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1.
Am J Med Genet A ; 179(8): 1408-1409, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31355539
2.
Mol Genet Metab ; 111(2): 63-72, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24388732

RESUMO

The mucopolysaccharidoses (MPS), a group of rare genetic disorders caused by defects in glycosaminoglycan (GAG) catabolism, are progressive, multi-systemic diseases with a high burden of morbidity. Enzyme replacement therapy (ERT) is available for MPS I, II, and VI, and may improve walking ability, endurance, and pulmonary function as evidenced by data from pivotal trials and extension studies. Despite these demonstrable benefits, cardiac valve disease, joint disease, and skeletal disease, all of which cause significant morbidity, do not generally improve with ERT if pathological changes are already established. Airway disease improves, but usually does not normalize. These limitations can be well understood by considering the varied functions of GAG in the body. Disruption of GAG catabolism has far-reaching effects due to the triggering of secondary pathogenic cascades. It appears that many of the consequences of these secondary pathogenic events, while they may improve on treatment, cannot be fully corrected even with long-term exposure to enzyme, thereby supporting the treatment of patients with MPS before the onset of clinical disease. This review examines the data from clinical trials and other studies in human patients to explore the limits of ERT as currently used, then discusses the pathophysiology, fetal tissue studies, animal studies, and sibling reports to explore the question of how early to treat an MPS patient with a firm diagnosis. The review is followed by an expert opinion on the rationale for and the benefits of early treatment.


Assuntos
Disostoses/tratamento farmacológico , Terapia de Reposição de Enzimas , Iduronato Sulfatase/uso terapêutico , Mucopolissacaridoses/tratamento farmacológico , N-Acetilgalactosamina-4-Sulfatase/uso terapêutico , Prevenção Secundária , Pré-Escolar , Ensaios Clínicos como Assunto , Disostoses/complicações , Disostoses/enzimologia , Disostoses/fisiopatologia , Glicosaminoglicanos/metabolismo , Valvas Cardíacas/efeitos dos fármacos , Valvas Cardíacas/enzimologia , Valvas Cardíacas/fisiopatologia , Humanos , Articulações/efeitos dos fármacos , Articulações/enzimologia , Articulações/fisiopatologia , Mucopolissacaridoses/complicações , Mucopolissacaridoses/enzimologia , Mucopolissacaridoses/fisiopatologia , Proteínas Recombinantes/uso terapêutico , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/enzimologia , Sistema Respiratório/fisiopatologia
3.
Tissue Eng Part A ; 19(21-22): 2339-48, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23672462

RESUMO

The immune response due to Galα1,3-Galß1-4GlcNAc-R(α-Gal) epitopes is an important factor in bioprosthetic heart valve failure. The aim of this study was to evaluate the immune reaction and anticalcification effect of α-galactosidase and decellularization for glutaraldehyde (GA)/genipin fixed bovine pericardium using α1,3-galactosyltransferase knockout(α-Gal KO) mouse(C57BL/6). Bovine pericardial tissues were decellularized and treated with α-galactosidase before fixation with 0.25% GA/0.4% genipin in organic solvent (75% ethanol and 5% octanol) and treatment with glycine. The removal of α-gal epitope from the bovine pericardium was analyzed by 3,3'-Diaminobenzidine staining intensity. The bovine pericardial tissues were subcutaneously implanted into wild type mice (n=19) and α-Gal KO mice (n=66), which had been presensitized with rabbit red blood cells to maximize immunologic response or not, and anti α-Gal antibodies were measured at various time intervals. Calcium contents of the explanted tissues (n=104) were measured 3 months after implantation. The treatment of α-galactosidase effectively removed the α-gal epitopes expressed on bovine pericardial tissues. In both GA and genipin groups, titers for both anti α-Gal IgM and IgG of α-Gal KO mice increased according to the duration of implantation, and were lower in the groups with decellularization than without decellularization, and were lower in the groups with α-galactosidase+decellularization than with decellularization. The calcium contents of GA/genipin fixed tissues were lower in the groups with decellularization than without decellularization, and were lower in the groups with α-galactosidase+decellularization than with decellularization. Treatment of α-galactosidase with decellularization is useful for removal of the immunogenicity, and reduced calcification in both GA and genipin fixed bovine pericardia, supporting the hypothesis that the immune reaction may cause the calcification. Treatment of α-galactosidase has possible promise to enhance durability of bioprosthetic heart valve. To our knowledge, this is the first report that demonstrates the in vivo efficacy of α-galactosidase using presensitized α-Gal KO mouse to mimic the human immunologic environment.


