RESUMO
Epidermolysis bullosa simplex (EBS) is a group of epidermal blistering diseases almost invariably transmitted as a dominant trait, which has recently been shown to arise from mutations in keratins 14 and 5 (K14 and K5). We describe a family with recessive EBS in which the disease is tightly linked to the substitution of the highly conserved glutamic acid-144 to alanine in the first helical segment of the rod domain of keratin 14. In contrast, linkage with keratin 5 was excluded. The loss of an ionic interaction with keratin 5 is likely to affect K14-K5 heterodimer formation. Our data suggest that this mutation underlies EBS in our family, and that mutations in keratin genes may impair the mechanical integrity of basal keratinocytes in a recessive as well as dominant fashion.
Assuntos
Epidermólise Bolhosa/genética , Genes Recessivos , Queratinas/genética , Alanina , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Sequência Conservada , Primers do DNA , Feminino , Genes Dominantes , Ligação Genética , Ácido Glutâmico , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinas/química , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Pele/metabolismo , Pele/patologiaRESUMO
The Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a life-threatening autosomal disease characterized by loss of dermal-epidermal adherence with abnormal anchoring fibrils (AF). We recently linked HS-RDEB to the type VII collagen gene (COL7A1) which encodes the major component of AF. We describe a patient who is homozygous for an insertion-deletion in the FN-4A domain of the COL7A1 gene. This defect causes a frameshift mutation which leads to a premature stop codon in the FN-5A domain, resulting in a marked diminution in mutated mRNA levels, with no detectable type VII collagen polypeptide in the patient. Our data suggest strongly that this null allele prevents normal anchoring fibril formation in homozygotes and is the underlying cause of HS-RDEB in this patient.
Assuntos
Colágeno/genética , Elementos de DNA Transponíveis , Epidermólise Bolhosa Distrófica/genética , Deleção de Sequência , Sequência de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Pré-Escolar , DNA Complementar , Epidermólise Bolhosa Distrófica/patologia , Imunofluorescência , Humanos , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Linhagem , Polimorfismo Genético , Pele/patologia , Pele/ultraestruturaRESUMO
We describe here eleven different mutations in SPINK5, encoding the serine protease inhibitor LEKTI, in 13 families with Netherton syndrome (NS, MIM256500). Most of these mutations predict premature termination codons. These results disclose a critical role of SPINK5 in epidermal barrier function and immunity, and suggest a new pathway for high serum IgE levels and atopic manifestations.
Assuntos
Anormalidades Múltiplas/genética , Proteínas de Transporte , Mutação/genética , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 5/genética , Códon de Terminação/genética , Análise Mutacional de DNA , Éxons/genética , Mutação da Fase de Leitura/genética , Genes Recessivos/genética , Humanos , Íntrons/genética , Proteínas Secretadas Inibidoras de Proteinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Inibidor de Serinopeptidase do Tipo Kazal 5 , SíndromeRESUMO
We present a 3D-printing technology allowing free-form fabrication of centimetre-scale injectable structures for minimally invasive delivery. They result from the combination of 3D printing onto a cryogenic substrate and optimisation of carboxymethylcellulose-based cryogel inks. The resulting highly porous and elastic cryogels are biocompatible, and allow for protection of cell viability during compression for injection. Implanted into the murine subcutaneous space, they are colonized with a loose fibrovascular tissue with minimal signs of inflammation and remain encapsulation-free at three months. Finally, we vary local pore size through control of the substrate temperature during cryogenic printing. This enables control over local cell seeding density in vitro and over vascularization density in cell-free scaffolds in vivo. In sum, we address the need for 3D-bioprinting of large, yet injectable and highly biocompatible scaffolds and show modulation of the local response through control over local pore size. STATEMENT OF SIGNIFICANCE: This work combines the power of 3D additive manufacturing with clinically advantageous minimally invasive delivery. We obtain porous, highly compressible and mechanically rugged structures by optimizing a cryogenic 3D printing process. Only a basic commercial 3D printer and elementary control over reaction rate and freezing are required. The porous hydrogels obtained are capable of withstanding delivery through capillaries up to 50 times smaller than their largest linear dimension, an as yet unprecedented compression ratio. Cells seeded onto the hydrogels are protected during compression. The hydrogel structures further exhibit excellent biocompatibility 3â¯months after subcutaneous injection into mice. We finally demonstrate that local modulation of pore size grants control over vascularization density in vivo. This provides proof-of-principle that meaningful biological information can be encoded during the 3D printing process, deploying its effect after minimally invasive implantation.
