RESUMO
OBJECTIVE: Diagnosing fetal heart failure remains challenging because it is difficult to know how well the fetal myocardium will perform as loading conditions change. In adult cardiology, natriuretic peptides (NPs) are established markers of heart failure. However, the number of studies investigating NP levels in fetuses is quite limited. The aim of this study was to evaluate the significance of plasma NP levels in the assessment of heart failure in fetuses with a congenital heart defect (CHD) and/or arrhythmia. METHODS: This was a prospective observational study conducted at a tertiary pediatric cardiac center. A total of 129 singletons with CHD and/or arrhythmia and 127 controls were analyzed between 2012 and 2015. Umbilical cord plasma atrial NP, brain NP and N-terminal pro-brain NP levels at birth were compared with ultrasonography findings indicating fetal heart failure, such as cardiovascular profile (CVP) score and morphological characteristics. RESULTS: Fetuses with CHD and/or arrhythmia had higher NP levels than did controls (P < 0.01). NP levels of fetuses with CHD and/or arrhythmia were correlated inversely with CVP score (P for trend < 0.01). No differences in NP levels were found in fetuses with CHD and/or arrhythmia and a CVP score of ≥ 8 in comparison to controls. Multivariate analysis showed that a CVP score of ≤ 5, tachy- or bradyarrhythmia at birth, preterm birth and umbilical artery pH < 7.15 were associated independently with high NP levels (P < 0.01). Among fetuses with a CVP score of ≤ 7, abnormal venous Doppler sonography findings were significantly more common and more severe in fetuses with tachy- or bradyarrhythmia than in those with CHD, and those with tachy- or bradyarrhythmia had higher NP levels than did those with CHD (P = 0.01). Fetuses with right-heart defect and moderate or severe tricuspid valve regurgitation had significantly higher NP levels than did fetuses with other types of CHD (P < 0.01). CONCLUSIONS: Plasma NP levels in fetuses with CHD and/or arrhythmia are correlated with the severity of fetal heart failure. Elevated NP levels are attributed mainly to an increase in central venous pressure secondary to arrhythmia or atrioventricular valve regurgitation due to CHD, rather than to the morphological abnormality itself. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.
Assuntos
Arritmias Cardíacas/sangue , Biomarcadores/sangue , Cardiopatias Congênitas/sangue , Insuficiência Cardíaca/sangue , Peptídeos Natriuréticos/sangue , Diagnóstico Pré-Natal , Adulto , Arritmias Cardíacas/congênito , Estudos de Coortes , Feminino , Insuficiência Cardíaca/congênito , Humanos , Valor Preditivo dos Testes , Gravidez , Resultado da Gravidez , Estudos ProspectivosRESUMO
OBJECTIVE: Recent studies have shown that plasma levels of brain natriuretic peptide (BNP)-32 and proBNP-108 are increased in heart failure (HF) and that the BNP-32 assay kit in current clinical use cross-reacts with proBNP-108. We investigated why proBNP is increased without processing in HF was investigated. DESIGN, SETTING AND PATIENTS: Plasma BNP-32 and proBNP-108 in normal individuals (n=10) and in patients with atrial fibrillation (AF) (n=18) and HF (n=132) was measured. BNP-32 and proBNP-108 in ventricular and atrial tissue and in pericardial fluid using a specific fluorescent enzyme immunoassay following Sep-Pak C18 (Waters, Milford, Massachusetts, USA) cartridge extraction and gel filtration was also measured. MAIN OUTCOME MEASURES: Levels of both BNP-32 and proBNP-108 were higher in HF than in control or AF (both p<0.01), and the levels of these peptides significantly correlated (r=0.94, p<0.001). The proBNP-108/total BNP (BNP-32+proBNP-108) ratio was widely distributed and lower in HF (0.33 (0.17)) than in control (0.41 (0.06), p<0.05) and AF (0.45 (0.04), p<0.002). The proBNP-108/total BNP ratio was higher in HF with ventricular than in HF with atrial overload (0.45 (0.10) vs 0.20 (0.11), p<0.001). Consistent with this finding, the major molecular form were proBNP-108 and BNP-32 in ventricular (n=6, 0.67 (0.04)) and atrial (n=7, 0.76 (0.05), p<0.0001) tissues, respectively. ProBNP-108 was also the major molecular form of BNP in pericardial fluid (n=8, 0.82 (0.05)). The proBNP-108/total BNP ratio increased and decreased with HF deterioration and improvement, respectively. CONCLUSION: These results suggest that BNP-32 and proBNP-108 is increased in HF and that the proBNP/total BNP ratio increases in association with pathophysiological conditions such as ventricular overload.