Assuntos
Bioprótese , Galactosiltransferases/deficiência , Valvas Cardíacas/enzimologia , alfa-Galactosidase/uso terapêutico , Animais , Cálcio , Bovinos , Ensaio de Imunoadsorção Enzimática , Galactosiltransferases/genética , Técnicas In Vitro , Iridoides/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pericárdio/citologia , Pericárdio/metabolismo
4.
J Mol Cell Cardiol ; 60: 107-15, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23624088

RESUMO

Neuronal nitric oxide synthase (NOS1 or nNOS) exerts negative inotropic and positive lusitropic effects through Ca(2+) handling processes in cardiac myocytes from healthy hearts. However, underlying mechanisms of NOS1 in diseased hearts remain unclear. The present study aims to investigate this question in angiotensin II (Ang II)-induced hypertensive rat hearts (HP). Our results showed that the systolic function of left ventricle (LV) was reduced and diastolic function was unaltered (echocardiographic assessment) in HP compared to those in shams. In isolated LV myocytes, contraction was unchanged but peak [Ca(2+)]i transient was increased in HP. Concomitantly, relaxation and time constant of [Ca(2+)]i decay (tau) were faster and the phosphorylated fraction of phospholamban (PLN-Ser(16)/PLN) was greater. NOS1 protein expression and activity were increased in LV myocyte homogenates from HP. Surprisingly, inhibition of NOS1 did not affect contraction but reduced peak [Ca(2+)]i transient; prevented faster relaxation without affecting the tau of [Ca(2+)]i transient or PLN-Ser(16)/PLN in HP, suggesting myofilament Ca(2+) desensitization by NOS1. Indeed, relaxation phase of the sarcomere length-[Ca(2+)]i relationship of LV myocytes shifted to the right and increased [Ca(2+)]i for 50% of sarcomere shortening (EC50) in HP. Phosphorylations of cardiac myosin binding protein-C (cMyBP-C(282) and cMyBP-C(273)) were increased and cardiac troponin I (cTnI(23/24)) was reduced in HP. Importantly, NOS1 or PKG inhibition reduced cMyBP-C(273) and cTnI(23/24) and reversed myofilament Ca(2+) sensitivity. These results reveal that NOS1 is up-regulated in LV myocytes from HP and exerts positive lusitropic effect by modulating myofilament Ca(2+) sensitivity through phosphorylation of key regulators in sarcomere.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Regulação Enzimológica da Expressão Gênica , Hipertensão/enzimologia , Miocárdio/enzimologia , Miofibrilas/enzimologia , Óxido Nítrico Sintase Tipo I/biossíntese , Animais , Células Cultivadas , Valvas Cardíacas/enzimologia , Valvas Cardíacas/patologia , Hipertensão/patologia , Camundongos , Contração Miocárdica , Miocárdio/patologia , Miofibrilas/patologia , Óxido Nítrico Sintase Tipo I/genética , Ratos , Ratos Sprague-Dawley
5.
Indian J Med Res ; 137(1): 203-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23481074