Assuntos
Elasticidade , Teste de Materiais , Impressão Tridimensional , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , Camundongos , PorosidadeRESUMO
We explored the role of CD18 (beta2 integrin) in platelet physiology, using mice genetically deficient in CD18 (CD18 -/-), or its main ligand CD54 (ICAM-1, CD54 -/-). CD18 and CD11a were evident in platelets from +/+, but not from CD18 -/- mice, as seen by immunofluorescence or Western blots. CD18 mRNA was also detectable by RT-PCR in platelets from +/+, but not from CD18 -/- mice. The life span of platelets was significantly shorter in CD18 -/- than in +/+ or CD54 -/- mice, as seen by in vivo biotinylation. When a local inflammation was elicited by the intra-tracheal injection of TNF, labeled platelets from +/+, but not from CD18 -/- donors, did localize in the lung. The content of Bcl-3 was about 20-fold higher in platelet from CD18 -/-, than in those from +/+ or CD54 -/- donors, as seen on Western blots or by immunofluorescence and flow cytometry, while the amount of pro-caspase-3 was decreased. An activation of caspases in platelets from CD18 -/- was also evidenced by protease assays. Accordingly, gelsolin, a protein cleaved by caspase-3, showed a low-molecular-weight band in platelets from CD18 -/- but not from +/+ donors. These results demonstrate that the beta2 integrin, present in mouse platelets, modulates caspase activation and consequently platelet life span and response to TNF.
Assuntos
Plaquetas/metabolismo , Antígenos CD18/metabolismo , Caspases/metabolismo , Animais , Anexina A5/metabolismo , Proteína 3 do Linfoma de Células B , Plaquetas/citologia , Plaquetas/fisiologia , Western Blotting , Antígenos CD18/biossíntese , Antígenos CD18/genética , Caspase 3 , Adesão Celular , Ativação Enzimática , Gelsolina/metabolismo , Expressão Gênica , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/fisiologia , Antígeno-1 Associado à Função Linfocitária/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Marie-Unna hypotrichosis (MU) is a rare autosomal dominant congenital alopecia characterised by progressive hair loss starting in early childhood, often aggravated at puberty and leading to scarring alopecia of variable severity. We have studied three multigeneration families of Belgian, British and French descent. The human genome was screened with microsatellite markers spaced at 10-cM intervals and significant evidence for linkage to the disease was observed on chromosome 8p21, with a maximum two-point lod score of 8.26 for D8S1786 at a recombination fraction of 0. Recombinants narrowed the region of interest to a genetic interval of about 12 cM flanked by markers D8S280 and D8S1839. This interval contains the hairless gene which is mutated in autosomal recessive congenital atrichia. Sequencing of the entire coding region and intronic splice sites of the hairless gene in these three families and in two unrelated familial cases revealed several polymorphic changes but failed to identify causative mutations. Nine other genes located within this region and expressed in skin were also excluded by mutation analysis. Together with a recent linkage study performed in a Dutch and a British family by van Steensel et al these results provide evidence for the presence of a gene distinct from hairless in chromosomal region 8p21 playing an important role in hair follicle biology.
Assuntos
Alopecia/genética , Cromossomos Humanos Par 8/genética , Hipotricose/genética , Mapeamento Cromossômico , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Ligação Genética , Humanos , Escore Lod , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Mutação , LinhagemRESUMO
Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.
Assuntos
Processamento Alternativo , Epidermólise Bolhosa Simples/genética , Mutação da Fase de Leitura , Queratinas/genética , Mutação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Feminino , Testes Genéticos , Humanos , Queratina-14 , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have investigated 8 patients from 7 unrelated families with lamellar ichthyosis (LI) for defects in the keratinocyte transglutaminase (TGK) gene. We have characterized three novel homozygous mutations and a previously reported splice acceptor site mutation. One patient showed a C-to-T change in the binding site for the transcription factor Sp1 within the promoter region. Another patient had a Gly 143-to-Glu mutation in exon 3 and a third patient, affected with a particular form of LI sparing the four limbs, demonstrated a Val382-to-Met mutation within exon 7. These three patients exhibited drastically reduced transglutaminase activity and an absence of detectable TGK polypeptide, as assessed by immunofluorescence and immunoblotting. Northern blot analysis showed that the Sp1 site mutation was associated with profound reduction of TGK transcript levels whereas normal transcript levels were observed for the two missense mutations. We hypothesize that the Sp1 site mutation impairs transcription of the TGK gene, whereas the two missense mutations induce structural changes leading to protein instability. Linkage to TGK was excluded in another family and no evidence for TGK defect was found in 3 other patients. These results further support the involvement of TGK in some patients with LI. They identify a TGK mutation as a cause for non-generalized LI and further delineate the molecular mechanisms underlying TGK deficiency in LI.
