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1.
Dev Dyn ; 252(3): 327-342, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-35434863

RESUMO

Pulmonary arterial hypertension (PAH) is an often fatal condition, the primary pathology of which involves loss of pulmonary vascular perfusion due to progressive aberrant vessel remodeling. The reduced capacity of the pulmonary circulation places increasing strain on the right ventricle of the heart, leading to death by heart failure. Currently, licensed therapies are primarily vasodilators, which have increased the median post-diagnosis life expectancy from 2.8 to 7 years. Although this represents a substantial improvement, the search continues for transformative therapeutics that reverse established disease. The genetics of human PAH heavily implicates reduced endothelial bone morphogenetic protein (BMP) signaling as a causal role for the disease pathobiology. Recent approaches have focused on directly enhancing BMP signaling or removing the inhibitory influence of pathways that repress BMP signaling. In this critical commentary, we review the evidence underpinning the development of two approaches: BMP-based agonists and inhibition of activin/GDF signaling. We also address the key considerations and questions that remain regarding these approaches.


Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Ligantes , Hipertensão Arterial Pulmonar/complicações , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Transdução de Sinais/fisiologia , Ativinas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Citocinas
2.
Am J Respir Crit Care Med ; 206(12): 1522-1533, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-35852389

RESUMO

Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV1, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.


Assuntos
Mutação com Ganho de Função , Pneumopatias , Humanos , Proteínas com Domínio T/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Fenótipo , Pneumopatias/genética , Mutação/genética , Genótipo
3.
J Cell Sci ; 133(14)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32576665

RESUMO

Bone morphogenetic protein 9 (BMP9) and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor ALK1 and the type II receptors activin type-IIA receptor (ACTR-IIA) and bone morphogenetic type II receptor (BMPR-II). We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and might influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. The repression was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.


Assuntos
Células Endoteliais , Fator 2 de Diferenciação de Crescimento , Receptores de Activinas Tipo II , Proteínas Morfogenéticas Ósseas , Quimiocina CCL2/genética , Fator 2 de Diferenciação de Crescimento/genética , Humanos , Fatores de Proteção
4.
Am J Respir Crit Care Med ; 203(11): 1419-1430, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33320799

RESUMO

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.


Assuntos
Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/patologia , Endotélio/patologia , Endotoxemia/sangue , Fator 2 de Diferenciação de Crescimento/sangue , Sepse/sangue , Lesão Pulmonar Aguda/etiologia , Animais , Estudos de Casos e Controles , Células Endoteliais/metabolismo , Endotoxemia/etiologia , Endotoxemia/patologia , Feminino , Humanos , Masculino , Camundongos , Edema Pulmonar/sangue , Edema Pulmonar/etiologia , Edema Pulmonar/patologia , Sepse/etiologia , Sepse/patologia
5.
Int J Mol Sci ; 22(20)2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34681942

RESUMO

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Endoglina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células CHO , Cricetulus , Endoglina/genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Recombinantes de Fusão/genética
6.
Am J Respir Cell Mol Biol ; 63(2): 160-171, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32255665

RESUMO

Mutations in the gene encoding BMPR2 (bone morphogenetic protein type 2 receptor) are the major cause of heritable pulmonary arterial hypertension (PAH). Point mutations in the BMPR2 ligand-binding domain involving cysteine residues (such as C118W) are causative of PAH and predicted to cause protein misfolding. Using heterologous overexpression systems, we showed previously that these mutations lead to retention of BMPR2 in the endoplasmic reticulum but are partially rescued by chemical chaperones. Here, we sought to determine whether the chemical chaperone 4-phenylbutyrate (4PBA) restores BMPR2 signaling in primary cells and in a knockin mouse harboring a C118W mutation. First, we confirmed dysfunctional BMP signaling in dermal fibroblasts isolated from a family with PAH segregating the BMPR2 C118W mutation. After BMP4 treatment, the induction of downstream signaling targets (Smad1/5, ID1 [inhibitor of DNA binding 1], and ID2) was significantly reduced in C118W mutant cells. Treatment with 4PBA significantly rescued Smad1/5, ID1, and ID2 expression. Pulmonary artery smooth muscle cells isolated from the lungs of heterozygous mice harboring the Bmpr2 C118W mutation exhibited significantly increased proliferation. In the presence of 4PBA, hyperproliferation was dramatically reduced. Furthermore, in vivo, 4PBA treatment of Bmpr2 C118W mice partially rescued Bmpr2 expression, restored downstream signaling, and improved vascular remodeling. These findings demonstrate in primary cells and in a knockin mouse that the repurposed small-molecule chemical chaperone 4PBA might be a promising precision medicine approach to treat PAH in patients with specific subtypes of BMPR2 mutation involving cysteine substitutions in the ligand-binding domain.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Cisteína/genética , Mutação/genética , Compostos Organofosforados/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Humanos , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/efeitos dos fármacos , Transdução de Sinais/genética , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/genética
7.
Am J Respir Crit Care Med ; 199(7): 891-902, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312106

RESUMO

RATIONALE: BMP9 (bone morphogenetic protein 9) is a circulating endothelial quiescence factor with protective effects in pulmonary arterial hypertension (PAH). Loss-of-function mutations in BMP9, its receptors, and downstream effectors have been reported in heritable PAH. OBJECTIVES: To determine how an acquired deficiency of BMP9 signaling might contribute to PAH. METHODS: Plasma levels of BMP9 and antagonist soluble endoglin were measured in group 1 PAH, group 2 and 3 pulmonary hypertension (PH), and in patients with severe liver disease without PAH. MEASUREMENTS AND MAIN RESULTS: BMP9 levels were markedly lower in portopulmonary hypertension (PoPH) versus healthy control subjects, or other etiologies of PAH or PH; distinguished PoPH from patients with liver disease without PAH; and were an independent predictor of transplant-free survival. BMP9 levels were decreased in mice with PH associated with CCl4-induced portal hypertension and liver cirrhosis, but were normal in other rodent models of PH. Administration of ALK1-Fc, a BMP9 ligand trap consisting of the activin receptor-like kinase-1 extracellular domain, exacerbated PH and pulmonary vascular remodeling in mice treated with hypoxia versus hypoxia alone. CONCLUSIONS: BMP9 is a sensitive and specific biomarker of PoPH, predicting transplant-free survival and the presence of PAH in liver disease. In rodent models, acquired deficiency of BMP9 signaling can predispose to or exacerbate PH, providing a possible mechanistic link between PoPH and heritable PAH. These findings describe a novel experimental model of severe PH that provides insight into the synergy between pulmonary vascular injury and diminished BMP9 signaling in the pathogenesis of PAH.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
8.
J Biol Chem ; 292(33): 13714-13726, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28646109

RESUMO

Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Endotélio Vascular/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/antagonistas & inibidores , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Aorta , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Selectina E/química , Selectina E/genética , Selectina E/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Fator 2 de Diferenciação de Crescimento , Humanos , Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Cinética , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/agonistas , Regulação para Cima/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/química , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
9.
Circulation ; 135(12): 1160-1173, 2017 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-28137936

RESUMO

BACKGROUND: Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. METHODS: In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr1]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. RESULTS: ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and ß-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in monocrotaline-exposed rats. CONCLUSIONS: These results show that ELA is an endogenous agonist of the human apelin receptor, exhibits a cardiovascular profile comparable to apelin, and is downregulated in human disease and rodent PAH models, and exogenous peptide can reduce the severity of cardiopulmonary remodeling and function in PAH in rats. This study provides additional proof of principle that an apelin receptor agonist may be of therapeutic use in PAH in humans.


Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Hormônios Peptídicos/uso terapêutico , Sequência de Aminoácidos , Animais , Apelina , Sítios de Ligação , Cateterismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Hipertensão Pulmonar/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/agonistas , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Masculino , Simulação de Dinâmica Molecular , Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Hormônios Peptídicos/farmacologia , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley
10.
Circulation ; 136(25): 2451-2467, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-28971999

RESUMO

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. METHODS: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. RESULTS: Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. CONCLUSIONS: Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.


Assuntos
Hipertensão Pulmonar Primária Familiar/patologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , MicroRNAs/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Piruvato Quinase/metabolismo , Animais , Antagomirs/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hipertensão Pulmonar Primária Familiar/genética , Hipertensão Pulmonar Primária Familiar/metabolismo , Glicólise , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Quinases Lim/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/antagonistas & inibidores , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Piruvato Quinase/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Simportadores/metabolismo
11.
J Pathol ; 243(3): 390-400, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28815607

RESUMO

Glomerular scarring, known as glomerulosclerosis, occurs in many chronic kidney diseases and involves interaction between glomerular endothelial cells (GECs), podocytes, and mesangial cells (MCs), leading to signals that promote extracellular matrix deposition and endothelial cell dysfunction and loss. We describe a 3D tri-culture system to model human glomerulosclerosis. In 3D monoculture, each cell type alters its phenotype in response to TGFß, which has been implicated as an important mediator of glomerulosclerosis. GECs form a lumenized vascular network, which regresses in response to TGFß. MCs respond to TGFß by forming glomerulosclerotic-like nodules with matrix deposition. TGFß treatment of podocytes does not alter cell morphology but increases connective tissue growth factor (CTGF) expression. BMP7 prevents TGFß-induced GEC network regression, whereas TGFß-induced MC nodule formation is prevented by SMAD3 siRNA knockdown or ALK5 inhibitors but not BMP7, and increased phospho-SMAD3 was observed in human glomerulosclerosis. In 3D tri-culture, GECs, podocytes, and MCs form a vascular network in which GECs and podocytes interact intimately within a matrix containing MCs. TGFß treatment induces formation of nodules, but combined inhibition of ALK5 and CTGF is required to prevent TGFß-induced nodule formation in tri-cellular cultures. Identification of therapeutic targets for glomerulosclerosis depends on the 3D culture of all three glomerular cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Glomérulos Renais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Matriz Extracelular/metabolismo , Humanos , Nefropatias/patologia , Glomérulos Renais/metabolismo , Células Mesangiais/citologia , Receptor do Fator de Crescimento Transformador beta Tipo I
12.
J Immunol ; 197(8): 3302-3314, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27647829

RESUMO

Bone morphogenetic protein (BMP)9 is a circulating growth factor that is part of the TGF-ß superfamily and is an essential regulator of vascular endothelial homeostasis. Previous studies have suggested a role for BMP9 signaling in leukocyte recruitment to the endothelium, but the directionality of this effect and underlying mechanisms have not been elucidated. In this study, we report that BMP9 upregulates TLR4 expression in human endothelial cells and that BMP9 pretreatment synergistically increases human neutrophil recruitment to LPS-stimulated human endothelial monolayers in an in vitro flow adhesion assay. BMP9 alone did not induce neutrophil recruitment to the endothelium. We also show that E-selectin and VCAM-1, but not ICAM-1, are upregulated in response to BMP9 in LPS-stimulated human endothelial cells. Small interfering RNA knockdown of activin receptor-like kinase 1 inhibited the BMP9-induced expression of TLR4 and VCAM-1 and inhibited BMP9-induced human neutrophil recruitment to LPS-stimulated human endothelial cells. BMP9 treatment also increased leukocyte recruitment within the pulmonary circulation in a mouse acute endotoxemia model. These results demonstrate that although BMP9 alone does not influence leukocyte recruitment, it primes the vascular endothelium to mount a more intense response when challenged with LPS through an increase in TLR4, E-selectin, and VCAM-1 and ultimately through enhanced leukocyte recruitment.


Assuntos
Endotélio Vascular/citologia , Fatores de Diferenciação de Crescimento/metabolismo , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Animais , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento , Humanos , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
J Biol Chem ; 291(6): 2954-66, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26631724

RESUMO

BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cardiomegalia/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Linhagem Celular , Células Endoteliais/patologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Humanos , Camundongos , Ratos
14.
Am J Hum Genet ; 93(3): 530-7, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23972370

RESUMO

Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-ß) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT.


Assuntos
Vasos Sanguíneos/anormalidades , Fatores de Diferenciação de Crescimento/genética , Mutação/genética , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/patologia , Adolescente , Adulto , Substituição de Aminoácidos/genética , Animais , Feminino , Predisposição Genética para Doença , Fator 2 de Diferenciação de Crescimento , Humanos , Ligantes , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Fenótipo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transdução de Sinais/genética , Síndrome , Fator de Crescimento Transformador beta/genética , Peixe-Zebra/genética
15.
Development ; 140(16): 3403-12, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23863480

RESUMO

Blood flow plays crucial roles in vascular development, remodeling and homeostasis, but the molecular pathways required for transducing flow signals are not well understood. In zebrafish embryos, arterial expression of activin receptor-like kinase 1 (alk1), which encodes a TGFß family type I receptor, is dependent on blood flow, and loss of alk1 mimics lack of blood flow in terms of dysregulation of a subset of flow-responsive arterial genes and increased arterial endothelial cell number. These data suggest that blood flow activates Alk1 signaling to promote a flow-responsive gene expression program that limits nascent arterial caliber. Here, we demonstrate that restoration of endothelial alk1 expression to flow-deprived arteries fails to rescue Alk1 activity or normalize arterial endothelial cell gene expression or number, implying that blood flow may play an additional role in Alk1 signaling independent of alk1 induction. To this end, we define cardiac-derived Bmp10 as the crucial ligand for endothelial Alk1 in embryonic vascular development, and provide evidence that circulating Bmp10 acts through endothelial Alk1 to limit endothelial cell number in and thereby stabilize the caliber of nascent arteries. Thus, blood flow promotes Alk1 activity by concomitantly inducing alk1 expression and distributing Bmp10, thereby reinforcing this signaling pathway, which functions to limit arterial caliber at the onset of flow. Because mutations in ALK1 cause arteriovenous malformations (AVMs), our findings suggest that an impaired flow response initiates AVM development.


Assuntos
Receptores de Ativinas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Artérias Carótidas/enzimologia , Embrião não Mamífero/irrigação sanguínea , Endotélio Vascular/enzimologia , Receptores de Ativinas/genética , Animais , Malformações Arteriovenosas/enzimologia , Malformações Arteriovenosas/patologia , Proteínas Morfogenéticas Ósseas/genética , Contagem de Células , Embrião não Mamífero/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Miocárdio/enzimologia , Miocárdio/patologia , Fosforilação , Transporte Proteico , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
J Biol Chem ; 289(45): 31150-9, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25237187

RESUMO

BMP9, a member of the TGFß superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.


Assuntos
Regulação da Expressão Gênica , Fatores de Diferenciação de Crescimento/metabolismo , Oxirredução , Animais , Linhagem Celular , Cristalização , Cristalografia por Raios X , DNA Complementar/metabolismo , Dimerização , Dissulfetos/química , Células Endoteliais/citologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Ligantes , Camundongos , Fenótipo , Proteólise , Proteínas Recombinantes/metabolismo , Transdução de Sinais
18.
Hum Mol Genet ; 22(18): 3667-79, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23669347

RESUMO

Pulmonary arterial hypertension (PAH) is characterized by dysregulated pulmonary artery endothelial cell (PAEC) proliferation, apoptosis and permeability. Loss-of-function mutations in the bone morphogenetic protein receptor type-II (BMPR-II) are the most common cause of heritable PAH, usually resulting in haploinsufficiency. We previously showed that BMPR-II expression is regulated via a lysosomal degradative pathway. Here, we show that the antimalarial drug, chloroquine, markedly increased cell surface expression of BMPR-II protein independent of transcription in PAECs. Inhibition of protein synthesis experiments revealed a rapid turnover of cell surface BMPR-II, which was inhibited by chloroquine treatment. Chloroquine enhanced PAEC expression of BMPR-II following siRNA knockdown of the BMPR-II transcript. Using blood outgrowth endothelial cells (BOECs), we confirmed that signalling in response to the endothelial BMPR-II ligand, BMP9, is compromised in BOECs from patients harbouring BMPR-II mutations, and in BMPR-II mutant PAECs. Chloroquine significantly increased gene expression of BMP9-BMPR-II signalling targets Id1, miR21 and miR27a in both mutant BMPR-II PAECs and BOECs. These findings provide support for the restoration of cell surface BMPR-II with agents such as chloroquine as a potential therapeutic approach for heritable PAH.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Cloroquina/farmacologia , Células Endoteliais/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Cloroquina/uso terapêutico , Hipertensão Pulmonar Primária Familiar , Fator 2 de Diferenciação de Crescimento , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Lisossomos/metabolismo , Mutação , Artéria Pulmonar/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Transcrição Gênica
19.
Biochem Soc Trans ; 43(3): 524-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26009202

RESUMO

Pulmonary arterial hypertension (PAH) is a complex, multi-factorial disorder characterized by both constriction and remodelling of the distal pulmonary vasculature. This leads to increased pulmonary pressures and eventually right heart failure. Current drugs, which primarily target the vasoconstriction, serve only to prolong life and novel therapies targeting both the vasoconstriction and the remodelling are required. Aberrant signalling between cells of the pulmonary vasculature has been associated with the development of PAH. In particular, endothelial dysfunction can lead to hyperplasia of the underlying medial layer. Connexins are a family of transmembrane proteins which can form intercellular communication channels known as gap junctions. This review will discuss recent evidence which shows that connexins play a role in regulation of the pulmonary vasculature and that dysregulation of connexins may contribute to PAH pathogenesis. Interaction of connexins with signalling pathways relevant to the pathogenesis of PAH, such as bone morphogenetic protein (BMP), serotonin and oestrogen are discussed.


Assuntos
Conexinas/metabolismo , Insuficiência Cardíaca/genética , Hipertensão Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Comunicação Celular/genética , Conexinas/genética , Estrogênios/metabolismo , Junções Comunicantes/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Serotonina/metabolismo , Transdução de Sinais/genética , Vasoconstrição/genética
20.
Vascul Pharmacol ; 155: 107381, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38795838

RESUMO

AIMS: Bone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence. METHODS AND RESULTS: We identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays. CONCLUSIONS: This study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction.


Assuntos
Movimento Celular , Células Endoteliais , Fator 2 de Diferenciação de Crescimento , Semaforinas , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Humanos , Movimento Celular/efeitos dos fármacos , Semaforinas/metabolismo , Semaforinas/genética , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Smad5/metabolismo , Proteína Smad5/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Proteína Smad1/metabolismo , Proteína Smad1/genética , Pulmão/metabolismo , Pulmão/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Células Cultivadas
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