Assuntos
Insuficiência Cardíaca/sangue , Peptídeo Natriurético Encefálico/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/sangue , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Átrios do Coração/metabolismo , Insuficiência Cardíaca/cirurgia , Ventrículos do Coração/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/metabolismo , Derrame Pericárdico/metabolismoRESUMO
Recently we identified and characterized porcine calcitonin receptor-stimulating peptide (CRSP) 1, CRSP2 and CRSP3 as members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family. In the present study, the genomic sequences and organization of CRSP1, 2, and 3 were determined, and the expression of the genes in the porcine brain was investigated using in situ hybridization. Analysis of 5'-upstream regions of the three CRSPs demonstrated that CRSP1 and CRSP2 have almost identical sequences (>98% similarity) and high sequence similarities including functional transcription binding sites with the corresponding region of human CALCA (CT/alpha CGRP), whereas CRSP3 retains less similarity with the above genes. RH mapping of CRSPs demonstrated that they resided in a region of swine chromosome 2 (SSC2). The arrangement of the genes in the region was found to be conserved in corresponding human and mouse regions. In situ hybridization demonstrated sense transcripts of the three genes in cerebrum, hippocampus, hypothalamus, pons/midbrain, and thalamus of 3-month-old pigs, and CRSP2 sense transcripts additionally in tractus opticus. The sense transcripts of alpha CGRP and CALCB (beta CGRP) were detected in cerebrum, hippocampus, and pons/midbrain of newborn mice, and to a lesser extent in pons/midbrain of 8-week-old mice. These results taken together with the chromosomal conservation and phylogenetic clustering of CT/CGRP family indicate that CRSP1, 2, and 3 may be functionally different from alpha CGRP and beta CGRP, though they are indicated to have a common progenitor gene.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores da Calcitonina/metabolismo , Suínos/genética , Suínos/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Citogenética , Primers do DNA/genética , DNA Complementar/genética , Evolução Molecular , Feminino , Expressão Gênica , Genoma , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Filogenia , Mapeamento de Híbridos Radioativos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Sítio de Iniciação de TranscriçãoRESUMO
Adrenomedullin 5 (AM5) is a new member of the calcitonin gene-related peptide (CGRP) family identified in teleost fish. Although its presence was suggested in the genome database of mammals, molecular identity and biological function of AM5 have not been examined yet. In this study, we cloned a cDNA encoding AM5 in the pig and examined its cardiovascular and renal effects. Putative mature AM5 was localized in the middle of prohormone and had potential signals for intermolecular ring formation and C-terminal amidation. The AM5 gene was expressed most abundantly in the spleen and thymus. Several AM5 genes were newly identified in the database of mammals, which revealed that the AM5 gene exists in primates, carnivores, and undulates but could not be identified in rodents. In primates, nucleotide deletion occurred in the mature AM5 sequence in anthropoids (human and chimp) during transition from the rhesus monkey. Synthetic mature AM5 injected intravenously into rats induced dose-dependent decreases in arterial pressure at 0.1-1 nmol/kg without apparent changes in heart rate. The decrease was maximal in 1 min and AM5 was approximately half as potent as AM. AM5 did not cause significant changes in urine flow and urine Na+ concentration at any dose. In contrast to the peripheral vasodepressor action, AM5 injected into the cerebral ventricle dose-dependently increased arterial pressure and heart rate at 0.1-1 nmol. The increase reached maximum more quickly after AM5 (5 min) than AM (15-20 min). AM5 added to the culture cells expressing calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of the receptor activity-modifying proteins (RAMPs), the combination of which forms major receptors for the CGRP family, did not induce appreciable increases in cAMP production in any combination, although AM increased it at 10(-)(10)-10(-)(9) M when added to the CLR and RAMP2/3 combination. These data indicate that AM5 seems to act on as yet unknown receptor(s) for AM5, other than CLR/CTR+RAMP, to exert central and peripheral cardiovascular actions in mammals.