RESUMO

BACKGROUND & OBJECTIVES: Studies have demonstrated the effect of CYP2C9 (cytochrome P450) and VKORC1 (vitamin K epoxide reductase complex) gene polymorphisms on the dose of acenocoumarol. The data from India about these gene polymorphisms and their effects on acenocoumarol dose are scarce. The aim of this study was to determine the occurrence of CYP2C9*2,*3 and VKORC 1 -1639G>A gene polymorphisms and to study their effects on the dose of acenocoumarol required to maintain a target International Normalized Ratio (INR) in patients with mechanical heart valve replacement. METHODS: Patients from the anticoagulation clinic of a tertiary care hospital in north India were studied. The anticoagulation profile, INR (International Normalized Ratio) values and administered acenocoumarol dose were obtained from the clinical records of patients. Determination of the CYP2C9*2,*3 and VKORC1 -1639G>A genotypes was done by PCR-RFLP (restriction fragment length polymorphism). RESULTS: A total of 111 patients were studied. The genotype frequencies of CYP2C9 *1/*1,*1/*2,*1/*3 were as 0.883, 0.072, 0.036 and that of VKORC1 -1639G>A for GG, AG, and AA genotypes were 0.883, 0.090, and 0.027, respectively. The percentage of patients carrying any of the variant alleles of CYP2C9 and VKORC1 in heterozygous or homozygous form was 34% among those receiving a low dose of ≤20 mg/wk while it was 13.8 per cent in those receiving >20 mg/wk (P=0.014). A tendency of lower dose requirements was seen among carriers of the studied polymorphisms. There was considerable variability in the dose requirements of patients with and without variant alleles. INTERPRETATION & CONCLUSIONS: The study findings point towards the role of CYP2C9 and VKORC1 gene polymorphisms in determining the inter-individual dose variability of acenocoumarol in the Indian patients with mechanical heart valve replacement.


Assuntos
Acenocumarol/administração & dosagem , Anticoagulantes/administração & dosagem , Hidrocarboneto de Aril Hidroxilases/genética , Vitamina K Epóxido Redutases/genética , Adulto , Alelos , Biomarcadores Farmacológicos , Citocromo P-450 CYP2C9 , Feminino , Genótipo , Implante de Prótese de Valva Cardíaca , Valvas Cardíacas/efeitos dos fármacos , Valvas Cardíacas/enzimologia , Valvas Cardíacas/cirurgia , Humanos , Índia , Coeficiente Internacional Normatizado , Masculino , Polimorfismo Genético
6.
Am J Pathol ; 182(5): 1922-31, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23499458

RESUMO

Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC-VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and α-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology.


Assuntos
Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/fisiopatologia , Valva Aórtica/patologia , Calcinose/patologia , Calcinose/fisiopatologia , Células Endoteliais/patologia , Hemodinâmica , Óxido Nítrico/metabolismo , Transdução de Sinais , Animais , Valva Aórtica/enzimologia , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/enzimologia , Calcinose/enzimologia , Diferenciação Celular , Géis , Valvas Cardíacas/enzimologia , Valvas Cardíacas/patologia , Humanos , Imuno-Histoquímica , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Reação em Cadeia da Polimerase em Tempo Real , Coloração e Rotulagem , Sus scrofa
7.
J Biol Chem ; 287(39): 32708-16, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22815472

RESUMO

Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects.


Assuntos
Cardiopatias Congênitas/enzimologia , Doenças das Valvas Cardíacas/enzimologia , Proteínas Musculares/metabolismo , Mutação de Sentido Incorreto , Polimorfismo Genético , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Estabilidade Enzimática/genética , Escherichia coli , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Doenças das Valvas Cardíacas/embriologia , Doenças das Valvas Cardíacas/genética , Valvas Cardíacas/embriologia , Valvas Cardíacas/enzimologia , Proteínas Musculares/genética , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uridina Difosfato Glucose Desidrogenase , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
8.
J Proteome Res ; 11(3): 1537-50, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22276806

RESUMO

Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In this work, aortic valve samples were collected from 20 patients that underwent aortic valve replacement (55% males, mean age of 74 years) and 20 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by quantitative differential electrophoresis (2D-DIGE) and mass spectrometry, and 35 protein species were clearly increased in aortic valves, including apolipoprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alfa-1-glycoprotein, vimentin, superoxide dismutase Cu-Zn, serum amyloid P-component, glutathione S-transferase-P, fatty acid-binding protein, transthyretin, and fibrinogen gamma. By contrast, 8 protein species were decreased (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, and calreticulin). All of the proteins identified play a significant role in cardiovascular processes, such as fibrosis, homeostasis, and coagulation. The significant changes observed in the abundance of key cardiovascular proteins strongly suggest that they can be involved in the pathogenesis of degenerative aortic stenosis. Further studies are warranted to better understand this process before we can attempt to modulate it.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valvas Cardíacas/metabolismo , Proteoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/enzimologia , Estenose da Valva Aórtica/patologia , Western Blotting , Estudos de Casos e Controles , Proteínas do Citoesqueleto/metabolismo , Feminino , Valvas Cardíacas/enzimologia , Valvas Cardíacas/patologia , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Componente Principal , Proteômica , Eletroforese em Gel Diferencial Bidimensional
9.
Acta Biomater ; 7(4): 1728-34, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21118731

RESUMO

Decellularized xenograft heart valves might be the ideal scaffolds for tissue engineered heart valves as the alternative to the currently used biological and mechanical prostheses. However, removal of the alpha-Gal epitope is a prerequisite to avoid hyperacute rejection of untreated xenograft material. The aim of this study was to develop an ELISA soft-tissue assay for alpha-Gal quantification in xenograft heart valves before and after a detergent-based (TriCol) or equivalent cell removal procedure. Leaflets from porcine valves were enzymatically digested to expose the epitope and reacted with the alpha-Gal monoclonal antibody M86 for its recognition. Rabbit erythrocytes were used as a reference for the quantification of alpha-Gal. Native aortic and pulmonary leaflets exhibited different epitope concentration: 4.33×10(11) vs. 7.12×10(11)/10 mg wet tissue (p<0.0001). Sampling of selected zones in native valves revealed a different alpha-Gal distribution within and among different leaflets. The pattern was consistent with immunofluorescence analysis and was unrelated to microvessel density distribution. After TriCol treatment alpha-Gal was no longer detectable in both pulmonary and aortic decellularized valves, confirming the ability of this method to remove both cells and alpha-Gal antigen. These results hold promise for a reliable quantitative evaluation of alpha-Gal in decellularized valves obtained from xenograft material for tissues engineering purposes. Additionally, this method is applicable to further evaluate currently used xenograft bioprostheses.


Assuntos
Ensaios Enzimáticos/métodos , Epitopos/imunologia , Galactosiltransferases/imunologia , Valvas Cardíacas/citologia , Valvas Cardíacas/enzimologia , Transplante Heterólogo , Animais , Anticorpos/imunologia , Antígenos/imunologia , Separação Celular , Imunofluorescência , Lectinas/metabolismo , Coelhos , Sus scrofa
10.
Mol Cell Biol ; 30(14): 3711-21, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20457809

RESUMO

One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.


Assuntos
Coração Fetal/embriologia , Coração Fetal/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Apoptose , Nó Atrioventricular/anormalidades , Nó Atrioventricular/embriologia , Nó Atrioventricular/enzimologia , Sequência de Bases , Primers do DNA/genética , Técnicas de Cultura Embrionária , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Epitélio/embriologia , Epitélio/enzimologia , Feminino , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/enzimologia , Cardiopatias Congênitas/genética , Valvas Cardíacas/anormalidades , Valvas Cardíacas/embriologia , Valvas Cardíacas/enzimologia , Mesoderma/embriologia , Mesoderma/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/metabolismo , Gravidez , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 297(4): H1354-60, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19666839

RESUMO

Heart valve disease and pulmonary hypertension, in patients with carcinoid tumors and people who used the fenfluramine-phentermine combination for weight control, have been associated with high levels of serotonin in blood. The mechanism by which serotonin induces valvular changes is not well understood. We recently reported that increased oxidative stress is associated with valvular changes in aortic valve stenosis in humans and mice. In this study, we tested the hypothesis that serotonin induces oxidative stress in human heart valves, and examined mechanisms by which serotonin may increase reactive oxygen species. Superoxide (O2*.-) was measured in heart valves from explanted human hearts that were not used for transplantation. (O2*.-) levels (lucigenin-enhanced chemoluminescence) were increased in homogenates of cardiac valves and blood vessels after incubation with serotonin. A nonspecific inhibitor of flavin-oxidases (diphenyliodonium), or inhibitors of monoamine oxidase [MAO (tranylcypromine and clorgyline)], prevented the serotonin-induced increase in (O2*.-). Dopamine, another MAO substrate that is increased in patients with carcinoid syndrome, also increased (O2*.-) levels in heart valves, and this effect was attenuated by clorgyline. Apocynin [an inhibitor of NAD(P)H oxidase] did not prevent increases in (O2*.-) during serotonin treatment. Addition of serotonin to recombinant human MAO-A generated (O2*.-), and this effect was prevented by an MAO inhibitor. In conclusion, we have identified a novel mechanism whereby MAO-A can contribute to increased oxidative stress in human heart valves and pulmonary artery exposed to serotonin and dopamine.


Assuntos
Valvas Cardíacas/enzimologia , Monoaminoxidase/metabolismo , Estresse Oxidativo , Serotonina/metabolismo , Superóxidos/metabolismo , Antioxidantes/farmacologia , Dopamina/metabolismo , Valvas Cardíacas/efeitos dos fármacos , Humanos , Inibidores da Monoaminoxidase/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes/metabolismo
12.
Eur Heart J ; 30(18): 2254-65, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19561339

RESUMO

AIMS: To increase the supply, many countries harvest allograft valves from explanted hearts of transplant recipients with ischaemic (ICM) or dilated cardiomyopathy (DCM). This study determines the structural integrity of valves from cardiomyopathic hearts. METHODS AND RESULTS: Extracellular matrix (ECM) was examined in human valves obtained from normal, ICM, and DCM hearts. To confirm if ECM changes were directly related to the cardiomyopathy, we developed a porcine model of chronic ICM. Histology and immunohistostaining, as well as non-invasive multiphoton and second harmonic generation (SHG) imaging revealed marked disruption of ECM structures in human valves from ICM and DCM hearts. The ECM was unaffected in valves from normal and acute ICM pigs, whereas chronic ICM specimens showed ECM alterations similar to those seen in ICM and DCM patients. Proteins and proteinases implicated in ECM remodelling, including Tenascin C, TGFbeta1, Cathepsin B, MMP2, were upregulated in human ICM and DCM, and porcine chronic ICM specimens. CONCLUSION: Valves from cardiomyopathic hearts showed significant ECM deterioration with a disrupted collagen and elastic fibre network. It will be important to determine the impact of this ECM damage on valve durability and calcification in vivo if allografts are to be used from these donors.


Assuntos
Cardiomiopatias/patologia , Matriz Extracelular/patologia , Valvas Cardíacas/patologia , Idoso , Animais , Cardiomiopatias/enzimologia , Doença Crônica , Matriz Extracelular/enzimologia , Feminino , Gelatinases/metabolismo , Valvas Cardíacas/enzimologia , Humanos , Masculino , Microscopia de Fluorescência por Excitação Multifotônica , Pessoa de Meia-Idade , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Suínos
13.
Proc Natl Acad Sci U S A ; 106(12): 4736-41, 2009 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-19251646

