2078-95, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25524704

RESUMO

Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the disease's main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB.


Assuntos
Acetilglucosaminidase/genética , Terapia Genética/métodos , Mucopolissacaridose III/genética , Mucopolissacaridose III/terapia , Acetilglucosaminidase/líquido cefalorraquidiano , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucopolissacaridose III/líquido cefalorraquidiano , Mucopolissacaridose III/enzimologia
8.
J Biol Chem ; 290(27): 16772-85, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-25971976

RESUMO

The human insulin-like growth factor 2 (IGF2) and insulin genes are located within the same genomic region. Although human genomic studies have demonstrated associations between diabetes and the insulin/IGF2 locus or the IGF2 mRNA-binding protein 2 (IGF2BP2), the role of IGF2 in diabetes pathogenesis is not fully understood. We previously described that transgenic mice overexpressing IGF2 specifically in ß-cells (Tg-IGF2) develop a pre-diabetic state. Here, we characterized the effects of IGF2 on ß-cell functionality. Overexpression of IGF2 led to ß-cell dedifferentiation and endoplasmic reticulum stress causing islet dysfunction in vivo. Both adenovirus-mediated overexpression of IGF2 and treatment of adult wild-type islets with recombinant IGF2 in vitro further confirmed the direct implication of IGF2 on ß-cell dysfunction. Treatment of Tg-IGF2 mice with subdiabetogenic doses of streptozotocin or crossing these mice with a transgenic model of islet lymphocytic infiltration promoted the development of overt diabetes, suggesting that IGF2 makes islets more susceptible to ß-cell damage and immune attack. These results indicate that increased local levels of IGF2 in pancreatic islets may predispose to the onset of diabetes. This study unravels an unprecedented role of IGF2 on ß-cells function.


Assuntos
Diabetes Mellitus/genética , Fator de Crescimento Insulin-Like II/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Desdiferenciação Celular , Linhagem Celular Tumoral , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos
9.
Exp Eye Res ; 138: 6-21, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26122048

RESUMO

The mouse retina is a commonly used animal model for the study of pathogenesis and treatment of blinding retinal vascular diseases such as diabetic retinopathy. In this study, we aimed to characterize normal and pathological variations in vascular anatomy in the mouse retina using fluorescein angiography visualized with scanning laser ophthalmoscopy and optical coherence tomography (SLO-OCT). We examined eyes from C57BL/6J wild type mice as well as the Ins2(Akita) and Akimba mouse models of diabetic retinopathy using the Heidelberg Retinal Angiography (HRA) and OCT system. Angiography was performed on three focal planes to examine distinct vascular layers. For comparison with angiographic data, ex vivo analyses, including Indian ink angiography, histology and 3D confocal scanning laser microscopy were performed in parallel. All layers of the mouse retinal vasculature could be readily visualized during fluorescein angiography by SLO-OCT. Blood vessel density was increased in the deep vascular plexus (DVP) compared with the superficial vascular plexus (SVP). Arteriolar and venular typologies were established and structural differences were observed between venular types. Unexpectedly, the hyaloid artery was found to persist in 15% of C57BL/6 mice, forming anastomoses with peripheral retinal capillaries. Fluorescein leakage was easily detected in Akimba retinae by angiography, but was not observed in Ins2(Akita) mice. Blood vessel density was increased in the DVP of 6 month old Ins2(Akita) mice, while the SVP displayed reduced branching in precapillary arterioles. In summary, we present the first comprehensive characterization of the mouse retinal vasculature by SLO-OCT fluorescein angiography. Using this clinical imaging technique, we report previously unrecognized variations in C57BL/6J vascular anatomy and novel features of vascular retinopathy in the Ins2(Akita) mouse model of diabetes.


