RESUMO
Targeting Meis1 and Hoxb13 transcriptional activity could be a viable therapeutic strategy for heart regeneration. In this study, we performd an in silico screening to identify FDA-approved drugs that can inhibit Meis1 and Hoxb13 transcriptional activity based on the resolved crystal structure of Meis1 and Hoxb13 bound to DNA. Paromomycin (Paro) and neomycin (Neo) induced proliferation of neonatal rat ventricular myocytes in vitro and displayed dose-dependent inhibition of Meis1 and Hoxb13 transcriptional activity by luciferase assay and disruption of DNA binding by electromobility shift assay. X-ray crystal structure revealed that both Paro and Neo bind to Meis1 near the Hoxb13-interacting domain. Administration of Paro-Neo combination in adult mice and in pigs after cardiac ischemia/reperfusion injury induced cardiomyocyte proliferation, improved left ventricular systolic function and decreased scar formation. Collectively, we identified FDA-approved drugs with therapeutic potential for induction of heart regeneration in mammals.
Assuntos
Proliferação de Células , Proteínas de Homeodomínio , Proteína Meis1 , Miócitos Cardíacos , Regeneração , Animais , Regeneração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proliferação de Células/efeitos dos fármacos , Proteína Meis1/metabolismo , Proteína Meis1/genética , Neomicina/farmacologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Modelos Animais de Doenças , Aprovação de Drogas , Camundongos , Função Ventricular Esquerda/efeitos dos fármacos , United States Food and Drug Administration , Ratos , Estados Unidos , Cristalografia por Raios X , Masculino , Camundongos Endogâmicos C57BL , Suínos , Células Cultivadas , Transcrição Gênica/efeitos dos fármacosRESUMO
The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.
Assuntos
Células Endoteliais , Proteômica , Humanos , Bioensaio , Microvasos , Organoides , Monoéster Fosfórico HidrolasesRESUMO
Myocardial regeneration in patients with cardiac damage is a long-sought goal of clinical medicine. In animal species in which regeneration occurs spontaneously, as well as in neonatal mammals, regeneration occurs through the proliferation of differentiated cardiomyocytes, which re-enter the cell cycle and proliferate. Hence, the reprogramming of the replicative potential of cardiomyocytes is an achievable goal, provided that the mechanisms that regulate this process are understood. Cardiomyocyte proliferation is under the control of a series of signal transduction pathways that connect extracellular cues to the activation of specific gene transcriptional programmes, eventually leading to the activation of the cell cycle. Both coding and non-coding RNAs (in particular, microRNAs) are involved in this regulation. The available information can be exploited for therapeutic purposes, provided that a series of conceptual and technical barriers are overcome. A major obstacle remains the delivery of pro-regenerative factors specifically to the heart. Improvements in the design of AAV vectors to enhance their cardiotropism and efficacy or, alternatively, the development of non-viral methods for nucleic acid delivery in cardiomyocytes are among the challenges ahead to progress cardiac regenerative therapies towards clinical application.
Assuntos
MicroRNAs , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Coração/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais , Ciclo Celular , Proliferação de Células , MamíferosRESUMO
Drug repositioning continues to be the most effective, practicable possibility to treat COVID-19 patients. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters target cells by binding to the ACE2 receptor via its spike (S) glycoprotein. We used molecular docking-based virtual screening approaches to categorize potential antagonists, halting ACE2-spike interactions by utilizing 450 FDA-approved chemical compounds. Three drug candidates (i.e., anidulafungin, lopinavir, and indinavir) were selected, which show high binding affinity toward the ACE2 receptor. The conformational stability of selected docked complexes was analyzed through molecular dynamics (MD) simulations. The MD simulation trajectories were assessed and monitored for ACE2 deviation, residue fluctuation, the radius of gyration, solvent accessible surface area, and free energy landscapes. The inhibitory activities of the selected compounds were eventually tested in-vitro using Vero and HEK-ACE2 cells. Interestingly, besides inhibiting SARS-CoV-2 S glycoprotein induced syncytia formation, anidulafungin and lopinavir also blocked S-pseudotyped particle entry into target cells. Altogether, anidulafungin and lopinavir are ranked the most effective among all the tested drugs against ACE2 receptor-S glycoprotein interaction. Based on these findings, we propose that anidulafungin is a novel potential drug targeting ACE2, which warrants further investigation for COVID-19 treatment.