RESUMO

Noonan syndrome (NS), the most common single-gene cause of congenital heart disease, is an autosomal dominant disorder that also features proportionate short stature, facial abnormalities, and an increased risk of myeloproliferative disease. Germline-activating mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, cause about half of NS cases; other causative alleles include KRAS, SOS1, and RAF1 mutants. We showed previously that knock-in mice bearing the NS mutant Ptpn11(D61G) on a mixed 129S4/SvJae X C57BL6/J background exhibit all major NS features, including a variety of cardiac defects, with variable penetrance. However, the cellular and molecular mechanisms underlying NS cardiac defects and whether genetic background and/or the specific NS mutation contribute to the NS phenotype remained unclear. Here, using an inducible knock-in approach, we show that all cardiac defects in NS result from mutant Shp2 expression in the endocardium, not in the myocardium or neural crest. Furthermore, the penetrance of NS defects is affected by genetic background and the specific Ptpn11 allele. Finally, ex vivo assays and pharmacological approaches show that NS mutants cause cardiac valve defects by increasing Erk MAPK activation, probably downstream of ErbB family receptor tyrosine kinases, extending the interval during which cardiac endocardial cells undergo endocardial-mesenchymal transformation. Our data provide a mechanistic underpinning for the cardiac defects in this disorder.


Assuntos
Endocárdio/enzimologia , Endocárdio/patologia , Cardiopatias Congênitas/enzimologia , Mesoderma/enzimologia , Mesoderma/patologia , Síndrome de Noonan/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Alelos , Animais , Coxins Endocárdicos/enzimologia , Coxins Endocárdicos/patologia , Ativação Enzimática , Técnicas de Introdução de Genes , Cardiopatias Congênitas/patologia , Valvas Cardíacas/anormalidades , Valvas Cardíacas/embriologia , Valvas Cardíacas/enzimologia , Camundongos , Mutação/genética , Organogênese , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo
15.
Dev Dyn ; 235(1): 94-104, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16342113

RESUMO

Rho-associated kinase (ROCK) is a downstream effector of small Rho-GTPases, and phosphorylates several substrates to regulate cell functions, including actin cytoskeletal reorganization and cellular motility. Endothelial-mesenchymal transformation (EMT) is a critical event in the formation of valves and septa during cardiogenesis. It has been reported that ROCK plays an important role in the regulation of endocardial cell differentiation and migration during mouse cardiogenesis (Zhao and Rivkees [2004] Dev. Biol. 275:183-191). Immunohistochemistry showed that, during chick cardiogenesis, ROCK1 and -2 were expressed in the transforming and migrating endothelial/mesenchymal cells in the outflow tract (OT) and atrioventricular (AV) canal regions from which valvuloseptal endocardial cushion tissue would later develop. Treatment with Y27632, a specific ROCK inhibitor, of cultured AV explants or AV endothelial monolayers of stage 14-minus heart (preactivated stage for EMT) on three-dimensional collagen gel perturbed the seeding of mesenchymal cells into the gel lattice. In these experiments, Y27632 did not suppress the expression of an early transformation marker, smooth muscle alpha-actin. Moreover, Y27632 inhibited the mesenchymal invasion in stage 14-18 AV explants, in which endothelial cells had committed to undergo EMT. ML-9, a myosin light chain kinase inhibitor, also inhibited the mesenchymal invasion in cultured AV explants. These results suggest that ROCKs have a critical role in the mesenchymal cell invasion/migration that occurs at the late onset of EMT.


Assuntos
Movimento Celular/fisiologia , Endocárdio/enzimologia , Septos Cardíacos/enzimologia , Valvas Cardíacas/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Biomarcadores , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Endocárdio/citologia , Endocárdio/embriologia , Septos Cardíacos/citologia , Septos Cardíacos/embriologia , Valvas Cardíacas/citologia , Valvas Cardíacas/embriologia , Peptídeos e Proteínas de Sinalização Intracelular , Mesoderma/metabolismo , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Quinases Associadas a rho
16.
J Histochem Cytochem ; 53(5): 583-92, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15872051

