Differences in the thoracic aorta by region and sex in a murine model of Marfan Syndrome

Marfan syndrome (MFS) is a hereditary disorder of the connective tissue that causes life-threatening aortic aneurysm, which initiates at the aortic root and can progress into the ascending portion. However, analysis of ascending aorta reactivity in animal models of MFS has remained elusive. Epidemiologic evidence suggests that although MFS is equally prevalent in men and women, men are at a higher risk of aortic complications than non-pregnant women. Nevertheless, there is no experimental evidence to support this hypothesis. The aim of this study was to explore whether there are regional and sex differences in the thoracic aorta function of mice heterozygous for the fibrillin 1 (Fbn1) allele encoding a missense mutation (Fbn1C1039G/+), the most common class of mutation in MFS. Ascending and descending thoracic aorta reactivity was evaluated by wire myography. Ascending aorta mRNA and protein levels, and elastic fiber integrity were assessed by qRT-PCR, Western blotting, and Verhoeff-Van Gieson histological staining, respectively. MFS differently altered reactivity in the ascending and descending thoracic aorta by either increasing or decreasing phenylephrine contractions, respectively. When mice were separated by sex, contractions to phenylephrine increased progressively from 3 to 6 months of age in MFS ascending aortas of males, whereas contractions in females were unchanged. Endothelium-dependent relaxation was unaltered in the MFS ascending aorta of either sex; an effect related to augmented endothelium-dependent hyperpolarization-type dilations. In MFS males, the non-selective cyclooxygenase (COX) inhibitor indomethacin prevented the MFS-induced enhancement of phenylephrine contractions linked to increased COX-2 expression. In MFS mice of both sexes, the non-selective nitric oxide synthase inhibitor L-NAME revealed negative feedback of nitric oxide on phenylephrine contractions, which was associated with upregulation of eNOS in females. Finally, MFS ascending aortas showed a greater number of elastic fiber breaks than the wild-types, and males exhibited more breaks than females. These results show regional and sex differences in Fbn1C1039G/+ mice thoracic aorta contractility and aortic media injuries. The presence of more pronounced aortic alterations in male mice provides experimental evidence to support that male MFS patients are at increased risk of suffering aortic complications

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-β, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose, short-term experimental model

<p>Abstract</p> <p>Background</p> <p>The dust mite <it>Blomia tropicalis </it>is an important source of aeroallergens in tropical areas. Although a mouse model for <it>B. tropicalis </it>extract (<it>Bt</it>E)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol.</p> <p>Objectives</p> <p>This work had two objectives. The first was to study the anti-<it>B. tropicalis </it>allergic responses in different mouse strains using a short-term model of respiratory allergy to <it>Bt</it>E. This study included the comparison of the allergic responses elicited by <it>Bt</it>E with those elicited by ovalbumin in mice of the strain that responded better to <it>Bt</it>E sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low <it>Bt</it>E doses.</p> <p>Methods</p> <p>Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 μg of <it>Bt</it>E on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 μg of <it>Bt</it>E. A/J mice, that were the best responders to <it>Bt</it>E sensitization, were used to compare the <it>B. tropicalis</it>-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of <it>Bt</it>E.</p> <p>Results</p> <p>Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-<it>Bt</it>E IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with <it>Bt</it>E induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low <it>Bt</it>E dose (10 μg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose <it>Bt</it>E immunization, for the development of allergy-associated immune and lung inflammatory responses.</p> <p>Conclusions</p> <p>The described short-term model of <it>Bt</it>E-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and <it>Bt</it>E induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of <it>Bt</it>E.</p