RESUMO
Thrombosis of the lung microvasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis that is endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing Niclosamide for COVID-19 therapy.
RESUMO
COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.
Assuntos
Anoctaminas/antagonistas & inibidores , COVID-19/patologia , Fusão Celular , Avaliação Pré-Clínica de Medicamentos , Células Gigantes/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Anoctaminas/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Canais de Cloreto/metabolismo , Chlorocebus aethiops , Feminino , Células Gigantes/metabolismo , Células Gigantes/virologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Masculino , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
A growing body of evidence indicates that cardiac regeneration after myocardial infarction can be achieved by stimulating the endogenous capacity of cardiomyocytes (CMs) to replicate. This process is controlled, both positively and negatively, by a large set of non-coding RNAs (ncRNAs). Some of the microRNAs (miRNAs) that can stimulate CM proliferation is expressed in embryonic stem cells and is required to maintain pluripotency (e.g. the miR-302â¼367 cluster). Others also govern the proliferation of different cell types, including cancer cells (e.g. the miR-17â¼92 cluster). Additional miRNAs were discovered through systematic screenings (e.g. miR-199a-3p and miR-590-3p). Several miRNAs instead suppress CM proliferation and are involved in the withdrawal of CMs from the cell cycle after birth (e.g. the let-7 and miR-15 families). Similar regulatory roles on CM proliferation are also exerted by a few long ncRNAs. This body of information has obvious therapeutic implications, as miRNAs with activator function or short antisense oligonucleotides against inhibitory miRNAs or lncRNAs can be administered to stimulate cardiac regeneration. Expression of miRNAs can be achieved by gene therapy using adeno-associated vectors, which transduce CMs with high efficiency. More effective and safer for therapeutic purposes, small nucleic acid therapeutics can be obtained as chemically modified, synthetic molecules, which can be administered through lipofection or inclusion in lipid or polymer nanoparticles for efficient cardiac delivery. The notion that it is possible to reprogramme CMs into a regenerative state and that this property can be enhanced by ncRNA therapeutics remains exciting, however extensive experimentation in large mammals and rigorous assessment of safety are required to advance towards clinical application.
Assuntos
Proliferação de Células/efeitos dos fármacos , Terapia Genética , Cardiopatias/terapia , MicroRNAs/uso terapêutico , Miócitos Cardíacos/efeitos dos fármacos , RNA Longo não Codificante/uso terapêutico , Regeneração/efeitos dos fármacos , Animais , Regulação da Expressão Gênica , Terapia Genética/efeitos adversos , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Humanos , MicroRNAs/efeitos adversos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Longo não Codificante/efeitos adversos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Recuperação de Função Fisiológica , Transdução de SinaisRESUMO
Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition1, particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue2. Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled.
Assuntos
Morte Súbita Cardíaca/etiologia , MicroRNAs/efeitos adversos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , Sus scrofa/genética , Animais , Proliferação de Células/genética , Coração/fisiologia , Coração/fisiopatologia , Masculino , MicroRNAs/administração & dosagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Regeneração/genéticaRESUMO
Cardiospheres (CSps) are self-assembling clusters of a heterogeneous population of poorly differentiated cells outgrowing from in vitro cultured cardiac explants. Scanty information is available on the molecular pathways regulating CSp growth and their differentiation potential towards cardiac and vascular lineages. Here we report that Notch1 stimulates a massive increase in both CSp number and size, inducing a peculiar gene expression programme leading to a cardiovascular molecular signature. These effects were further enhanced using Adeno-Associated Virus (AAV)-based gene transfer of activated Notch1-intracellular domain (N1-ICD) or soluble-Jagged1 (sJ1) ligand to CSp-forming cells. A peculiar effect was exploited by selected pro-proliferating miRNAs: hsa-miR-590-3p induced a cardiovascular gene expression programme, while hsa-miR-199a-3p acted as the most potent stimulus for the activation of the Notch pathway, thus showing that, unlike in adult cardiomyocytes, these miRNAs involve Notch signalling activation in CSps. Our results identify Notch1 as a crucial regulator of CSp growth and differentiation along the vascular lineage, raising the attracting possibility that forced activation of this pathway might be exploited to promote in vitro CSp expansion as a tool for toxicology screening and cell-free therapeutic strategies.
