RESUMO
C-type natriuretic peptide (CNP)-knockout (KO) rats exhibit impaired skeletal growth, with long bones shorter than those in wild-type (WT) rats. This study compared craniofacial morphology in the CNP-KO rat with that in the Spontaneous Dwarf Rat (SDR), a growth hormone (GH)-deficient model. The effects of subcutaneous administration of human CNP with 53 amino acids (CNP-53) from 5 weeks of age for 4 weeks on craniofacial morphology in CNP-KO rats were also investigated. Skulls of CNP-KO rats at 9 weeks of age were longitudinally shorter and the foramen magnum was smaller than WT rats. There were no differences in foramen magnum stenosis and midface hypoplasia between CNP-KO rats at 9 and 33 weeks of age. These morphological features were the same as those observed in CNP-KO mice and activated fibroblast growth factor receptor 3 achondroplasia-phenotype mice. In contrast, SDR did not exhibit foramen magnum stenosis and midface hypoplasia, despite shorter stature than in control rats. After administration of exogenous CNP-53, the longitudinal skull length and foramen magnum size in CNP-KO rats were significantly greater, and full or partial rescue was confirmed. The synchondrosis at the cranial base in CNP-KO rats is closed at 9 weeks, but not at 4 weeks of age. In contrast, synchondrosis closure in CNP-KO rats treated with CNP-53 was incomplete at 9 weeks of age. Administration of exogenous CNP-53 accelerated craniofacial skeletogenesis, leading to improvement in craniofacial morphology. As these findings in CNP-KO rats are similar to those in patients with achondroplasia, treatment with CNP-53 or a CNP analog may be able to restore craniofacial morphology and foramen magnum size as well as short stature.
Assuntos
Constrição Patológica , Face/anormalidades , Forame Magno/patologia , Peptídeo Natriurético Tipo C/deficiência , Peptídeo Natriurético Tipo C/uso terapêutico , Acondroplasia/tratamento farmacológico , Animais , Desenvolvimento Ósseo , Humanos , Ratos , Fatores de TempoRESUMO
Signaling by C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B, is a pivotal stimulator of endochondral bone growth. We recently developed CNP knockout (KO) rats that exhibit impaired skeletal growth with early growth plate closure. In the current study, we further characterized the phenotype and growth plate morphology in CNP-KO rats, and the effects of exogenous CNP in rats. We used CNP-53, an endogenous form of CNP consisting of 53 amino acids, and administered it for four weeks by continuous subcutaneous infusion at 0.15 or 0.5 mg/kg/day to four-week old CNP-KO and littermate wild type (WT) rats. We demonstrated that CNP-KO rats were useful as a reproducible animal model for skeletal dysplasia, due to their impairment in endochondral bone growth. There was no significant difference in plasma bone-turnover markers between the CNP-KO and WT rats. At eight weeks of age, growth plate closure was observed in the distal end of the tibia and the calcaneus of CNP-KO rats. Continuous subcutaneous infusion of CNP-53 significantly, and in a dose-dependent manner, stimulated skeletal growth in CNP-KO and WT rats, with CNP-KO rats being more sensitive to the treatment. CNP-53 also normalized the length of long bones and the growth plate thickness, and prevented growth plate closure in the CNP-KO rats. Using organ culture experiment of fetal rat tibia, gene set enrichment analysis indicated that CNP might have a negative influence on mitogen activated protein kinase signaling cascades in chondrocyte. Our results indicated that CNP-KO rats might be a valuable animal model for investigating growth plate physiology and the mechanism of growth plate closure, and that CNP-53, or its analog, may have the potential to promote growth and to prevent early growth plate closure in the short stature.
Assuntos
Lâmina de Crescimento/crescimento & desenvolvimento , Peptídeo Natriurético Tipo C/deficiência , Peptídeo Natriurético Tipo C/farmacologia , Animais , Biomarcadores/sangue , Peso Corporal/efeitos dos fármacos , Remodelação Óssea , Feminino , Técnicas de Inativação de Genes , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Humanos , Hipertrofia , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Tíbia/efeitos dos fármacos , Tíbia/patologiaRESUMO
Recent studies revealed C-type natriuretic peptide (CNP) and its receptor, guanylyl cyclase-B (GC-B) are potent stimulators of endochondral bone growth. As they exist ubiquitously in body, we investigated the physiological role of the local CNP/GC-B in the growth plate on bone growth using cartilage-specific knockout mice. Bones were severely shorter in cartilage-specific CNP or GC-B knockout mice and the extent was almost the same as that in respective systemic knockout mice. Cartilage-specific GC-B knockout mice were shorter than cartilage-specific CNP knockout mice. Hypertrophic chondrocyte layer of the growth plate was drastically reduced and proliferative chondrocyte layer, along with the proliferation of chondrocytes there, was moderately reduced in either cartilage-specific knockout mice. The survival rate of cartilage-specific CNP knockout mice was comparable to that of systemic CNP knockout mice. The local CNP/GC-B system in growth plate is responsible for physiological endochondral bone growth and might further affect mortality via unknown mechanisms.