Assuntos
Adrenomedulina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Adrenomedulina/química , Adrenomedulina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Feminino , Dados de Sequência Molecular , SuínosRESUMO
BACKGROUND: Plasma levels of adrenomedullin, a potent vasodilator peptide, are increased in cirrhotic patients, whereas its role in vascular hyporeactivity in cirrhosis has not been clarified. METHODS: Adrenomedullin expression was evaluated by radioimmunoassay and reverse-transcription polymerase chain reaction. Vascular reactivity to phenylephrine, alpha-adrenoceptor agonist, was investigated in the aortic rings from control rats and CCl-induced cirrhotic rats with ascites in the presence of the neutralizing antibody against adrenomedullin, human adrenomedullin and/or N-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor. RESULTS: Plasma adrenomedullin levels were significantly higher in cirrhotic rats than in controls (16.3 +/- 2.9 versus 7.4 +/- 1.7 fmol/mL, P < 0.05) and correlated negatively with systemic arterial pressure (r = -0.62, P < 0.05). Gene expression of adrenomedullin in various organs (liver, kidney, lung) and vessels (portal vein, aorta) was enhanced in cirrhotic rats compared with controls. Neutralizing antibody against adrenomedullin ameliorated the blunted contractile response to phenylephrine in cirrhotic aorta (Rmax: 1.5 +/- 0.1 versus 1.0 +/- 0.1 g/mg tissue, P < 0.05), whereas contraction remained unchanged in control aorta (Rmax: 1.9 +/- 0.2 versus 1.9 +/- 0.2 g/mg tissue). Intravenous infusion of human adrenomedullin induced a reduction of mean arterial pressure together with an increase of serum nitrate levels, which was abolished by neutralizing antibody against adrenomedullin. Human adrenomedullin caused a blunted contractile response to phenylephrine in both control and cirrhotic aortas, which was not observed in the presence of N-nitro-L-arginine methyl ester. CONCLUSIONS: These findings indicate that the overproduction of adrenomedullin may contribute to vascular hyporeactivity in cirrhosis via a release of nitric oxide.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Aorta Torácica/efeitos dos fármacos , Cirrose Hepática/metabolismo , Peptídeos/metabolismo , Fenilefrina/farmacologia , Vasodilatação/efeitos dos fármacos , Adrenomedulina , Animais , Ascite/metabolismo , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Óxido Nítrico/sangue , Peptídeos/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. Levels of AM are markedly increased in the fetoplacental circulation during pregnancy, although its function there remains unknown. To clarify the physiological functions of AM, we chose a gene-targeting strategy in mice. METHODS AND RESULTS: Targeted null mutation of the AM gene is lethal in utero: the mortality rate among AM(-/-) embryos was >80% at E13.5. The most apparent abnormality in surviving AM(-/-) embryos at E13.5 to E14.0 was severe hemorrhage, readily observable under the skin and in visceral organs. Hemorrhage was not detectable at E12.5 to E13.0, although the yolk sac lacked well-developed vessels. Electron microscopic examination showed endothelial cells to be partially detached from the basement structure at E12.5 in vitelline vessels and hepatic capillaries, which allowed efflux of protoerythrocytes through the disrupted barrier. The basement membrane was not clearly recognizable in the aorta and cervical artery, and the endothelial cells stood out from the wall of the lumen, only partially adhering to the basement structure. AM(+/-) mice survived to adulthood but exhibited elevated blood pressures with diminished nitric oxide production. CONCLUSIONS: AM is indispensable for the vascular morphogenesis during embryonic development and for postnatal regulation of blood pressure by stimulating nitric oxide production.
Assuntos
Vasos Sanguíneos/anormalidades , Anormalidades Cardiovasculares/patologia , Hipertensão/patologia , Peptídeos/deficiência , Adrenomedulina , Animais , Vasos Sanguíneos/patologia , Vasos Sanguíneos/ultraestrutura , Perda do Embrião/etiologia , Perda do Embrião/patologia , Endotélio Vascular/embriologia , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Marcação de Genes , Genes Letais , Genótipo , Hemodinâmica/genética , Hemorragia/embriologia , Hemorragia/genética , Hemorragia/patologia , Heterozigoto , Homozigoto , Hipertensão/genética , Hipertensão/fisiopatologia , Endogamia , Bombas de Infusão , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Peptídeos/administração & dosagem , Peptídeos/genética , Fenótipo , Proteínas Recombinantes/administração & dosagem , Membrana Vitelina/irrigação sanguínea , Membrana Vitelina/embriologia , Membrana Vitelina/patologiaRESUMO
A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.
