RESUMO
BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.
Assuntos
Cálcio , Miocárdio , Proteínas de Ligação a RNA , Proteínas de Peixe-Zebra , Animais , Humanos , Cálcio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Inflammation triggered by the deposition of LDL (low-density lipoprotein) in the arterial wall leads to the development of atherosclerosis. Regulatory T (Treg) cells inhibit vascular inflammation through the induction of immune tolerance toward LDL-related antigens. However, tolerogenic mechanisms that promote the generation of LDL-specific Treg cells in vivo remain unclear. METHODS: We identified LDL-specific T cells by activation-induced marker expression and analyzed expression profiles and suppressive functions of TCR (T-cell antigen receptor)-transgenic T cells upon repetitive transfer into antigen-transgenic mice via flow cytometry. RESULTS: We investigated the naturally occurring Treg-cell response against human LDL in standard chow diet-fed mice that are transgenic for human ApoB100 (apolipoprotein B100). We found that IL (interleukin)-10 expression in LDL-specific T cells from spleen increases with age, albeit LDL-specific populations do not enlarge in older mice. To investigate the generation of IL-10-producing LDL-specific T cells, we transferred naive CD4+ T cells recognizing human ApoB100 from TCR-transgenic mice into human ApoB100-transgenic mice. Adoptive transfer of human ApoB100-specific T cells induced immune tolerance in recipient mice and effectively inhibited activation of subsequently transferred naive T cells of the same specificity in vivo. Moreover, repetitive transfers increased the population of Treg type 1 cells that suppress ApoB100-specific responses via IL-10. In a translational approach, LDL-specific Treg type 1 cells from blood of healthy donors suppressed the activation of monocytic THP-1 cells in an IL-10-dependent manner. CONCLUSIONS: We show that repetitive transfer of naive ApoB100-specific T cells and recurrent LDL-specific T-cell stimulation induces Treg type 1 cell-mediated immune tolerance against LDL in vivo. Our results provide insight into the generation of autoantigen-specific anti-inflammatory T cells under tolerogenic conditions.
Assuntos
Linfócitos T CD4-Positivos , Linfócitos T Reguladores , Camundongos , Humanos , Animais , Interleucina-10/genética , Camundongos Transgênicos , Tolerância Imunológica , Receptores de Antígenos de Linfócitos T/metabolismo , Inflamação/metabolismoRESUMO
[Figure: see text].
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Artéria Femoral/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/metabolismo , Neovascularização Fisiológica , Fatores de Transcrição/metabolismo , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Circulação Colateral , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Artéria Femoral/fisiopatologia , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Fluxo Sanguíneo Regional , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.
Assuntos
Fibrilação Atrial , Mutação , Miócitos Cardíacos , Cadeias Leves de Miosina , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Linhagem Celular , Conexina 43/genética , Conexina 43/metabolismo , Técnicas de Inativação de Genes , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.
Assuntos
Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Fígado/citologia , Animais , Marcadores Genéticos , Humanos , Fígado/metabolismo , Masculino , Especificidade de Órgãos , Ratos , TranscriptomaRESUMO
Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 familyâ 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.
Assuntos
Cardiomiopatias/prevenção & controle , Família 1 do Citocromo P450/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/patologia , Família 1 do Citocromo P450/antagonistas & inibidores , Família 1 do Citocromo P450/genética , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Insuficiência Cardíaca , Mutagênese , Fenótipo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
Endothelial cell (EC) identity is in part genetically predetermined. Transcription factor NR2F2 (also known as chicken ovalbumin upstream promoter transcription factor II, COUP-TFII) plays a key role in EC fate decision making; however, many of the underlying mechanisms remain enigmatic. In the present study, we demonstrate that NR2F2 differentially regulates gene expression of venous versus lymphatic ECs (LECs) and document a novel paradigm whereby NR2F2 homodimers induce a venous EC fate, while heterodimers with the LEC-specific transcription factor PROX1 instruct LEC lineage specification. NR2F2 homodimers inhibit arterial differentiation in venous ECs through direct binding to the promoter regions of the Notch target genes HEY1 and HEY2 (HEY1/2), whereas NR2F2/PROX1 heterodimers lack this inhibitory effect, resulting at least in part in non-canonical HEY1/2 expression in LECs. Furthermore, NR2F2/PROX1 heterodimers actively induce or are permissive for the expression of a major subset of LEC-specific genes. In addition to NR2F2/PROX1 heterodimerisation, the expression of HEY1 and some of these LEC-specific genes is dependent on PROX1 DNA binding. Thus, NR2F2 homodimers in venous ECs and NR2F2/PROX1 heterodimers in LECs differentially regulate EC subtype-specific genes and pathways, most prominently the Notch target genes HEY1/2. This novel mechanistic insight could pave the way for new therapeutic interventions for vascular-bed-specific disorders.
Assuntos
Fator II de Transcrição COUP/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Homeodomínio/metabolismo , Vasos Linfáticos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Veias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Humanos , Multimerização Proteica , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genéticaRESUMO
Endothelial cells (ECs) lining arteries and veins have distinct molecular/functional signatures. The underlying regulatory mechanisms are incompletely understood. Here, we established a specific fingerprint of freshly isolated arterial and venous ECs from human umbilical cord comprising 64 arterial and 12 venous genes, representing distinct functions/pathways. Among the arterial genes were 8 transcription factors (TFs), including Notch target HEY2, the current "gold standard" determinant for arterial EC (aEC) specification. Culture abrogated differential gene expression in part due to gradual loss of canonical Notch activity and HEY2 expression. Notably, restoring HEY2 expression or Delta-like4-induced Notch signaling in cultured ECs only partially reinstated the aEC gene signature, whereas combined overexpression of the 8 TFs restored this fingerprint more robustly. Whereas some TFs stimulated few genes, others boosted a large proportion of arterial genes. Although there was some overlap and cross-regulation, the TFs largely complemented each other in regulating the aEC gene profile. Finally, overexpression of the 8 TFs in human umbilical vein ECs conveyed an arterial-like behavior upon their implantation in a Matrigel plug in vivo. Thus, our study shows that Notch signaling determines only part of the aEC signature and identifies additional novel and complementary transcriptional players in the complex regulation of human arteriovenous EC identity.
Assuntos
Artérias/citologia , Células Endoteliais/metabolismo , Fatores de Transcrição/genética , Transcriptoma , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Redes Reguladoras de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Vasos Linfáticos , Animais , Humanos , Camundongos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Regulação para Baixo , Células Endoteliais/metabolismo , Vasos Linfáticos/metabolismo , Transdução de SinaisRESUMO
Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.
Assuntos
Fibrilação Atrial , Peixe-Zebra , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.
Assuntos
Endodesoxirribonucleases , Armadilhas Extracelulares , Camundongos , Animais , Endodesoxirribonucleases/genética , Armadilhas Extracelulares/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Cromatina , DNA/metabolismo , Desoxirribonucleases/genéticaRESUMO
Even though VEGF-B is a homologue of the potent angiogenic factor VEGF, its angiogenic activities have been controversial. Intrigued by findings that VEGF-B may also affect neuronal cells, we assessed the neuroprotective and vasculoprotective effects of VEGF-B in the skin, in which vessels and nerves are functionally intertwined. Although VEGF-B and its FLT1 receptor were prominently expressed in dorsal root ganglion (DRG) neurons innervating the hindlimb skin, they were not essential for nerve function or vascularization of the skin. However, primary DRG cultures lacking VEGF-B or FLT1 exhibited increased neuronal stress and were more susceptible to paclitaxel-induced cell death. Concomitantly, mice lacking VEGF-B or a functional FLT1 developed more retrograde degeneration of sensory neurons in a model of distal neuropathy. On the other hand, the addition of the VEGF-B isoform, VEGF-B(186), to DRG cultures antagonized neuronal stress, maintained the mitochondrial membrane potential and stimulated neuronal survival. Mice overexpressing VEGF-B(186) or FLT1 selectively in neurons were protected against the distal neuropathy, whereas exogenous VEGF-B(186), either delivered by gene transfer or as a recombinant factor, was protective by directly affecting sensory neurons and not the surrounding vasculature. Overall, this indicates that VEGF-B, instead of acting as an angiogenic factor, exerts direct neuroprotective effects through FLT1. These findings also suggest a clinically relevant role for VEGF-B in preventing distal neuropathies.
Assuntos
Degeneração Retrógrada/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Imuno-Histoquímica , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polineuropatias/genética , Polineuropatias/metabolismo , Ratos , Ratos Sprague-Dawley , Degeneração Retrógrada/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator B de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Thrombosis is the leading complication of common human disorders including diabetes, coronary heart disease, and infection and remains a global health burden. Current anticoagulant therapies that target the general clotting cascade are associated with unpredictable adverse bleeding effects, because understanding of hemostasis remains incomplete. Here, using perturbational screening of patient peripheral blood samples for latent phenotypes, we identified dysregulation of the major mechanosensory ion channel Piezo1 in multiple blood lineages in patients with type 2 diabetes mellitus (T2DM). Hyperglycemia activated PIEZO1 transcription in mature blood cells and selected high Piezo1expressing hematopoietic stem cell clones. Elevated Piezo1 activity in platelets, red blood cells, and neutrophils in T2DM triggered discrete prothrombotic cellular responses. Inhibition of Piezo1 protected against thrombosis both in human blood and in zebrafish genetic models, particularly in hyperglycemia. Our findings identify a candidate target to precisely modulate mechanically induced thrombosis in T2DM and a potential screening method to predict patient-specific risk. Ongoing remodeling of cell lineages in hematopoiesis is an integral component of thrombotic risk in T2DM, and related mechanisms may have a broader role in chronic disease.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Trombose , Animais , Humanos , Hiperglicemia/complicações , Canais Iônicos/metabolismo , Mecanotransdução Celular , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.