Assuntos
Proteína Jagged-1/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Receptor Notch1/genética , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Dependovirus , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Humanos , Miócitos Cardíacos/fisiologia , Transdução de Sinais/genética , TransfecçãoRESUMO
Aims: The Notch signalling pathway regulates the balance between proliferation and differentiation in several tissues, including the heart. Our previous work has demonstrated that the proliferative potential of neonatal cardiomyocytes relies on Notch1 activity. A deep investigation on the biochemical regulation of the Notch signalling in cardiomyocytes is the focus of the current research. Methods and results: We show that the Notch1 intracellular domain is acetylated in proliferating neonatal rat cardiomyocytes and that acetylation tightly controls the amplitude and duration of Notch signalling. We found that acetylation extends the half-life of the protein, and enhanced its transcriptional activity, therefore counteracting apoptosis and sustaining cardiomyocyte proliferation. Sirt1 acted as a negative modulator of Notch1 signalling; its overexpression in cardiomyocytes reverted Notch acetylation and dampened its stability. A constitutively acetylated fusion protein between Notch1 and the acetyltransferase domain of p300 promoted cardiomyocyte proliferation, which was remarkably sustained over time. Viral vector-mediated expression of this protein enhanced heart regeneration after apical resection in neonatal mice. Conclusion: These results identify the reversible acetylation of Notch1 as a novel mechanism to modulate its signalling in the heart and tune the proliferative potential of cardiomyocytes.
Assuntos
Proliferação de Células , Miócitos Cardíacos/metabolismo , Processamento de Proteína Pós-Traducional , Receptor Notch1/metabolismo , Acetilação , Animais , Proteína p300 Associada a E1A/metabolismo , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Domínios Proteicos , Estabilidade Proteica , Ratos Wistar , Receptor Notch1/genética , Regeneração , Transdução de Sinais , Sirtuína 1/metabolismo , Fatores de TempoRESUMO
We performed whole exome sequencing in individuals from a family with autosomal dominant gastropathy resembling Ménétrier disease, a premalignant gastric disorder with epithelial hyperplasia and enhanced EGFR signalling. Ménétrier disease is believed to be an acquired disorder, but its aetiology is unknown. In affected members, we found a missense p.V742G variant in MIB2, a gene regulating NOTCH signalling that has not been previously linked to human diseases. The variant segregated with the disease in the pedigree, affected a highly conserved amino acid residue, and was predicted to be deleterious although it was found with a low frequency in control individuals. The purified protein carrying the p.V742G variant showed reduced ubiquitination activity in vitro and white blood cells from affected individuals exhibited significant reductions of HES1 and NOTCH3 expression reflecting alteration of NOTCH signalling. Because mutations of MIB1, the homolog of MIB2, have been found in patients with left ventricle non-compaction (LVNC), we investigated members of our family with Ménétrier-like disease for this cardiac abnormality. Asymptomatic left ventricular hypertrabeculation, the mildest end of the LVNC spectrum, was detected in two members carrying the MIB2 variant. Finally, we identified an additional MIB2 variant (p.V984L) affecting protein stability in an unrelated isolated case with LVNC. Expression of both MIB2 variants affected NOTCH signalling, proliferation and apoptosis in primary rat cardiomyocytes.In conclusion, we report the first example of left ventricular hypertrabeculation/LVNC with germline MIB2 variants resulting in altered NOTCH signalling that might be associated with a gastropathy clinically overlapping with Ménétrier disease.