Assuntos
Guanilato Ciclase/genética , Rana catesbeiana/genética , Receptores do Fator Natriurético Atrial/genética , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/farmacologia , Sequência de Bases , Northern Blotting , Células COS , Clonagem Molecular , GMP Cíclico/metabolismo , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Expressão Gênica , Guanilato Ciclase/farmacologia , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Adrenomedullin is a recently discovered 52-amino acid peptide that is a potent vasodilator and is produced in the brain in experimental models of cerebral ischemia. Infusion of adrenomedullin increases regional cerebral blood flow and reduces infarct volume after vascular occlusion in rats, and thus may represent an endogenous neuroprotectant. Disturbances in cerebral blood flow (CBF), including hypoperfusion and hyperemia, frequently occur after severe traumatic brain injury (TBI) in infants and children. We hypothesized that cerebrospinal fluid (CSF) adrenomedullin concentration would be increased after severe TBI in infants and children, and that increases in adrenomedullin would be associated with alterations in CBF. We also investigated whether posttraumatic CSF adrenomedullin concentration was associated with relevant clinical variables (CBF, age, Glasgow Coma Scale [GCS] score, mechanism of injury, and outcome). Total adrenomedullin concentration was measured using a radioimmunometric assay. Sixty-six samples of ventricular CSF from 21 pediatric patients were collected during the first 10 days after severe TBI (GCS score < 8). Control CSF was obtained from children (n = 10) undergoing lumbar puncture without TBI or meningitis. Patients received standard neurointensive care, including CSF drainage. CBF was measured using Xenon computed tomography (CT) in 11 of 21 patients. Adrenomedullin concentration was markedly increased in CSF of infants and children after severe TBI vs control (median 4.5 versus 1.0 fmol/mL, p < 0.05). Sixty-two of 66 CSF samples (93.9%) from head-injured infants and children had a total adrenomedullin concentration that was greater than the median value for controls. Increases in CSF adrenomedullin were most commonly observed early after TBI. CBF was positively correlated with CSF adrenomedullin concentration (p < 0.001), but this relationship was not significant when controlling for the effect of time. CSF adrenomedullin was not significantly associated with other selected clinical variables. We conclude adrenomedullin is markedly increased in the CSF of infants and children early after severe TBI. We speculate that adrenomedullin participates in the regulation of CBF after severe TBI.
Assuntos
Lesões Encefálicas/líquido cefalorraquidiano , Peptídeos/líquido cefalorraquidiano , Adrenomedulina , Circulação Cerebrovascular , Criança , Pré-Escolar , Escala de Coma de Glasgow , Humanos , Lactente , Valor Preditivo dos TestesRESUMO
In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR METHOD: Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0.005), while AM mRNA levels were decreased (P < 0.05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.
Assuntos
Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Nefrite Lúpica/sangue , Peptídeos/metabolismo , Adrenomedulina , Adulto , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Mesângio Glomerular/metabolismo , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-6/genética , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Linfocinas/genética , Linfocinas/metabolismo , Pessoa de Meia-Idade , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Peptídeos/genética , Prednisolona/uso terapêutico , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family. Cultured endothelial cells secrete CNP, and its secretion rate from the endothelial cells is augmented by lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha, which participate in the pathophysiology of inflammation. In this study, we investigated the regulation of CNP secretion from monocytes and macrophages to estimate its contribution to the progression of inflammation. MATERIALS AND METHODS: CNP secretion rates from two human leukemia cell lines (THP-1 and HL-60), human peripheral blood lymphocytes, granulocytes, monocytes, monocyte-derived macrophages, and mouse peritoneal macrophages were measured under conditions with or without stimulation. Immunoreactive CNP levels in the culture media of these cells were measured by a specific radioimmunoassay. RESULTS: The secretion rates of CNP from THP-1 and HL-60 cells were augmented according to the degree of their differentiation into macrophage-like cells under the stimulation with phorbol ester. Peripheral blood monocytes also increased the CNP secretion rate after their differentiation into macrophages. Retinoic acid elicited synergistic effects on the CNP secretion rate from HL-60 cells when administered with lipopolysaccharide, interferon-gamma, interleukin-1beta, tumor necrosis factor-alpha, or phorbol ester. In contrast, the phorbol ester-stimulated CNP secretion rate from THP-1 cells was suppressed with dexamethasone, which inhibits monocyte differentiation into macrophage. CONCLUSIONS: The secretion rate of CNP from monocytes was shown to be regulated based on the degree of their differentiation. This study provides evidence that the monocyte/macrophage system is one of the sources of CNP, especially under inflammatory conditions.