Assuntos
Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Vasos Retinianos/patologia , Envelhecimento/patologia , Animais , Arteríolas/patologia , Biomarcadores/metabolismo , Permeabilidade Capilar , Angiofluoresceinografia , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Oftalmoscopia , Neovascularização Retiniana/patologia , Vasos Retinianos/anatomia & histologia , Tomografia de Coerência Óptica , Vênulas/patologia
10.
Clin Immunol ; 151(2): 114-26, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24576619

RESUMO

Interleukin 2 (IL2) is the key cytokine supporting survival and function of regulatory T cells (Tregs). We recently reported that low-dose IL2 safely expands/stimulates Tregs and improves autoimmune conditions in humans. Further development of IL2 in autoimmune diseases will require chronic IL2 administration, which could affect beneficial effector immune responses regulated by Tregs. We used recombinant adeno-associated viral vector (rAAV)-mediated gene transfer to continuously release IL2 in mice and assessed its long-term effects on immune responses. A single rAAV-IL2 injection enabled sustained stimulation and expansion of Tregs without inducing Teff activation and prevented diabetes in NOD mice. After several weeks of IL2 production, mice responded normally to a viral challenge and to vaccination, and had pregnancies with offspring that developed normally. They showed no change in the occurrence and growth of chemically-induced tumors. Altogether, chronic low-dose IL2 treatment does not affect beneficial effector immune responses at doses that prevent autoimmune diabetes.


Assuntos
Autoimunidade/imunologia , Infecções/imunologia , Interleucina-2/metabolismo , Neoplasias/imunologia , Linfócitos T Reguladores/fisiologia , Vacinação , Animais , Feminino , Regulação da Expressão Gênica/imunologia , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Interleucina-2/efeitos adversos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Fatores de Tempo
11.
Mamm Genome ; 25(9-10): 539-48, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24838824

RESUMO

Visualization of important disease-driving tissues in their native morphological state, such as the pancreas, given its importance in glucose homeostasis and diabetes, provides critical insight into the etiology and progression of disease and our understanding of how cellular changes impact disease severity. Numerous challenges to maintaining tissue morphology exist when one attempts to preserve or to recreate such tissues for histological evaluation. We have overcome many of these challenges and have developed new methods for visualizing the whole murine pancreas and single islets of Langerhans in an effort to gain a better understanding of how islet cell volume, spatial distribution, and vascularization are altered as diabetes progresses. These methods are readily adaptable without requirement for costly specialized equipment, such as magnetic resonance imaging, positron emission tomography, or computed tomography, and can be used to provide additional robust analysis of diabetes susceptibility in mouse models of Type 1 and Type II diabetes.


Assuntos
Imageamento Tridimensional/métodos , Imagem Molecular , Pâncreas/metabolismo , Animais , Diabetes Mellitus Experimental/diagnóstico , Glucose/metabolismo , Teste de Tolerância a Glucose , Imuno-Histoquímica , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Citometria de Varredura a Laser , Masculino , Camundongos , Tamanho do Órgão , Pâncreas/patologia
12.
Mol Metab ; 81: 101899, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38346589

RESUMO

OBJECTIVE: Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. METHODS: PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. RESULTS: PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. CONCLUSIONS: This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.


Assuntos
Doença de Depósito de Glicogênio Tipo II , Camundongos , Ratos , Humanos , Animais , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Doença de Depósito de Glicogênio Tipo II/patologia , Músculo Esquelético/metabolismo , Glicogênio/metabolismo , Terapia Genética/métodos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/terapia
13.
Mol Ther ; 20(2): 254-66, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22008915

RESUMO

Mucopolysaccharidosis type IIIA (MPSIIIA) is an inherited lysosomal storage disease caused by deficiency of sulfamidase, resulting in accumulation of the glycosaminoglycan (GAG) heparan sulfate. It is characterized by severe progressive neurodegeneration, together with somatic alterations, which lead to death during adolescence. Here, we tested the ability of adeno-associated virus (AAV) vector-mediated genetic modification of either skeletal muscle or liver to revert the already established disease phenotype of 2-month-old MPSIIIA males and females. Intramuscular administration of AAV-Sulfamidase failed to achieve significant therapeutic benefit in either gender. In contrast, AAV8-mediated liver-directed gene transfer achieved high and sustained levels of circulating active sulfamidase, which reached normal levels in females and was fourfold higher in males, and completely corrected lysosomal GAG accumulation in most somatic tissues. Remarkably, a 50% reduction of GAG accumulation was achieved throughout the entire brain of males, which correlated with a partial improvement of the pathology of cerebellum and cortex. Liver-directed gene transfer expanded the lifespan of MPSIIIA males, underscoring the importance of reaching supraphysiological plasma levels of enzyme for maximal therapeutic benefit. These results show how liver-directed gene transfer can reverse somatic and ameliorate neurological pathology in MPSIIIA.


