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1.
PLoS Biol ; 18(11): e3000885, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33170835

RESUMO

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase (Nox) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5-but not NOX1, NOX2, or NOX4-with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human Nox5 in endothelial cells developed-upon aging-severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.


Assuntos
Hipertensão/fisiopatologia , NADPH Oxidase 5/genética , Óxido Nítrico/metabolismo , Adulto , Fatores Etários , Idoso , Animais , Células Endoteliais , Endotélio Vascular , Feminino , Técnicas de Introdução de Genes/métodos , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , NADPH Oxidase 5/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio
2.
Proc Natl Acad Sci U S A ; 116(14): 7129-7136, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894481

RESUMO

Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein-protein interactions but also metabolite-dependent interactions. Based on this protein-metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4 Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood-brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein-metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Descoberta de Drogas , NADPH Oxidase 4/metabolismo , Óxido Nítrico Sintase/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Masculino , Camundongos , NADPH Oxidase 4/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Pirazóis/farmacologia , Piridonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acidente Vascular Cerebral/prevenção & controle
3.
Proc Natl Acad Sci U S A ; 114(46): 12315-12320, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29087944

RESUMO

Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood-brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.


Assuntos
Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/enzimologia , Isquemia Miocárdica/enzimologia , NADPH Oxidase 4/genética , Animais , Benzoxazóis/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Isquemia Encefálica/prevenção & controle , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Feminino , Artéria Femoral/lesões , Regulação da Expressão Gênica , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Membro Posterior/metabolismo , Membro Posterior/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Isquemia Miocárdica/prevenção & controle , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Especificidade de Órgãos , Pirazóis/farmacologia , Piridonas/farmacologia , Ratos , Transdução de Sinais , Triazóis/farmacologia
4.
Cell Mol Life Sci ; 69(14): 2327-43, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22648375

RESUMO

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.


Assuntos
NADPH Oxidases/metabolismo , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Animais , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Estresse Oxidativo/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Sci Rep ; 12(1): 11570, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35798762

RESUMO

Atherosclerosis and its complications are major causes of cardiovascular morbidity and death. Apart from risk factors such as hypercholesterolemia and inflammation, the causal molecular mechanisms are unknown. One proposed causal mechanism involves elevated levels of reactive oxygen species (ROS). Indeed, early expression of the ROS forming NADPH oxidase type 5 (Nox5) in vascular endothelial cells correlates with atherosclerosis and aortic aneurysm. Here we test the pro-atherogenic Nox5 hypothesis using mouse models. Because Nox5 is missing from the mouse genome, a knock-in mouse model expressing human Nox5 in its physiological location of endothelial cells (eNOX5ki/ki) was tested as a possible new humanised mouse atherosclerosis model. However, whether just on a high cholesterol diet or by crossing in aortic atherosclerosis-prone ApoE-/- mice with and without induction of diabetes, Nox5 neither induced on its own nor aggravated aortic atherosclerosis. Surprisingly, however, diabetic ApoE-/- x eNOX5ki/ki mice developed aortic aneurysms more than twice as often correlating with lower vascular collagens, as assessed by trichrome staining, without changes in inflammatory gene expression, suggesting that endothelial Nox5 directly affects extracellular matrix remodelling associated with aneurysm formation in diabetes. Thus Nox5-derived reactive oxygen species are not a new independent mechanism of atherosclerosis but may enhance the frequency of abdominal aortic aneurysms in the context of diabetes. Together with similar clinical findings, our preclinical target validation opens up a first-in-class mechanism-based approach to treat or even prevent abdominal aortic aneurysms.


Assuntos
Aneurisma da Aorta Abdominal , Aterosclerose , Diabetes Mellitus , NADPH Oxidase 5 , Animais , Aterosclerose/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Camundongos , Camundongos Knockout para ApoE , NADPH Oxidase 5/metabolismo , Oxigênio , Espécies Reativas de Oxigênio/metabolismo
7.
NPJ Syst Biol Appl ; 4: 8, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29423274

RESUMO

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease-disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy.