Assuntos
Células Sanguíneas/metabolismo , Leucemia/patologia , Macrófagos Peritoneais/metabolismo , Peptídeo Natriurético Tipo C/biossíntese , Proteínas de Neoplasias/biossíntese , Animais , Células Sanguíneas/patologia , Diferenciação Celular/efeitos dos fármacos , Dexametasona/farmacologia , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Células HL-60/patologia , Humanos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C3H , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Peptídeo Natriurético Tipo C/metabolismo , Proteínas de Neoplasias/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A protein possessing vascular smooth muscle cell (SMC) growth-promoting activity (VSGP) was purified from bovine ovarian follicular fluid. The purified protein showed a broad band on SDS-PAGE with an apparent molecular mass of 90-100 kDa. The purified protein was characterized by amino acid sequence analysis of its N-terminal and internal peptides. Based on the information of the peptide sequences, bovine ovarian cDNA library was screened and cDNA clones encoding the protein were isolated. Human homolog of the protein was also cloned from human ovarian cDNA library. Nucleotide sequence analysis revealed that bovine VSGP transcript has a 2421-bp open reading frame, which encodes a protein of 807 amino acid residues. A homology search indicated that bovine and human VSGP are counterparts of rat F-spondin, which has been previously identified as a promoter molecule of neurite extension in rat fetal floor plate. RNA blot analysis showed wide distribution of VSGP/F-spondin transcripts in fetal and adult human tissues. Especially the expression was highest in the adult human ovary. The purified bovine VSGP/F-spondin showed vascular SMC growth promoting activity with an ED(50) value of 10(-8) M. Together with these findings, we demonstrated here that VSGP/F-spondin is a major factor for vascular SMC proliferation in the ovary. In conclusion, our present study provides a distinct and important function of VSGP/F-spondin as a strong VSMC proliferation promoting factor, in addition to the previously proposed function in neuronal system, and also provides insight into mechanisms underlying vascular SMC proliferation during ovarian folliculogenesis.
Assuntos
DNA Complementar/genética , Líquido Folicular/química , Substâncias de Crescimento/genética , Substâncias de Crescimento/isolamento & purificação , Músculo Liso Vascular/citologia , Peptídeos , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Primers do DNA/genética , Proteínas da Matriz Extracelular , Feminino , Substâncias de Crescimento/farmacologia , Humanos , Dados de Sequência Molecular , Ovário/química , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Adrenomedullin (AM), a potent vasodilator peptide, has natriuretic effects, and its plasma concentration is elevated in cardiovascular diseases. In the present study, we investigated the induction of AM expression due to interactions between THP-1 cells (human monocytic cell line) and human umbilical cord vein endothelial cells (HUVECs). AM levels in the culture medium were measured by radioimmunoassay. The luciferase vector containing the 5'-flanking region of the human AM gene was transfected into either HUVECs or THP-1 cells. Addition of THP-1 cells to HUVECs for 48 h induced marked increases in AM levels, which were 16-fold higher than those of HUVECs alone. Luciferase vectors containing the 5'-flanking region of human AM gene (pLCF-1534) were transferred into THP-1 cells or HUVECs. Addition of THP-1 cells to pLCF-1534-transfected HUVECs induced an increase in luciferase activity in cell lysates, which was 5-fold higher than that of the transfected HUVECs alone. In contrast, the luciferase activity of lysates from pLCF-1534-transfected THP-1 cells was not affected by coculture with HUVECs. A separate coculture experiment revealed that direct contact of THP-1 cells and HUVECs contributed to enhanced AM production in the cocoulture. Co-incubation of the cell membrane fraction from THP-1 cells augmented AM production by HUVECs. Both anti-interleukin (IL)-1alpha antibody and IL-1 receptor antagonist significantly inhibited AM production in the cocultures. The cell-to-cell interaction between monocytes and HUVECs induces AM production by HUVECs, which may play an important role in the pathogenesis of vascular disorders.