Assuntos
Ictiose Lamelar/genética , Mutação Puntual , Transglutaminases/genética , Adulto , Northern Blotting , Criança , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Ligação Genética , Humanos , Ictiose Lamelar/enzimologia , Immunoblotting , Masculino , Linhagem , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Transglutaminases/análiseRESUMO
INTRODUCTION: Plasminogen activators (PA) and plasmin are known to affect platelets but little is known of their role in platelet kinetics. We took advantage of genetically deficient mice to explore the role of urokinase (uPA) and tissue type (tPA) PAs, as well as the uPA receptor (uPAR, CD87) in platelet kinetics. MATERIALS AND METHODS: Platelet shape and number were investigated by flow cytometry. Platelet kinetics was investigated by the in vivo biotinylation and FACS analysis. Platelet production was investigated by counting megakaryocytes in bone marrow. RESULTS: Platelets counts were within the same range in wild type (+/+), uPA, tPA and uPAR-deficient mice. Platelet survival was similar in +/+, uPA-/-, tPA-/- but markedly reduced in uPAR-/- mice. The number of megakaryocytes in bone marrow and spleen was increased 2-3-fold in uPAR-/- compared to +/+ mice. TGF-beta mRNA level within the bone marrow was also significantly increased in uPAR-/- mice. Consistent with an increased platelet production, platelets from uPAR-/- mice had a higher RNA content, as seen by Propidium Iodide (PI) labeling and FACS analysis. Since uPAR is detectable in both hemopoietic and non-hemopoietic cells, radiation chimera were prepared. Investigation of platelet kinetics in chimera showed that platelet survival is reduced with a deficit in either bone marrow-derived, or non-hemopoietic, host cells. CONCLUSION: These results demonstrate that uPAR, but not uPA or tPA, is essential for maintaining normal platelet survival. In addition, uPAR-/- mice maintain normal platelet numbers through increased production.
Assuntos
Plaquetas/fisiologia , Ativadores de Plasminogênio/sangue , Receptores de Superfície Celular/sangue , Animais , Fibrinolisina/metabolismo , Cinética , Megacariócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tecidual/sangueRESUMO
Investigating the signalling pathways that regulate heart development is essential if stem cells are to become an effective source of cardiomyocytes that can be used for studying cardiac physiology and pharmacology and eventually developing cell-based therapies for heart repair. Here, we briefly describe current understanding of heart development in vertebrates and review the signalling pathways thought to be involved in cardiomyogenesis in multiple species. We discuss how this might be applied to stem cells currently thought to have cardiomyogenic potential by considering the factors relevant for each differentiation step from the undifferentiated cell to nascent mesoderm, cardiac progenitors and finally a fully determined cardiomyocyte. We focus particularly on how this is being applied to human embryonic stem cells and provide recent examples from both our own work and that of others.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Transdução de Sinais , Animais , Coração/embriologia , Humanos , MorfogêneseRESUMO
The epidermis and its related appendages such as the hair follicle constitute the epithelial compartment of the skin. The exact location and distribution of the keratinocyte colony-forming cells within the epidermis or its appendages are unknown. We report that in the rat vibrissa, keratinocyte colony-forming cells are highly clustered in the bulge-containing region. Approximately 95% of the total colonies formed in culture from fractionated vibrissae were in this location and fewer than 4% were located in the matrix area of the follicle. Finer dissection of the bulge-containing region located the colony-forming cells in the small part containing the bulge itself. The segregation of keratinocyte colony-forming cells in the bulge confirms the hypothesis that the bulge is the reservoir of the stem cells responsible for the long-term growth of the hair follicle and perhaps of the epidermis as well.