8.
J Cereb Blood Flow Metab ; 36(9): 1508-12, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27354091

RESUMO

Promising results have been reported in preclinical stroke target validation for pharmacological principles that disrupt the N-methyl-D-aspartate receptor-post-synaptic density protein-95-neuronal nitric oxide synthase complex. However, post-synaptic density protein-95 is also coupled to potentially neuroprotective mechanisms. As post-synaptic density protein-95 inhibitors may interfere with potentially neuroprotective mechanisms and sufficient validation has often been an issue in translating basic stroke research, we wanted to close that gap by comparing post-synaptic density protein-95 inhibitors with NOS1(-/-) mice and a NOS inhibitor. We confirm the deleterious role of NOS1 in stroke both in vivo and in vitro, but find three pharmacological post-synaptic density protein-95 inhibitors to be therapeutically ineffective.


Assuntos
Lesões Encefálicas/prevenção & controle , Isquemia Encefálica/prevenção & controle , Guanilato Quinases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Animais , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Ligação Proteica
9.
Antioxid Redox Signal ; 23(5): 406-27, 2015 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-24383718

RESUMO

SIGNIFICANCE: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. RECENT ADVANCES: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. CRITICAL ISSUES: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. FUTURE DIRECTIONS: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition.


Assuntos
Inibidores Enzimáticos/metabolismo , Evolução Molecular , NADPH Oxidases/antagonistas & inibidores , Animais , Inibidores Enzimáticos/química , Humanos , Ligantes , NADPH Oxidases/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo
10.
Sci Rep ; 5: 13428, 2015 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-26310318

RESUMO

Biomedical research suffers from a dramatically poor translational success. For example, in ischemic stroke, a condition with a high medical need, over a thousand experimental drug targets were unsuccessful. Here, we adopt methods from clinical research for a late-stage pre-clinical meta-analysis (MA) and randomized confirmatory trial (pRCT) approach. A profound body of literature suggests NOX2 to be a major therapeutic target in stroke. Systematic review and MA of all available NOX2(-/y) studies revealed a positive publication bias and lack of statistical power to detect a relevant reduction in infarct size. A fully powered multi-center pRCT rejects NOX2 as a target to improve neurofunctional outcomes or achieve a translationally relevant infarct size reduction. Thus stringent statistical thresholds, reporting negative data and a MA-pRCT approach can ensure biomedical data validity and overcome risks of bias.


Assuntos
Terapia de Alvo Molecular , NADPH Oxidases/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Animais , Infarto Encefálico/complicações , Infarto Encefálico/patologia , Feminino , Camundongos Endogâmicos C57BL , Viés de Publicação , Reprodutibilidade dos Testes , Relatório de Pesquisa , Estatística como Assunto , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/enzimologia
11.
J Mol Med (Berl) ; 90(12): 1391-406, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23090009

RESUMO

Ischemia/reperfusion injury (IRI) is crucial in the pathology of major cardiovascular diseases, such as stroke and myocardial infarction. Paradoxically, both the lack of oxygen during ischemia and the replenishment of oxygen during reperfusion can cause tissue injury. Clinical outcome is also determined by a third, post-reperfusion phase characterized by tissue remodeling and adaptation. Increased levels of reactive oxygen species (ROS) have been suggested to be key players in all three phases. As a second paradox, ROS seem to play a double-edged role in IRI, with both detrimental and beneficial effects. These Janus-faced effects of ROS may be linked to the different sources of ROS or to the different types of ROS that exist and may also depend on the phase of IRI. With respect to therapeutic implications, an untargeted application of antioxidants may not differentiate between detrimental and beneficial ROS, which might explain why this approach is clinically ineffective in lowering cardiovascular mortality. Under some conditions, antioxidants even appear to be harmful. In this review, we discuss recent breakthroughs regarding a more targeted and promising approach to therapeutically modulate ROS in IRI. We will focus on NADPH oxidases and their catalytic subunits, NOX, as they represent the only known enzyme family with the sole function to produce ROS. Similar to ROS, NADPH oxidases may play a dual role as different NOX isoforms may mediate detrimental or protective processes. Unraveling the precise sequence of events, i.e., determining which role the individual NOX isoforms play in the various phases of IRI, may provide the crucial molecular and mechanistic understanding to finally effectively target oxidative stress.


Assuntos
NADPH Oxidases/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/metabolismo , Animais , Humanos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo
12.
Ned Tijdschr Geneeskd ; 154: A205, 2010.
Artigo em Holandês | MEDLINE | ID: mdl-20170579

RESUMO

A premature neonate had hypoplastic thumbnails with triangular lunulae and an aplastic left patella, due tot nail-patella-syndrome.


Assuntos
Síndrome da Unha-Patela/diagnóstico , Trigêmeos , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Unhas Malformadas/diagnóstico
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