Assuntos
Endotélio Vascular/citologia , Regulação da Expressão Gênica , Monócitos/fisiologia , Peptídeos/metabolismo , Adrenomedulina , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/fisiologia , Arteriosclerose/etiologia , Arteriosclerose/patologia , Adesão Celular , Comunicação Celular , Membrana Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/antagonistas & inibidores , Leucemia Monocítica Aguda/patologia , Luciferases/biossíntese , Luciferases/genética , Camundongos , Peptídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Sialoglicoproteínas/farmacologia , Transfecção , Células Tumorais Cultivadas , Vasodilatação/fisiologiaRESUMO
OBJECTIVE: Adrenomedullin (AM) is a potent vasodilator peptide. Plasma AM concentration is increased in patients with various heart diseases, and both myocytes (MCs) and non-myocytes (NMCs) secrete AM and express its receptors. These facts suggest that cardiac cells possess an autocrine/paracrine capability mediated by AM. METHODS: MCs and NMCs were prepared from cardiac ventricles of neonatal rats. AM and endothelin-1 concentrations were measured by radioimmunoassays, and interleukin-6 level by a specific bioassay. Total nitrite/nitrate contents were measured with a fluorescence assay kit. RESULTS: A basal secretion rate of AM from NMCs was 2.8-fold higher than that from MCs. Interleukin-1beta, tumor necrosis factor-alpha and lipopolysaccharide stimulated AM secretion from NMCs but not from MCs. AM stimulated interleukin-6 production in the presence of these cytokines or lipopolysaccharide, which was more prominent in NMCs. In the presence of interleukin-1beta, AM augmented nitric oxide synthesis 2.7-fold in NMCs, but slightly in MCs. NMCs secreted endothelin-1 at a rate nine times higher than MCs, and AM inhibited endothelin-1 secretion from NMCs. CONCLUSION: This in vitro study suggests that AM in the heart is mainly produced in NMCs and exerts its effects through NMCs, especially under inflammatory conditions.
Assuntos
Citocinas/farmacologia , Miocárdio/metabolismo , Peptídeos/metabolismo , Adrenomedulina , Análise de Variância , Animais , Animais Recém-Nascidos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Endotelina-1/análise , Endotelina-1/metabolismo , Fibroblastos/metabolismo , Interleucina-1/farmacologia , Interleucina-6/análise , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Estimulação Química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-beta1 (TGF-beta), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-beta and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET- 1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET- 1, whereas approximately 90% of that from ECs was ET-1. By TGF-beta stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.
Assuntos
Endotelina-1/metabolismo , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Peptídeos/metabolismo , Adrenomedulina , Animais , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotelina-1/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Peptídeos/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Characterization of immunoreactive adrenomedullin (AM) secreted from cultured human vascular smooth muscle cells and 7 other cells indicates that AM is synthesized and secreted from all cultured cells we surveyed. The secretion rate of AM measured ranges from 0.001-6.83 fmol/10(5) cells/h, and endothelial cells, vascular smooth muscle cells and fibroblasts generally secrete AM at high rates. Based on the results of regulation of AM secretion from vascular wall cells, fibroblasts, macrophages and other cells measured in this and previous studies, AM secretion is found to be generally stimulated by inflammatory cytokines, lipopolysaccharide (LPS) and hormones. Especially, vascular smooth muscle cells and fibroblasts elicited uniform and strong stimulatory responses of AM secretion to tumor necrosis factor (TNF), interleukin-1 (IL-1), LPS and glucocorticoid, but endothelial cells did not elicit such prominent responses. AM secretion of monocyte-macrophage was mainly regulated by the degree of differentiation into macrophage and activation by LPS and inflammatory cytokines including interferon-gamma. The other examined cells showed weaker responses to LPS and IL-1. Although cultured cells may have been transformed as compared with those in the tissue, these data indicate that AM is widely synthesized and secreted from most of the cells in the body and functions as a local factor regulating inflammation and related reactions in addition to as a potent vasodilator. The responses of AM secretion to LPS and inflammatory cytokines suggest that fibroblasts, vascular smooth muscle cells and macrophage are the major sources of AM in the septic shock.