Early impairment of lung mechanics in a murine model of Marfan syndrome

Early morbidity and mortality in patients with Marfan syndrome (MFS) -a connective tissue disease caused by mutations in fibrillin-1 gene- are mainly caused by aorta aneurysm and rupture. However, the increase in the life expectancy of MFS patients recently achieved by reparatory surgery promotes clinical manifestations in other organs. Although some studies have reported respiratory alterations in MFS, our knowledge of how this connective tissue disease modifies lung mechanics is scarce. Hence, we assessed whether the stiffness of the whole lung and of its extracellular matrix (ECM) is affected in a well-characterized MFS mouse model (FBN1 C1039G/+ ). The stiffness of the whole lung and of its ECM were mea- sured by conventional mechanical ventilation and atomic force microscopy, respectively. We studied 5-week and 9-month old mice, whose ages are representative of early and late stages of the disease. At both ages, the lungs of MFS mice were significantly more compli- ant than in wild type (WT) mice. By contrast, no significant differences were found in local lung ECM stiffness. Moreover, histopathological lung evaluation showed a clear emphyse- matous-like pattern in MFS mice since alveolar space enlargement was significantly increased compared with WT mice. These data suggest that the mechanism explaining the increased lung compliance in MFS is not a direct consequence of reduced ECM stiffness, but an emphysema-like alteration in the 3D structural organization of the lung. Since lung alterations in MFS are almost fully manifested at an early age, it is suggested that respira- tory monitoring could provide early biomarkers for diagnosis and/or follow-up of patients with the Marfan syndrom

Effect of the Antioxidant Lipoic Acid in Aortic Phenotype in a Marfan Syndrome Mouse Model

Marfan syndrome (MFS) cardiovascular manifestations such as aortic aneurysms and cardiomyopathy carry substantial morbidity/mortality. We investigated the effects of lipoic acid, an antioxidant, on ROS production and aortic remodeling in a MFS mgΔloxPneo mouse model. MFS and WT (wild-type) 1-month-old mice were allocated to 3 groups: untreated, treated with losartan, and treated with lipoic acid. At 6 months old, echocardiography, ROS production, and morphological analysis of aortas were performed. Aortic ROS generation in 6-month-old MFS animals was higher at advanced stages of disease in MFS. An unprecedented finding in MFS mice analyzed by OCT was the occurrence of focal inhomogeneous regions in the aortic arch, either collagen-rich extremely thickened or collagen-poor hypotrophic regions. MFS animals treated with lipoic acid showed markedly reduced ROS production and lower ERK1/2 phosphorylation; meanwhile, aortic dilation and elastic fiber breakdown were unaltered. Of note, lipoic acid treatment associated with the absence of focal inhomogeneous regions in MFS animals. Losartan reduced aortic dilation and elastic fiber breakdown despite no change in ROS generation. In conclusion, oxidant generation by itself seems neutral with respect to aneurysm progression in MFS; however, lipoic acid-mediated reduction of inhomogeneous regions may potentially associate with less anisotropy and reduced chance of dissection/rupture

Micropartículas derivadas de monócitos como potenciais direcionadoras de mieloperoxidase para superfície endotelial