Assuntos
Queratinócitos/citologia , Células-Tronco/citologia , Vibrissas/citologia , Animais , Células Cultivadas , Células Epiteliais , Técnicas In Vitro , Ratos , Ratos Endogâmicos F344 , Vibrissas/anatomia & histologiaRESUMO
We have examined the growth capacity of keratinocytes isolated from human scalp hair follicles. Like the keratinocytes of glabrous epidermis, most of the colony-forming cells are classified as holoclones or meroclones when analyzed in a clonal assay. Some of them have extensive growth potential, as they are able to undergo at least 130 doublings. Therefore, the hair follicle, like the epidermis, contains keratinocytes with the expected property of stem cells: an extensive proliferative capacity permitting the generation of a large amount of epithelium. We have also examined the distribution of clonogenic keratinocytes within the hair follicle. Several hundred colony-forming cells are concentrated at a region below the midpoint of the follicle and outside the hair bulb. This region lies deeper than the site of insertion of the arrector pili muscle, which corresponds with the position of the bulge when the latter can be identified. In contrast, few colony-forming cells are present in the hair bulb, where most of the mitotic activity is observed during the active growth phase of the follicle. Paraclones, which are present both in the midregion and in the bulb of hair follicles, are unlikely to be the transient amplifying cells expected from kinetic studies.
Assuntos
Cabelo/citologia , Queratinócitos/citologia , Células-Tronco/citologia , Animais , Divisão Celular , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Feminino , Cabelo/crescimento & desenvolvimento , Humanos , Recém-Nascido , Queratinócitos/transplante , Camundongos , Camundongos Nus , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
The aim of this study was to investigate the influence of collagen or polyornithine substrates on cell migration in explant cultures of dorsal root ganglia (DRG) by means of light microscopy and immunocytochemistry. Myelin-associated glycoprotein (MAG) immunoreactivity was used to characterize the subpopulation of small B ganglion cells, whereas neuron-specific enolase (NSE) immunoreactivity acted as a general neuronal cell marker. After a few days in culture, DRG explants grown on collagen substrate showed a flattened shape consisting of a core surrounded by a crown of neurites, which were mixed up with migrating cells of different types. These migrating cells were immunostained for both MAG and NSE and were observed after 7 days in the vicinity of the explant core, then after 14 days also at a distance from the explant core. In contrast, even after 14 days in culture, explants grown on polyornithine substrate maintained a globular shape. The MAG-positive ganglion cells were confined to the explant core and no cell migration was observed on this type of substrate. MAG immunoprecipitates located at the ganglion cell surface were observed in explants cultured on polyornithine, but rarely on collagen substrate. In conclusion, it is suggested that this pattern of intracellular distribution of MAG immunoreactive material could reflect interactions between cell surface and extracellular matrix, and could condition the migratory ability of small ganglion cells.
Assuntos
Colágeno , Gânglios Espinais/citologia , Proteínas da Mielina/metabolismo , Peptídeos , Animais , Movimento Celular , Células Cultivadas , Embrião de Galinha , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Glicoproteína Associada a MielinaRESUMO
We explored the role of CD40-CD40L (CD154) in the severe malaria elicited by Plasmodium berghei anka infection in mice. Mortality was >90% by day 8 after infection in +/+ mice, but markedly decreased in CD40-/- or in CD40L-/- mice, as well as in +/+ mice treated with anti-CD40L monoclonal antibody. Parasitemia was similar in the different conditions. Breakdown of the blood-brain barrier was evident in infected +/+, but not in CD40-/- mice. Thrombocytopenia was less severe in CD40-/- mice than in the +/+ controls. Sequestration of macrophages in brain venules and alveolar capillaries was reduced in CD40-/- or in CD40L-/- mice, whereas sequestration of parasitized red blood cells or polymorphonuclear leukocytes in alveolar capillaries was CD40-CD40L-independent. CD40 mRNA was increased in the brain and lung of infected mice whereas CD40L was increased in the lung. Tumor necrosis factor plasma levels were similarly increased in infected +/+ or CD40-/- mice. Expression of CD54 and its mRNA levels in the brain were moderately decreased in CD40-deficient mice. Thus the mortality associated with severe malaria requires CD40-CD40L interaction that contributes to the breakdown of the blood-brain barrier, macrophage sequestration, and platelet consumption.
Assuntos
Antígenos CD40/fisiologia , Ligante de CD40/fisiologia , Malária/imunologia , Plasmodium berghei , Animais , Barreira Hematoencefálica , Encéfalo/imunologia , Encéfalo/patologia , Antígenos CD40/genética , Ligante de CD40/genética , Regulação da Expressão Gênica/imunologia , Molécula 1 de Adesão Intercelular/genética , Macrófagos/fisiologia , Malária/sangue , Malária/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contagem de Plaquetas , RNA Mensageiro/genética , Trombocitopenia , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Electrical pacing at physiological rate induces myocardial remodeling associated with regional changes in workload, blood flow and oxygen consumption. However, to what extent energy-producing pathways are also modified within the paced heart remains to be investigated. Pacing could particularly affect glycogen metabolism since hypertrophy stimulates glycolysis and increased workload favors glucose over fat oxidation. In order to test this hypothesis, we used the embryonic chick heart model in which ventricular pacing rapidly resulted in thinning of the ventricle wall and thickening of the atrial wall. Hearts of stage 22HH chick embryos were submitted in ovo to asynchronous and intermittent ventricular pacing delivered at physiological rate during 24 h. The resulting alterations of glycogen content were determined in atrium, ventricle and conotruncus of paced and sham-operated hearts. Hemodynamic parameters of the paced and spontaneously beating hearts were derived from computerized image analysis of video recordings. With respect to sham, paced hearts showed a significant decrease in glycogen content (nmoles glucose units/microg protein; mean+/-S.D.) only in atrium (1.48+/-0.40 v 0.84+/-0.34, n=8) and conotruncus (0.75+/-0.28 v 0.42+/-0.23, n=8). Pacing decreased the end diastolic and stroke volumes by 34 and 44%, respectively. Thus, the rapid glycogen depletion in regions remote from the stimulation site appears to be associated with regional changes in workload and remodeling. These findings underscore the importance of the coupling mechanisms between metabolic pathways and myocardial remodeling in the ectopically paced heart.
Assuntos
Relógios Biológicos/fisiologia , Glicogênio/metabolismo , Ventrículos do Coração/metabolismo , Coração/embriologia , Animais , Embrião de Galinha , Estimulação Elétrica , Coração/fisiopatologia , Frequência Cardíaca , Hemodinâmica , Miocárdio/metabolismo , Marca-Passo ArtificialRESUMO
The upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands, and epidermis, i.e., all the lineages of the hairy skin. At the time when hair production ceases and when the lower region of the follicle undergoes major structural changes, the lower region contains a significant number of clonogenic keratinocytes, and can then respond to morphogenetic signals. This demonstrates that multipotent stem cells migrate to the root of the follicle to produce whisker growth. Moreover, our results indicate that the clonogenic keratinocytes are closely related, if not identical, to the multipotent stem cells, and that the regulation of whisker growth necessitates a precise control of stem cell trafficking.
Assuntos
Folículo Piloso/crescimento & desenvolvimento , Células-Tronco/fisiologia , Vibrissas/crescimento & desenvolvimento , Animais , Linhagem da Célula , Movimento Celular , Células Cultivadas , Quimera , Ensaio de Unidades Formadoras de Colônias , Embrião de Mamíferos/fisiologia , Células Epidérmicas , Epiderme/crescimento & desenvolvimento , Feminino , Genes Reporter , Folículo Piloso/citologia , Folículo Piloso/transplante , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Morfogênese , Ratos , Ratos Endogâmicos F344 , Vibrissas/anatomia & histologia , Vibrissas/fisiologiaRESUMO
Cardiac morphogenesis and function are known to depend on both aerobic and anaerobic energy-producing pathways. However, the relative contribution of mitochondrial oxidation and glycogenolysis, as well as the determining factors of oxygen demand in the distinct chambers of the embryonic heart, remains to be investigated. Spontaneously beating hearts isolated from stage 11, 20, and 24HH chick embryos were maintained in vitro under controlled metabolic conditions. O(2) uptake and glycogenolytic rate were determined in atrium, ventricle, and conotruncus in the absence or presence of glucose. Oxidative capacity ranged from 0.2 to 0.5 nmol O(2)/(h.microg protein), did not depend on exogenous glucose, and was the highest in atria at stage 20HH. However, the highest reserves of oxidative capacity, assessed by mitochondrial uncoupling, were found at the youngest stage and in conotruncus, representing 75 to 130% of the control values. At stage 24HH, glycogenolysis in glucose-free medium was 0.22, 0.17, and 0.04 nmol glucose U(h.microg protein) in atrium, ventricle, and conotruncus, respectively. Mechanical loading of the ventricle increased its oxidative capacity by 62% without altering glycogenolysis or lactate production. Blockade of glycolysis by iodoacetate suppressed lactate production but modified neither O(2) nor glycogen consumption in substrate-free medium. These findings indicate that atrium is the cardiac chamber that best utilizes its oxidative and glycogenolytic capacities and that ventricular wall stretch represents an early and major determinant of the O(2) uptake. Moreover, the fact that O(2) and glycogen consumptions were not affected by inhibition of glyceraldehyde-3-phosphate dehydrogenase provides indirect evidence for an active glycerol-phosphate shuttle in the embryonic cardiomyocytes.
Assuntos
Glicogênio/metabolismo , Coração/embriologia , Miocárdio/metabolismo , Animais , Embrião de Galinha , Morfogênese , OxirreduçãoRESUMO
Unlike in adult heart, embryonic myocardium works at low PO2 and depends preferentially on glucose. Therefore, activity of the embryonic heart during anoxia and reoxygenation should be particularly affected by changes in glucose availability. Hearts excised from 4-d-old chick embryos were submitted in vitro to strictly controlled anoxia-reoxygenation transitions at glucose concentrations varying from 0 to 20 mmol/L. Spontaneous and regular heart contractions were detected optically as movements of the ventricle wall and instantaneous heart rate, amplitude of contraction, and velocities of contraction and relaxation were determined. Anoxia induced transient tachycardia and rapidly depressed contractile activity, whereas reoxygenation provoked a temporary and complete cardioplegia (oxygen paradox). In the presence of glucose, atrial rhythm became irregular during anoxia and chaotic-periodic during reoxygenation. The incidence of these arrhythmias depended on duration of anoxia, and no ventricular ectopic beats were observed. Removal of glucose or blockade of glycolysis suppressed arrhythmias. These results show similarities but also differences with respect to the adult heart. Indeed, glucose 1) delayed and anoxic contractile failure, shortened the reoxygenation-induced cardiac arrest, and improved the recovery of contractile activity; 2) attenuated stunning at 20 mmol/L but worsened it at 8 mmol/L; and 3) paradoxically, was arrhythmogenic during anoxia and reoxygenation, especially when present at the physiologic concentration of 8 mmol/L. The last named phenomenon seems to be characteristic of the young embryonic heart, and our findings underscore that fluctuations of glycolytic activity may play a role in the reactivity of the embryonic myocardium to anoxiareoxygenation transitions.
Assuntos
Arritmias Cardíacas/etiologia , Glucose/farmacologia , Coração/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/complicações , Animais , Arritmias Cardíacas/metabolismo , Embrião de Galinha , Glucose/metabolismo , Hipóxia/complicações , Hipóxia/metabolismo , Técnicas In Vitro , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismoRESUMO
Mice were exposed to pure oxygen for various times to explore the pulmonary platelet trapping associated with alveolar damage, its mechanism, and its role in the lesions. Platelet sequestration, evaluated by electron microscopy and by injection of radiolabeled platelets, was detectable after 72 h and reached a maximum after 96 h of exposure (i.e., shortly before death). Circulating platelets (analyzed by Facscan) showed some increase in the expression of CD11a and CD62, but little change in CD31 and CD61. Both platelet activation and lung sequestration were dependent on TNF-alpha, since antibody against TNF-alpha reduced the expression of CD11a on circulating platelets and their sequestration in the lung. Lung platelet sequestration was also decreased by anti-CD11a MoAb. Northern blot analysis of lung mRNA isolated at 96 h of oxygen exposure revealed a 7-fold increase in CD54 (intercellular adhesion molecule-1 [ICAM-1]) and a 2.5-fold increase in TNF-alpha mRNAs respectively. These results demonstrate that the platelet pulmonary trapping induced by hyperoxia is dependent upon TNF-alpha and the CD11a-CD54 adhesion molecules. However, platelet trapping does not appear to play an important pathogenic role in acute oxygen injury, since treatments that decrease trapping (anti-TNF-alpha, anti-CD11a, or antibody-induced thrombocytopenia) did not markedly attenuate the alveolar damage.
Assuntos
Sequestro Broncopulmonar/etiologia , Hiperóxia/complicações , Ativação Plaquetária/fisiologia , Animais , Anticorpos Monoclonais , Antígenos CD18/imunologia , Citometria de Fluxo , Hiperóxia/fisiopatologia , Molécula 1 de Adesão Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neutrófilos/fisiologia , Oxigênio/toxicidade , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/fisiopatologia , RNA Mensageiro/análise , Trombocitopenia/fisiopatologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.