Assuntos
Desenvolvimento Ósseo/fisiologia , Lâmina de Crescimento/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Animais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Cartilagem/metabolismo , Lâmina de Crescimento/patologia , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeo Natriurético Tipo C/deficiência , Peptídeo Natriurético Tipo C/genética , Fenótipo , Radiografia , Receptores do Fator Natriurético Atrial/deficiência , Receptores do Fator Natriurético Atrial/genética , Taxa de SobrevidaRESUMO
Nos últimos anos, o sistema do peptídeo natriurético do tipo C (CNP) e seu receptor (NPR-B) foi apontado como um importante regulador do processo de ossificação endocondral. Vários estudos em animais evidenciam o seu papel de estímulo à proliferação e diferenciação de condrócitos e secreção de matriz extracelular. Mutações bialélicas com perda de função do gene do NPR-B (NPR2) levam a uma doença denominada displasia acromesomélica do tipo Maroteaux (AMDM), uma displasia esquelética caracterizada por baixa estatura extrema. Observa-se que familiares de pacientes com AMDM carreadores de mutação no NPR2 têm estatura abaixo da média da população a qual pertencem, sugerindo um papel de mutações em heterozigose do NPR2 como causadoras de baixa estatura idiopática (BEI). Os objetivos deste estudo foram avaliar a presença de mutações no gene NPR2 em um grupo de pacientes com BEI e correlacionar os achados moleculares com o fenótipo dos pacientes e familiares. A região codificadora do gene NPR2 foi sequenciada pelo método de Sanger em 60 pacientes com diagnóstico de BEI. Foram identificadas cinco diferentes variantes alélicas missense em heterozigose no NPR2, cada uma em um único paciente. Essas variantes foram submetidas à análise funcional in vitro para avaliação da atividade da guanililciclase e microscopia confocal para localização intracelular dos receptores NPR-B. As variantes c.226T > C / p.Ser76Pro, c.788G > C / p.Arg263Pro e c.2455C > T / p.Arg819Cys segregam com o fenótipo de baixa estatura dentro das famílias e determinam um prejuízo funcional ao NPR-B. As três variantes geram proteínas que exercem efeito dominante negativo e os receptores NPR-B com as mutações p.Ser76Pro e p.Arg263Pro não se localizam na membrana celular. As variantes c.491C > G / p.Ala164Gly e c.1636A > T / p.Asn546Tyr não segregam com o fenótipo de baixa estatura nas famílias e não se evidenciou um efeito dominante negativo. O escore-Z da altura dos indivíduos carreadores das variantes...
Over the past several years, C-type natriuretic peptide (CNP) and its receptor (NPR-B) system has emerged as an important regulator of endochondral bone growth. Animal models showed a CNP/NPR-B role in promoting chondrocyte proliferation and differentiation and matrix synthesis. Biallelic loss-of-function mutations in NPR-B gene (NPR2) cause acromesomelic dysplasia type Maroteux (AMDM), a skeletal dysplasia with extreme short stature. Relatives of patients with AMDM, heterozygous for NPR2 mutations, were noted to be shorter than expected for their population of origin, suggesting that heterozygous mutations in NPR2 could be a cause of idiopathic short stature (ISS). The objective of this study was to investigate the presence of NPR2 mutations in a group of patients with ISS and to correlate molecular findings with phenotype. The NPR2 coding region was sequenced by Sanger's method in 60 patients with ISS. Five different heterozygous missense variants in NPR2 were identified in five patients. The functional consequences of those variants were established using in vitro cell-based assay to determine guanylate cyclase activity and confocal microscopy to determine intracellular localization of NPR-B. The variants c.226T > C / p.Ser76Pro, c.788G > C / p.Arg263Pro and c.2455C > T / p.Arg819Cys segregated with short stature phenotype and were functionally deleterious. NPR-B receptors with these three variants have a dominantnegative effect and p.Ser76Pro and p.Arg263Pro NPR-B were not localized in the cell membrane. Cosegregation analysis of the variants c.491C > G / p.Ala164Gly and c.1636A > T / p.Asn546Tyr was inconclusive and they did not have a dominant negative effect. Carriers of functionally deleterious variants have a height SD score that ranged from -4.5 to -1.7. One of these patients and two relatives have disproportionate short stature and one has shortened metacarpal. In conclusion, heterozygous mutations in NPR2 gene are cause of short stature in...
Assuntos
Humanos , Animais , Masculino , Feminino , Camundongos , Biologia Molecular/métodos , Estatura/genética , Insuficiência de Crescimento/genética , Lâmina de Crescimento/crescimento & desenvolvimento , Nanismo/genética , Peptídeo Natriurético Tipo C/deficiência , Peptídeo Natriurético Tipo C/genéticaRESUMO
BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder. EXPERIMENTAL APPROACH: Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro. KEY RESULTS: Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFß-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients. CONCLUSIONS AND IMPLICATIONS: These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.
Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Peptídeos Natriuréticos/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Animais , Fator Natriurético Atrial , Bleomicina , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Hipertensão Pulmonar/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Peptídeo Natriurético Tipo C/deficiência , Peptídeo Natriurético Tipo C/metabolismo , Precursores de Proteínas/deficiência , Precursores de Proteínas/metabolismo , Fibrose Pulmonar/induzido quimicamente , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismoAssuntos
Desenvolvimento Ósseo , Cartilagem/fisiologia , Peptídeo Natriurético Tipo C/genética , Animais , Remodelação Óssea , Condrócitos/fisiologia , Lâmina de Crescimento/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptídeo Natriurético Tipo C/deficiência , Peptídeo Natriurético Tipo C/fisiologiaRESUMO
Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc(-/-) mice). The Nppc(-/-) mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc(-/-) mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia.