RESUMO
Several genetic defects, including a mutation in myosin heavy chain 11 (Myh11), are reported to cause familial thoracic aortic aneurysm and dissection (FTAAD). We recently showed that mice lacking K1256 of Myh11 developed aortic dissection when stimulated with angiotensin II, despite the absence of major pathological phenotypic abnormalities prior to stimulation. In this study, we used a comprehensive, data-driven, unbiased, multi-omics approach to find underlying changes in transcription and metabolism that predispose the aorta to dissection in mice harboring the Myh11 K1256del mutation. Pathway analysis of transcriptomes showed that genes involved in membrane transport were downregulated in homozygous mutant (Myh11ΔK/ΔK) aortas. Furthermore, expanding the analysis with metabolomics showed that two mechanisms that raise the cytosolic Ca2+ concentration-multiple calcium channel expression and ADP-ribose synthesis-were attenuated in Myh11ΔK/ΔK aortas. We suggest that the impairment of the Ca2+ influx attenuates aortic contraction and that suboptimal contraction predisposes the aorta to dissection.
Assuntos
Aneurisma da Aorta Torácica , Cálcio , Camundongos , Animais , Cálcio/metabolismo , Multiômica , Aorta/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Aorta Torácica/patologiaRESUMO
Chronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1-driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1ß secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1ß secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.
Assuntos
Síndromes Periódicas Associadas à Criopirina/patologia , Descoberta de Drogas/métodos , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Teóricos , Mosaicismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Células Cultivadas , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Síndromes Periódicas Associadas à Criopirina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Lactente , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
Heart failure with reduced ejection fraction (HFrEF) is characterized not only by reduced left ventricular ejection fraction (EF) but is also combined with symptoms such as dyspnea, fatigue, and edema. Several pharmacological interventions have been established. However, a treatment targeting a novel pathophysiological mechanism is still needed. Evidence indicating that inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may be cardioprotective has been accumulating. Thus, we focused on vitamin K3 and used its framework as a new PDK4 inhibitor skeleton to synthesize new PDK4 inhibitors that show higher activity than the existing PDK4 inhibitor, dichloroacetic acid, and tested their cardioprotective effects on a mouse heart failure model. Among these inhibitors, PDK4 inhibitor 8 improved EF the most, even though it did not reverse cardiac fibrosis or wall thickness. This novel, potent PDK4 inhibitor may improve EF of failing hearts by regulating bioenergetics via activation of the tricarboxylic acid cycle.
Assuntos
Insuficiência Cardíaca , Proteínas Quinases , Animais , Camundongos , Insuficiência Cardíaca/tratamento farmacológico , Volume Sistólico , Função Ventricular Esquerda , Coração , Modelos Animais de DoençasRESUMO
Pathogenic variants in myosin heavy chain (Myh11) cause familial thoracic aortic aneurysms and dissections (FTAAD). However, the underlying pathological mechanisms remain unclear because of a lack of animal models. In this study, we established a mouse model with Myh11 K1256del, the pathogenic variant we found previously in two FTAAD families. The Myh11∆K/∆K aorta showed increased wall thickness and ultrastructural abnormalities, including weakened cell adhesion. Notably, the Myh11∆K/+ mice developed aortic dissections and intramural haematomas when stimulated with angiotensin II. Mechanistically, integrin subunit alpha2 (Itga2) was downregulated in the Myh11∆K/∆K aortas, and the smooth muscle cell lineage cells that differentiated from Myh11∆K/∆K induced pluripotent stem cells. The contractility of the Myh11∆K/∆K aortas in response to phenylephrine was also reduced. These results imply that the suboptimal cell adhesion indicated by Itga2 downregulation causes a defect in the contraction of the aorta. Consequently, the defective contraction may increase the haemodynamic stress underlying the aortic dissections.
Assuntos
Aneurisma da Aorta Torácica , Dissecção Aórtica , Dissecção Aórtica/genética , Dissecção Aórtica/metabolismo , Animais , Aorta/patologia , Aneurisma da Aorta Torácica/patologia , Lisina/metabolismo , CamundongosRESUMO
In heart failure (HF) caused by hypertension, the myocyte size increases, and the cardiac wall thickens. A low-molecular-weight compound called ICG001 impedes ß-catenin-mediated gene transcription, thereby protecting both the heart and kidney. However, the HF-preventive mechanisms of ICG001 remain unclear. Hence, we investigated how ICG001 can prevent cardiac hypertrophy and fibrosis induced by transverse aortic constriction (TAC). Four weeks after TAC, ICG001 attenuated cardiac hypertrophy and fibrosis in the left ventricular wall. The TAC mice treated with ICG001 showed a decrease in the following: mRNA expression of brain natriuretic peptide (Bnp), Klf5, fibronectin, ß-MHC, and ß-catenin, number of cells expressing the macrophage marker CD68 shown in immunohistochemistry, and macrophage accumulation shown in flow cytometry. Moreover, ICG001 may mediate the substrates in the glycolysis pathway and the distinct alteration of oxidative stress during cardiac hypertrophy and HF. In conclusion, ICG001 is a potential drug that may prevent cardiac hypertrophy and fibrosis by regulating KLF5, immune activation, and the Wnt/ß-catenin signaling pathway and inhibiting the inflammatory response involving macrophages.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Cardiomegalia/tratamento farmacológico , Cardiomiopatias/tratamento farmacológico , Insuficiência Cardíaca/prevenção & controle , Proteínas de Membrana/antagonistas & inibidores , Miocárdio/patologia , Fosfoproteínas/antagonistas & inibidores , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , Animais , Cardiomegalia/etiologia , Cardiomiopatias/etiologia , Fibrose , Glicólise/efeitos dos fármacos , Insuficiência Cardíaca/etiologia , Inflamação , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/imunologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Aortic dissection is a life-threatening condition, which is characterised by separation of the constituent layers of the aortic wall. We have recently shown that monocyte/macrophage infiltration into the aortic wall is a pathogenic mechanism of the condition. In the present study, we investigated whether the anti-inflammatory agent, indomethacin, could inhibit monocyte/macrophage accumulation in the aortic wall and ensuing dissection. Indomethacin was administered (from 3 days prior with daily oral administration) to mice in which aortic dissection was induced using beta-aminopropionitrile (BAPN) and angiotensin II (Ang II) infusion (2 weeks). Indomethacin prevented death from abdominal aortic dissection and decreased incidence of aortic dissection by as high as 40%. Histological and flow cytometry analyses showed that indomethacin administration resulted in inhibition of monocyte transendothelial migration and monocyte/macrophage accumulation in the aortic wall. These results indicate that indomethacin administration reduces rate of onset of aortic dissection in a murine model of the condition.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Aneurisma da Aorta Abdominal/tratamento farmacológico , Dissecção Aórtica/prevenção & controle , Ruptura Aórtica/prevenção & controle , Indometacina/uso terapêutico , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BLRESUMO
AIMS: Doxorubicin (Dox) is a potent anticancer agent that is widely used in the treatment of a variety of cancers, but its usage is limited by cumulative dose-dependent cardiotoxicity mainly due to oxidative damage. Ataxia telangiectasia mutated (ATM) kinase is thought to play a role in mediating the actions of oxidative stress. Here, we show that ATM in cardiac fibroblasts is essential for Dox-induced cardiotoxicity. METHODS AND RESULTS: ATM knockout mice showed attenuated Dox-induced cardiotoxic effects (e.g. cardiac dysfunction, apoptosis, and mortality). As ATM was expressed and activated predominantly in cardiac fibroblasts, fibroblast-specific Atm-deleted mice (Atm(fl/fl);Postn-Cre) were generated to address cell type-specific effects, which showed that the fibroblast is the key lineage mediating Dox-induced cardiotoxicity through ATM. Mechanistically, ATM activated the Fas ligand, which subsequently regulated apoptosis in cardiomyocytes at later stages. Therapeutically, a potent and selective inhibitor of ATM, KU55933, when administered systemically was able to prevent Dox-induced cardiotoxicity. CONCLUSION: ATM-regulated effects within cardiac fibroblasts are pivotal in Dox-induced cardiotoxicity, and antagonism of ATM and its functions may have potential therapeutic implications.
Assuntos
Ataxia Telangiectasia/genética , Cardiotoxicidade/etiologia , Doxorrubicina/farmacologia , Fibroblastos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacosRESUMO
Aortic dissection and intramural haematoma comprise an aortopathy involving separation of the aortic wall. Underlying mechanisms of the condition remain unclear. Here we show that granulocyte macrophage colony-stimulating factor (GM-CSF) is a triggering molecule for this condition. Transcription factor Krüppel-like factor 6 (KLF6)-myeloid-specific conditional deficient mice exhibit this aortic phenotype when subjected to aortic inflammation. Mechanistically, KLF6 downregulates expression and secretion of GM-CSF. Administration of neutralizing antibody against GM-CSF prevents the condition in these mice. Conversely, administration of GM-CSF in combination with aortic inflammation to wild-type mice is sufficient to induce the phenotype, suggesting the general nature of effects. Moreover, patients with this condition show highly increased circulating levels of GM-CSF, which is also locally expressed in the dissected aorta. GM-CSF is therefore a key regulatory molecule causative of this aortopathy, and modulation of this cytokine might be an exploitable treatment strategy for the condition.
Assuntos
Aneurisma Aórtico/etiologia , Dissecção Aórtica/etiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Hematoma/etiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Dissecção Aórtica/sangue , Animais , Aneurisma Aórtico/sangue , Estudos de Casos e Controles , Feminino , Hematoma/sangue , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/genética , Regulação para CimaRESUMO
AIMS: Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. METHODS AND RESULTS: Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. CONCLUSION: Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts.
Assuntos
Fibroblastos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Trombospondinas/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Trombospondinas/genética , Transativadores/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.