Assuntos
Sistema Nervoso Central/patologia , Terapia Genética , Hidrolases/genética , Fígado/metabolismo , Mucopolissacaridose III/terapia , Animais , Cerebelo/ultraestrutura , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Ordem dos Genes , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Hidrolases/metabolismo , Injeções Intramusculares , Injeções Intravenosas , Fígado/ultraestrutura , Lisossomos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose III/genética , Mucopolissacaridose III/mortalidade , Músculo Esquelético/metabolismo , Análise de Sobrevida , Transdução Genética , Córtex Visual/patologia , Córtex Visual/ultraestrutura
14.
Invest Ophthalmol Vis Sci ; 64(3): 22, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36912597

RESUMO

Purpose: Iron overload causes oxidative damage in the retina, and it has been involved in the pathogeny of diabetic retinopathy, which is one of the leading causes of blindness in the adult population worldwide. However, how systemic iron enters the retina during diabetes and the role of blood retinal barrier (BRB) in this process remains unclear. Methods: The db/db mouse, a well-known model of type 2 diabetes, and a model of systemic iron overload induced by iron dextran intraperitoneal injection, were used. Perls staining and mass spectrophotometry were used to study iron content. Western blot and immunohistochemistry of iron handling proteins were performed to study systemic and retinal iron metabolism. BRB function was assessed by analyzing vascular leakage in fundus angiographies, whole retinas, and retinal sections and by studying the status of tight junctions using transmission electron microscopy and Western blot analysis. Results: Twenty-week-old db/db mice with systemic iron overload presented ferritin overexpression without iron increase in the retina and did not show any sign of BRB breakdown. These findings were also observed in iron dextran-injected mice. In those animals, after BRB breakdown induced by cryopexy, iron entered massively in the retina. Conclusions: Our results suggested that BRB protects the retina from excessive iron entry in early stages of diabetic retinopathy. Furthermore, ferritin overexpression before iron increase may prepare the retina for a potential BRB breakdown and iron entry from the systemic circulation.


Assuntos
Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Sobrecarga de Ferro , Camundongos , Animais , Retinopatia Diabética/metabolismo , Dextranos/metabolismo , Ferro/metabolismo , Ferritinas/metabolismo , Diabetes Mellitus Tipo 2/patologia , Retina/metabolismo , Barreira Hematorretiniana/metabolismo , Complexo Ferro-Dextran/toxicidade , Sobrecarga de Ferro/metabolismo
15.
Exp Eye Res ; 98: 79-87, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22484557

RESUMO

Intervascular bridges are fibrous strands that connect neighboring capillaries. These strands present associated cells, intervascular bridging cells (IBCs), whose nature and functional significance remains controversial. The aim of this study was to characterize the immunophenotype of IBCs, and contribute to understand their mechanical and intercellular communication properties in the retina. Quantification and retinal distribution of IBCs were also determined. For this purpose, C57BL/6N and nestin-GFP transgenic mice, as well as human retinas, were used. Whole-mount retinas were studied by means of immunohistochemistry and cytochemistry, and isolation of retinal vasculature was achieved by trypsin/pepsin digest technique. PAS reaction and the immunolabeling with anti-collagen IV and laminin antibodies revealed that IBCs were completely surrounded by a basement membrane, connecting two or more neighboring capillaries. IBCs were scarce and their number decreased with age. They were preferentially localized in the deep vascular plexus. In a murine model of experimental glaucoma, methylcellulose injected eyes showed retinal neovascularization and increased number of IBCs in the deep vascular plexus. IBCs were marked with anti-NG2, anti-PDGFR-ß and anti-CD34 antibodies, and with tomato lectin, and were negative for PECAM-1. IBCs expressed nestin and filamentous actin, but desmin and α-smooth muscle actin were not detected. Moreover, these cells expressed the gap junction protein connexin 43. These results showed that IBCs had a pericytic nature since they expressed NG2 and the receptor for PDGF-B, and they were negative for PECAM-1. However, they were marked with CD34 and the tomato lectin, suggesting that they constitute a special subtype of pericytes, sharing characteristics with endothelial cells. IBCs presumably present mechanical functions due to the presence of filamentous actin. Connexin 43 was found in IBCs, suggesting that these cells allow intercellular communication between adjacent capillaries. This may represent an advantage for vasomotor tone integration and coordination in blood vessels without innervation, such as those of the retina.