Submitted by Repositório Arca ([email protected]) on 2019-08-06T11:28:06Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-08-20T13:49:26Z (GMT) No. of bitstreams: 2 Thayna Meirelles Santos. Microparticulas derivadas de monocitos como potenciais direcionadoras de mieloperoxidase....pdf: 696920 bytes, checksum: a90b6637d582c9ea3e8f39896a504689 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-20T13:49:26Z (GMT). No. of bitstreams: 2 Thayna Meirelles Santos. Microparticulas derivadas de monocitos como potenciais direcionadoras de mieloperoxidase....pdf: 696920 bytes, checksum: a90b6637d582c9ea3e8f39896a504689 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.A Mieloperoxidase (MPO) é uma enzima catiônica presente nos grânulos azurófilos de neutrófilos e monócitos responsável pela formação de espécies reativas de oxigênio (ROS) envolvidas na diminuição da biodisponibilidade do NO, modulando a resposta inflamatória vascular. Micropartículas (MPs) são vesículas derivadas da membrana plasmática, durante ativação celular ou apoptose, e atuam como mensageiras intercelulares por interação específica com as células alvo. MPs de diversas origens parecem afetar a função vascular por mecanismos dependentes do estresse oxidativo. MPs derivadas de monócitos humanos (MoMPs) foram produzidas por ativação celular in vitro com ionóforo de cálcio e caracterizadas por microscopia eletrônica de transmissão como estruturas delimitadas por bimembrana lipídica e diâmetro característico menor que 111m. Detectamos por quimioluminescência, em reação dependente de luminol e H202, inibida por azida sódica, atividade de mieloperoxidase associada às MoMPs. A exposição das MoMPs a pH 10,7 ou ao aumento da força iônica do meio resultou em dissociação da MPO da sua superfície, enquanto nenhuma dissociação ocorreu após exposição a pH 3,0, demonstrando que esta associação depende de interações eletrostáticas. A MPO não foi capaz de religar-se nas MoMPs após tratamento com pH 10,7, indicando a necessidade de uma determinada distribuição de cargas na superfície das MoMPs. Além disso, não foi capaz de se ligar na superfície de linfócitos e hemácias, sugerindo a participação de molécula(s) específica(s) nas MoMPs responsáveis por esta interação. Identificamos a expressão das integrinas LF A-I e MAC-I na superfície das MoMPs ricas em MPO por citometria de fluxo, demonstrando seu potencial em aderir especificamente à superfície do endotélio ativado. Estes resultados sugerem que as MPs derivadas de monócitos podem atuar como carreadoras da MPO para superfície endotelial, sugerindo assim sua contribuição na disfunção do endotélio vascular.The myeloperoxidase (MPO) is a cationic enzyme present in azurophilic granules of neutrophils and monocytes. This enzyme is responsible for the formation of reactive oxygen species (ROS) involved in decreased NO bioavailability and modulates the vascular inflammatory response. Microparticles (MPs) are shed membrane vesicles arising from cell activation or apoptosis, and act as intercellular messengers, specifically interact with target cells. MPs from various cells could affect vascular function through mechanisms triggered by oxidative stress. Monocytes derived MPs (MoMPs) were obtained from calcium ionophore activated human monocytes in vitro and characterized by transmission electron microscopy as vesicles bounded by bilayer lipid membrane and diameter less than 1 Km. We detected by chemiluminescence MPO activity MoMPs associated, by luminol with added H2O2 and inhibited by sodium azide. Treating of MoMPs with buffer at pH 10.7 or increased ionic strength resulted in releasing of the MPO from the MPs surface, whereas no release occurred with buffer at pH 3.0, demonstrating that nature of association between MPO and these MPs is electrostatic. MPO was not able to re-attach to MoMPs after treatment with pH 10.7, indicating the need for a specific charge distribution on the MoMPs surface. Moreover, MPO was not able to bind on the lymphocytes and erythrocytes surface, suggesting the involvement of specific (s) molecule (s) in MoMPs responsible for this association. We identified the expression of integrins LFA-1 and MAC-1 on the MPO enrich MoMPs surface by flow cytometry, demonstrating their potential to adhere specifically to activated endothelium adding the MPO to them, suggesting their contribution to endothelial dysfunction

Mutated fibrillin-1 intracellular processing in Marfan syndrome: bypass of a protein disulfide isomerase-mediated quality control