Assuntos
Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Peptídeos/metabolismo , Adrenomedulina , Análise de Variância , Animais , Bovinos , Células Cultivadas , Fibroblastos/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Músculo Liso Vascular/citologia , Células PC12 , Ratos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismoRESUMO
We measured mRNA levels of adrenomedullin (AM), C-type natriuretic peptide (CNP), vascular endothelial growth factor (VEGF), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy. To evaluate these mRNA levels, we employed a real-time quantitative PCR method which was performed using a hybridization probe labeled with two fluorescence dyes. This strategy was found to afford the standard curves with a high correlation, suggesting that this method is useful for evaluations of mRNA levels. By this method, levels of AM, CNP, VEGF, IL-1beta and IL-6 mRNA in PBMC of 49 IgA nephropathy patients and 35 healthy volunteers were evaluated. Among the mRNAs examined, AM mRNA levels were significantly lower in severe-grade than in mild-grade IgA nephropathy patients. Furthermore, AM mRNA levels correlated with CNP mRNA levels in PBMC of patients with IgA nephropathy, and each peptide generated from these mRNAs has antiproliferative effects on mesangial cells. These data indicate that gene expression of AM in PBMC is regulated according to the pathophysiological states of IgA nephropathy and that decreased AM production may contribute to the progression of IgA nephropathy.
Assuntos
Glomerulonefrite por IGA/genética , Leucócitos Mononucleares/metabolismo , Peptídeos/genética , Adrenomedulina , Adulto , Idoso , Sequência de Bases , Estudos de Casos e Controles , Primers do DNA/genética , Fatores de Crescimento Endotelial/genética , Regulação da Expressão Gênica , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/metabolismo , Humanos , Interleucina-1/genética , Interleucina-6/genética , Leucócitos Mononucleares/imunologia , Linfocinas/genética , Pessoa de Meia-Idade , Peptídeo Natriurético Tipo C/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. To determine the extent to which chronic AM overproduction affects circulatory physiology under normal and pathological conditions, we used a preproendothelin-1 promoter to establish transgenic mouse lines overexpressing AM in their vasculature. METHODS AND RESULTS: Transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (BP) and higher plasma cGMP levels than their wild-type littermates. Blockade of NO synthase with N(G)-monomethyl-L-arginine elevated BP to a greater degree in AM transgenic mice, offsetting the BP difference between the 2 groups. Despite their lower basal BP, administration of bacterial lipopolysaccharide elicited smaller declines in BP and less severe organ damage in AM transgenic mice than in wild-type mice. Furthermore, the 24-hour survival rate after induction of lipopolysaccharide shock was significantly higher in the transgenic mice. CONCLUSIONS: A chronic increase in vascular AM production reduces BP at least in part via an NO-dependent pathway. In addition, smaller responses to LPS in transgenic mice suggest that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock.
Assuntos
Vasos Sanguíneos/metabolismo , Hipotensão/etiologia , Lipopolissacarídeos , Peptídeos/fisiologia , Choque/induzido quimicamente , Adrenomedulina , Animais , Pressão Sanguínea/efeitos dos fármacos , Suscetibilidade a Doenças , Endotelina-1 , Endotelinas/genética , Hipotensão/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Transgênicos/genética , Peptídeos/metabolismo , Precursores de Proteínas/genéticaAssuntos
Aneurisma Intracraniano/sangue , Aneurisma Intracraniano/líquido cefalorraquidiano , Peptídeos/sangue , Peptídeos/líquido cefalorraquidiano , Hemorragia Subaracnóidea/sangue , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Vasoespasmo Intracraniano/diagnóstico , Adrenomedulina , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vasoespasmo Intracraniano/sangue , Vasoespasmo Intracraniano/líquido cefalorraquidianoRESUMO
Adrenomedullin (AM) is a vasodilator peptide first identified in pheochromocytoma tissue, but endothelial cells and vascular smooth muscle cells actively secrete AM in addition to expressing AM receptors. AM dilates blood vessels through its direct action on the smooth muscle and the endothelial cell-mediated nitric oxide pathway. We have further demonstrated that AM is synthesized and secreted from macrophages, fibroblasts, cardiomyocytes and many other types of cells. AM secretion from these cells as well as the vascular wall cells are commonly stimulated with inflammatory cytokines and lipopolysaccharide. AM receptor is also widely distributed, and AM is shown to regulate production of inflammatory cytokines and cell growth. Based on these data, AM is deduced to be a multi-functional peptide participating in the regulation of vascular tone, inflammation and other physiologic events of the vasculature.