Assuntos
Comunicação Celular/fisiologia , Endotélio Vascular/citologia , Pericitos/citologia , Vasos Retinianos/citologia , Adulto , Idoso de 80 Anos ou mais , Animais , Antígenos/metabolismo , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Capilares/citologia , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glaucoma/patologia , Proteínas de Fluorescência Verde/genética , Humanos , Imunofenotipagem , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Nestina , Pericitos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Polissacarídeos/metabolismo , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia
16.
Nephrol Dial Transplant ; 27(8): 3296-305, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22555253

RESUMO

BACKGROUND: In renal transplantation, cold ischaemia (CI) determines acute rejection through innate immunity among others. Acute rejection episodes are a risk factor for late allograft dysfunction and proteinuria. This implies some alteration of the glomerular filtration barrier (GFB). Besides its effects on acute rejection, we hypothesized that CI might somehow damage the GFB being directly responsible for late proteinuria. METHODS: On rat kidney allografts suffering from antibody-mediated acute rejection with or without CI and compared with syngeneic grafts, we quantified the gene expression of innate and adaptive immune mediators and assessed the capillary glomerular basement membranes (CapBM) by immunostaining collagen-IV (ColIV). ColIV was also assessed in equivalent groups from a previous chronic study followed up for 24 weeks. RESULTS: CI up-regulated enzymes critical in the stabilization of collagen chains, increasing ColIV deposition and thickening the CapBM. CI increased the C4d and IgG deposits within grafts, amplified innate immunity (heat shock protein 70, fibronectin, Toll-like-receptor-4 and MyD88) and synergized with alloreactivity in triggering adaptive response through CD40. CONCLUSIONS: Initial CI increased the ColIV deposition in CapBM, damaging the GFB and being responsible for part of the proteinuria associated with late allograft dysfunction. This deterioration of the GFB is related to the early innate immunity activation and subsequent up-regulation of CD40 in acute rejected grafts. In chronic rejected allografts, thickened CapBM may be a consequence of an unresolved immune-inflammatory response worsened by CI.


Assuntos
Isquemia Fria/efeitos adversos , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , Doença Aguda , Imunidade Adaptativa , Animais , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Barreira de Filtração Glomerular/patologia , Barreira de Filtração Glomerular/fisiopatologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Rejeição de Enxerto/fisiopatologia , Imunidade Inata , Isoanticorpos/metabolismo , Transplante de Rim/imunologia , Transplante de Rim/patologia , Transplante de Rim/fisiologia , Masculino , Fator 88 de Diferenciação Mieloide/genética , Ratos , Ratos Endogâmicos BN , Ratos Wistar , Receptor 4 Toll-Like/genética , Transplante Homólogo , Transplante Isogênico
17.
PLoS Genet ; 5(8): e1000615, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19696889

RESUMO

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm(-/-)). Here, we show that Pfkm(-/-) mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm(-/-) mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm(-/-) mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm(-/-) mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies.


Assuntos
Cardiomegalia/etiologia , Doença de Depósito de Glicogênio Tipo VII/enzimologia , Doenças Hematológicas/etiologia , Músculo Esquelético/metabolismo , Fosfofrutoquinase-1/deficiência , Animais , Cardiomegalia/enzimologia , Cardiomegalia/metabolismo , Modelos Animais de Doenças , Eritrócitos/metabolismo , Feminino , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo VII/complicações , Doença de Depósito de Glicogênio Tipo VII/metabolismo , Doenças Hematológicas/enzimologia , Doenças Hematológicas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfofrutoquinase-1/genética
18.
Drug Deliv ; 29(1): 2357-2374, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35904152