RESUMO
Requirements for vesicle fusion within the heart remain poorly understood, despite the multitude of processes that necessitate proper intracellular trafficking within cardiomyocytes. Here, we show that Syntaxin 4 (STX4), a target-Soluble N-ethylmaleimide sensitive factor attachment receptor (t-SNARE) protein, is required for normal vertebrate cardiac conduction and vesicular transport. Two patients were identified with damaging variants in STX4. A patient with a homozygous R240W missense variant displayed biventricular dilated cardiomyopathy, ectopy, and runs of non-sustained ventricular tachycardia, sensorineural hearing loss, global developmental delay, and hypotonia, while a second patient displayed severe pleiotropic abnormalities and perinatal lethality. CRISPR/Cas9-generated stx4 mutant zebrafish exhibited defects reminiscent of these patients' clinical presentations, including linearized hearts, bradycardia, otic vesicle dysgenesis, neuronal atrophy, and touch insensitivity by 3 days post fertilization. Imaging of Vamp2+ vesicles within stx4 mutant zebrafish hearts showed reduced docking to the cardiomyocyte sarcolemma. Optical mapping of the embryonic hearts coupled with pharmacological modulation of Ca2+ handling together support that zebrafish stx4 mutants have a reduction in L-type Ca2+ channel modulation. Transgenic overexpression of zebrafish Stx4R241W, analogous to the first patient's STX4R240W variant, indicated that the variant is hypomorphic. Thus, these data show an in vivo requirement for SNAREs in regulating normal embryonic cardiac function and that variants in STX4 are associated with pleiotropic human disease, including cardiomyopathy.
RESUMO
Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.
Assuntos
Fator II de Transcrição COUP/fisiologia , Linfangiogênese/genética , Veias/embriologia , Xenopus laevis/embriologia , Peixe-Zebra/embriologia , Animais , Fator II de Transcrição COUP/genética , Anormalidades Cardiovasculares/genética , Movimento Celular , Endotélio Vascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas Supressoras de Tumor/genética , Xenopus laevis/genética , Peixe-Zebra/genéticaRESUMO
CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms.
Assuntos
Sistemas CRISPR-Cas , Testes Genéticos , Técnicas Analíticas Microfluídicas , Peixe-Zebra/genética , Animais , Sistema Cardiovascular/crescimento & desenvolvimento , Técnicas de Cultura de Células , Sequenciamento de Nucleotídeos em Larga Escala , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
AIMS: The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. METHODS AND RESULTS: We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands. CONCLUSION: Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.
Assuntos
Doença do Sistema de Condução Cardíaco/genética , Sequenciamento do Exoma , Variação Genética , Frequência Cardíaca/genética , Potenciais de Ação/genética , Adulto , Idade de Início , Idoso , Animais , Doença do Sistema de Condução Cardíaco/epidemiologia , Doença do Sistema de Condução Cardíaco/metabolismo , Doença do Sistema de Condução Cardíaco/fisiopatologia , Estudos de Casos e Controles , Simulação por Computador , Canal de Potássio ERG1/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Japão/epidemiologia , Lamina Tipo A/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Proteínas Nucleares/genética , Fenótipo , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Lymphatic capillary growth is an integral part of wound healing, yet, the combined effectiveness of stem/progenitor cells on lymphatic and blood vascular regeneration in wounds needs further exploration. Stem/progenitor cell transplantation also emerged as an approach to cure lymphedema, a condition caused by lymphatic system deficiency. While lymphedema treatment requires lymphatic system restoration from the capillary to the collector level, it remains undetermined whether stem/progenitor cells support a complex regenerative response across the entire anatomical spectrum of the system. Here, we demonstrate that, although multipotent adult progenitor cells (MAPCs) showed potential to differentiate down the lymphatic endothelial lineage, they mainly trophically supported lymphatic endothelial cell behaviour in vitro. In vivo, MAPC transplantation supported blood vessel and lymphatic capillary growth in wounds and restored lymph drainage across skin flaps by stimulating capillary and pre-collector vessel regeneration. Finally, human MAPCs mediated survival and functional reconnection of transplanted lymph nodes to the host lymphatic network by improving their (lymph)vascular supply and restoring collector vessels. Thus, MAPC transplantation represents a promising remedy for lymphatic system restoration at different anatomical levels and hence an appealing treatment for lymphedema. Furthermore, its combined efficacy on lymphatic and blood vascular growth is an important asset for wound healing.
Assuntos
Linfedema/patologia , Células-Tronco Multipotentes/metabolismo , Cicatrização/fisiologia , Animais , Técnicas de Cultura de Células , Células Endoteliais/fisiologia , Endotélio Linfático , Humanos , Linfa/fisiologia , Linfonodos/fisiopatologia , Linfangiogênese/fisiologia , Sistema Linfático/fisiopatologia , Vasos Linfáticos/patologia , Linfedema/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/fisiologia , Transplante de Células-Tronco/métodos , Células-TroncoRESUMO
AIMS: The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity. RESULTS: Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC α1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGCα1 allele [sGCα1-/-CM]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGCα1-/-CM than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGCα1 mutant (DNsGCα1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGCα1tg/+, but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGCα1tg/+ and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGCα1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGCα1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity. Antioxid. Redox Signal. 26, 153-164.