RESUMO

Murine arylamine N-acetyltransferase 2 (NAT2) is expressed in the developing heart and in the neural tube at the time of closure. Classically described as a xenobiotic metabolizing enzyme, there is increasing evidence for a distinct biological role for murine NAT2. We have characterized the expression of arylamine N-acetyltransferase 2 during cardiogenesis, mapping its expression in vivo, using a lacZ insertion deletion, and also in vitro, by measuring NAT2 enzyme activity. These findings show that cardiac Nat2 expression is both temporally and spatially regulated during development. In neonatal mice, cardiac Nat2 expression is most extensive in the central fibrous body and is evident in the atrioventricular valves and the valves of the great vessels. Whereas Nat2 expression is not detected in ventricular myocardial cells, Nat2 is strongly expressed in scattered cells in the region of the sinus node, the epicardium of the right atrial appendage, and in the pulmonary artery. Expression of active NAT2 protein is maximal when the developing heart attains the adult circulation pattern and moves from metabolizing glucose to fatty acids. NAT2 acetylating activity in cardiac tissue from Nat2(-/-) and Nat2(+/-) mice indicates a lack of compensating acetylating activity either from other acetylating enzymes or by NAT2 encoded by the wild-type Nat2 allele in Nat2(+/-) heterozygotes. The temporal and spatial control of murine Nat2 expression points to an endogenous role distinct from xenobiotic metabolism and indicates that Nat2 expression may be useful as a marker in cardiac development.


Assuntos
Sistemas de Transporte de Aminoácidos/biossíntese , Coração/embriologia , Coração/crescimento & desenvolvimento , Miocárdio/enzimologia , Acetilação , Sistema A de Transporte de Aminoácidos , Animais , Animais Recém-Nascidos , Feminino , Genótipo , Átrios do Coração/enzimologia , Valvas Cardíacas/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Artéria Pulmonar/enzimologia
17.
Science ; 293(5535): 1670-3, 2001 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-11533493

RESUMO

Cardiac valve formation is a complex process that involves cell signaling events between the myocardial and endocardial layers of the heart across an elaborate extracellular matrix. These signals lead to marked morphogenetic movements and transdifferentiation of the endocardial cells at chamber boundaries. Here we identify the genetic defect in zebrafish jekyll mutants, which are deficient in the initiation of heart valve formation. The jekyll mutation disrupts a homolog of Drosophila Sugarless, a uridine 5'-diphosphate (UDP)-glucose dehydrogenase required for heparan sulfate, chondroitin sulfate, and hyaluronic acid production. The atrioventricular border cells do not differentiate from their neighbors in jekyll mutants, suggesting that Jekyll is required in a cell signaling event that establishes a boundary between the atrium and ventricle.


Assuntos
Valvas Cardíacas/embriologia , Coração/embriologia , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Glucose Desidrogenase/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética) , Padronização Corporal , Proteínas Morfogenéticas Ósseas/genética , Endocárdio/embriologia , Endocárdio/metabolismo , Feminino , Expressão Gênica , Glicosaminoglicanos/metabolismo , Valvas Cardíacas/citologia , Valvas Cardíacas/enzimologia , Valvas Cardíacas/metabolismo , Masculino , Dados de Sequência Molecular , Morfogênese , Mutação , Miocárdio/citologia , Miocárdio/metabolismo , Fenótipo , Mapeamento Físico do Cromossomo , Transdução de Sinais , Peixe-Zebra/genética
18.
J Pathol ; 194(2): 225-31, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11400152

RESUMO

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.


Assuntos
Doenças das Valvas Cardíacas/enzimologia , Valvas Cardíacas/enzimologia , Metaloproteinases da Matriz/análise , Inibidores Teciduais de Metaloproteinases/análise , Valva Aórtica/enzimologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/análise , Metaloendopeptidases/genética , Pessoa de Meia-Idade , Valva Mitral/enzimologia , RNA Mensageiro/análise , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/genética
19.
Yonsei Med J ; 40(2): 184-90, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10333724

RESUMO

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.


Assuntos
Criopreservação , Valvas Cardíacas/enzimologia , Valvas Cardíacas/fisiologia , Sobrevivência de Tecidos/fisiologia , Animais , Suínos
20.
Yonsei Medical Journal ; : 184-190, 1999.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wpr-45254

RESUMO

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.


Assuntos
Animais , Criopreservação , Valvas Cardíacas/fisiologia , Valvas Cardíacas/enzimologia , Suínos , Sobrevivência de Tecidos/fisiologia
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