RESUMO

To create a chronic glaucoma animal model by a single intracameral injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (Ms) co-loaded with dexamethasone and fibronectin (MsDexaFibro). MsDexaFibro were prepared by a water-in-oil-in-water emulsion method including dexamethasone in the organic phase and fibronectin in the inner aqueous phase. To create the chronic glaucoma model, an interventionist and longitudinal animal study was performed using forty-five Long Evans rats (4-week-old). Rats received a single intracameral injection of MsDexafibro suspension (10%w/v) in the right eye. Ophthalmological parameters such as clinical signs, intraocular pressure (IOP), neuro-retinal functionality by electroretinography (ERG), retinal structural analysis by optical coherence tomography (OCT), and histology were evaluated up to six months. According to the results obtained, the model proposed was able to induce IOP increasing in both eyes over the study, higher in the injected eyes up to 6 weeks (p < 0.05), while preserving the ocular surface. OCT quantified progressive neuro-retinal degeneration (mainly in the retinal nerve fiber layer) in both eyes but higher in the injected eye. Ganglion cell functionality decreased in injected eyes, thus smaller amplitudes in PhNR were detected by ERG. In conclusion, a new chronic glaucoma animal model was created by a single injection of MsDexaFibro very similar to open-angle glaucoma occurring in humans. This model would impact in different fields such as ophthalmology, allowing long period of study of this pathology; pharmacology, evaluating the neuroprotective activity of active compounds; and pharmaceutical technology, allowing the correct evaluation of the efficacy of long-term sustained ocular drug delivery systems.


Assuntos
Modelos Animais de Doenças , Glaucoma de Ângulo Aberto , Glaucoma , Animais , Dexametasona , Fibronectinas , Glaucoma/induzido quimicamente , Glaucoma de Ângulo Aberto/induzido quimicamente , Glicóis , Humanos , Pressão Intraocular , Microesferas , Ratos , Ratos Long-Evans , Água
19.
Biomedicines ; 9(6)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208744

RESUMO

BACKGROUND: To compare two prolonged animal models of glaucoma over 24 weeks of follow-up. A novel pre-trabecular model of chronic glaucoma was achieved by injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (10-20 µm) (Ms20/10) into the ocular anterior chamber to progressively increase ocular hypertension (OHT). METHODS: Rat right eyes were injected to induce OHT: 50% received a suspension of Ms20/10 in the anterior chamber at 0, 2, 4, 8, 12, 16 and 20 weeks, and the other 50% received a sclerosing episcleral vein injection biweekly (EPIm). Ophthalmological clinical signs, intraocular pressure (IOP), neuroretinal functionality measured by electroretinography (ERG), and structural analysis of the retina, retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) protocols using optical coherence tomography (OCT) and histological exams were performed. RESULTS: Both models showed progressive neuroretinal degeneration (p < 0.05), and contralateral eye affectation. The Ms20/10 model showed a more progressive increase in IOP and better preservation of ocular surface. Although no statistical differences were found between models, the EPIm showed a tendency to produce thicker retinal and thinner GCL thicknesses, slower latency and smaller amplitude as measured using ERG, and more aggressive disturbances in retinal histology. In both models, while the GCL showed the greatest percentage loss of thickness, the RNFL showed the greatest and earliest rate of thickness loss. CONCLUSIONS: The intracameral model with biodegradable microspheres resulted more like the conditions observed in humans. It was obtained by a less-aggressive mechanism, which allows for adequate study of the pathology over longer periods.

20.
Pharmaceutics ; 13(2)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567776

RESUMO

Progressive degeneration of neuroretinal tissue with maintained elevated intraocular pressure (IOP) to simulate chronic glaucoma was produced by intracameral injections of poly (lactic-co-glycolic) acid (PLGA) microspheres (Ms) in rat eyes. The right eye of 39 rats received different sizes of PLGA-Ms (2 µL suspension; 10% w/v): 14 with 38-20 µm Ms (Ms38/20 model) and 25 with 20-10 µm particles (Ms20/10 model). This novel glaucoma animal model was compared to the episcleral vein sclerosis (EPI) model (25 eyes). Injections were performed at baseline, two, four and six weeks. Clinical signs, IOP, retina and optic nerve thicknesses (using in vivo optical coherence tomography; OCT), and histological studies were performed. An IOP increment was observed in all three groups, however, the values obtained from the PLGA-Ms injection resulted lower with a better preservation of the ocular surface. In fact, the injection of Ms20/10 created a gentler, more progressive, and more sustained increase in IOP. This IOP alteration was correlated with a significant decrease in most OCT parameters and in histological ganglion-cell count for the three conditions throughout the eight-week follow-up. In all cases, progressive degeneration of the retina, retinal ganglion cells and optic nerve, simulating chronic glaucoma, was detected by OCT and corroborated by histological study. Results showed an alternative glaucoma model to the well-known episcleral vein model, which was simpler to perform, more reproducible and easier to monitor in vivo.

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