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT, mas não SMF. Além disso, o silenciamento da PDI promoveu a desorganização da matriz extracelular depositada de fibrilina-1 nos MEFs WT, enquanto nos MEFs SMF, a desorganização basal da matriz não foi adicionalmente alterada. Em paralelo, investigações in vivo mostraram que o estresse do RE não é induzido em camundongos SMF com 1 ou 3 meses de idade, apesar de manifestações fenotípicas evidentes. Entretanto, concomitante à progressão da doença, detectamos a ocorrência de estresse do RE nas aortas ascendentes dos camundongos aos 6 meses. Esta detecção foi exclusiva desta região da aorta e não ocorreu em outros órgãos afetados ou não afetados pela SMF. Assim, a manifestação do fenótipo clássico da SMF não requer uma perda da homeostase do RE diretamente induzida pela fibrilina-1 mutada. Ao contrário, esta é capaz de evadir mecanismos de controle de qualidade mediados pela PDI, sendo secretada normalmente. Assim, esta evasão do controle de qualidade pela PDI é uma condição permissiva essencial para o fenótipo da SMF. Por outro lado, o estresse do RE é uma característica evolutiva do aneurisma da aorta ascendente na SMF concomitante ao agravamento do fenótipo neste tecidoMarfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT, but not MFS MEFs. Besides, PDI silencing promoted fibrillin-1 extracellular matrix disorganization in WT MEFs, whereas in MFS MEFs, the basal matrix disorganization was not further modified. Parallel in vivo evaluations demonstrated that ER stress is also not induced in 1 and 3 month-old mice MFS, despite evident phenotypical manifestations. However, concomitant to accelerated disease progression at 6 months, ER stress was detectable in ascendant aorta, but not in other disease-affected or unaffected organs. Thus, classic MFS phenotype manifestations do not require loss of ER homeostasis directly induced by mutated fibrillin-1. Contrarily, the latter can evade a PDI-mediated quality control mechanism to be normally secreted. Therefore, evading such PDI-mediated quality control is an essential permissive condition for enabling the MFS phenotype. On the other hand, ER stress is an evolutive feature of MFS ascendant aorta aneurysm concomitant to phenotype progression in this tissu

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-β, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS

Differences in the Thoracic Aorta by Region and Sex in a Murine Model of Marfan Syndrome

Marfan syndrome (MFS) is a hereditary disorder of the connective tissue that causes life-threatening aortic aneurysm, which initiates at the aortic root and can progress into the ascending portion. However, analysis of ascending aorta reactivity in animal models of MFS has remained elusive. Epidemiologic evidence suggests that although MFS is equally prevalent in men and women, men are at a higher risk of aortic complications than non-pregnant women. Nevertheless, there is no experimental evidence to support this hypothesis. The aim of this study was to explore whether there are regional and sex differences in the thoracic aorta function of mice heterozygous for the fibrillin 1 (Fbn1) allele encoding a missense mutation (Fbn1C1039G/+), the most common class of mutation in MFS. Ascending and descending thoracic aorta reactivity was evaluated by wire myography. Ascending aorta mRNA and protein levels, and elastic fiber integrity were assessed by qRT-PCR, Western blotting, and Verhoeff-Van Gieson histological staining, respectively. MFS differently altered reactivity in the ascending and descending thoracic aorta by either increasing or decreasing phenylephrine contractions, respectively. When mice were separated by sex, contractions to phenylephrine increased progressively from 3 to 6 months of age in MFS ascending aortas of males, whereas contractions in females were unchanged. Endothelium-dependent relaxation was unaltered in the MFS ascending aorta of either sex; an effect related to augmented endothelium-dependent hyperpolarization-type dilations. In MFS males, the non-selective cyclooxygenase (COX) inhibitor indomethacin prevented the MFS-induced enhancement of phenylephrine contractions linked to increased COX-2 expression. In MFS mice of both sexes, the non-selective nitric oxide synthase inhibitor L-NAME revealed negative feedback of nitric oxide on phenylephrine contractions, which was associated with upregulation of eNOS in females. Finally, MFS ascending aortas showed a greater number of elastic fiber breaks than the wild-types, and males exhibited more breaks than females. These results show regional and sex differences in Fbn1C1039G/+ mice thoracic aorta contractility and aortic media injuries. The presence of more pronounced aortic alterations in male mice provides experimental evidence to support that male MFS patients are at increased risk of suffering aortic complications