RESUMO
BACKGROUND: Cardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential. RESULT: In this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-ß to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3's recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-ß-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration. CONCLUSION: Collectively, our findings demonstrate TET3's pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.
Assuntos
Dioxigenases , Fibroblastos , Fibrose , Animais , Fibrose/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Camundongos , Humanos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Dano ao DNA/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Miocárdio/patologia , Miocárdio/metabolismo , Masculino , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
BACKGROUND: Cardiac fibrosis plays a major pathophysiological role in any form of chronic heart disease, and high levels are associated with poor outcome. Diffuse and focal cardiac fibrosis are different subtypes, which have different pathomechanisms and prognostic implications. The total fibrosis burden in endomyocardial biopsy tissue was recently proved to play an independent prognostic role in aortic stenosis patients after transcatheter aortic valve implantation (TAVI). AIMS: Here, for the first time, we aim to assess the specific impact of different fibrosis subtypes on sudden cardiac death (SCD) as a primary reason for cardiovascular mortality after TAVI. METHODS: The fibrosis pattern was assessed histologically in the left ventricular biopsies obtained during TAVI interventions in 161 patients, who received a structured follow-up thereafter. RESULTS: Receiver operating characteristic analyses, performed 6, 12, 24 and 48 months after TAVI, showed diffuse, but not focal, fibrosis as a significant predictor for SCD at all timepoints, with the highest area under the curve at the first time point and a decrease in its SCD predictivity over time. In both multivariate Cox proportional hazards and Fine-Gray competing risk models, including both fibrosis subtypes, as well as age, sex and ejection fraction, high diffuse fibrosis remained statistically significant. Accordingly, it represents an independent SCD predictor, most importantly for the occurrence of early events. CONCLUSIONS: The burden of diffuse cardiac fibrosis plays an important and independent prognostic role regarding SCD early after TAVI. Therefore, the histological evaluation of fibrosis topography has value as a prognostic tool for TAVI patients and may help to tailor individualised approaches to optimise their postinterventional management.
Assuntos
Estenose da Valva Aórtica , Morte Súbita Cardíaca , Fibrose , Substituição da Valva Aórtica Transcateter , Humanos , Substituição da Valva Aórtica Transcateter/efeitos adversos , Masculino , Feminino , Idoso , Morte Súbita Cardíaca/etiologia , Estenose da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/complicações , Estenose da Valva Aórtica/mortalidade , Idoso de 80 Anos ou mais , Fatores de Risco , Miocárdio/patologia , PrognósticoRESUMO
BACKGROUND: Aortic stenosis (AS) is one of the most common cardiac diseases and major cause of morbidity and mortality in the elderly. Transcatheter aortic valve implantation (TAVI) is performed in such patients with symptomatic severe AS and reduces mortality for the majority of these patients. However, a significant percentage dies within the first two years after TAVI, such that there is an interest to identify parameters, which predict outcome and could guide pre-TAVI patient selection. High levels of cardiac fibrosis have been identified as such independent predictor of cardiovascular mortality after TAVI. Promoter hypermethylation commonly leads to gene downregulation, and the Iroquois homeobox 3 (IRX3) gene was identified in a genome-wide transcriptome and methylome to be hypermethylated and downregulated in AS patients. In a well-described cohort of 100 TAVI patients in which cardiac fibrosis levels were quantified histologically in cardiac biopsies, and which had a follow-up of up to two years, we investigated if circulating methylated DNA of IRX3 in the peripheral blood is associated with cardiac fibrosis and/or mortality in AS patients undergoing TAVI and thus could serve as a biomarker to add information on outcome after TAVI. RESULTS: Patients with high levels of methylation in circulating IRX3 show a significantly increased survival as compared to patients with low levels of IRX3 methylation indicating that high peripheral IRX3 methylation is associated with an improved outcome. In the multivariable setting, peripheral IRX3 methylation acts as an independent predictor of all-cause mortality. While there is no significant correlation of levels of IRX3 methylation with cardiac death, there is a significant but very weak inverse correlation between circulating IRX3 promoter methylation level and the amount of cardiac fibrosis. Higher levels of peripheral IRX3 methylation further correlated with decreased cardiac IRX3 expression and vice versa. CONCLUSIONS: High levels of IRX3 methylation in the blood of AS patients at the time of TAVI are associated with better overall survival after TAVI and at least partially reflect myocardial IRX3 expression. Circulating methylated IRX3 might aid as a potential biomarker to help guide both pre-TAVI patient selection and post-TAVI monitoring.
Assuntos
Estenose da Valva Aórtica , Ácidos Nucleicos Livres , Substituição da Valva Aórtica Transcateter , Humanos , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/cirurgia , Biópsia , Metilação de DNA , Proteínas de Homeodomínio/genética , Substituição da Valva Aórtica Transcateter/efeitos adversos , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Deficiências de Ferro , Humanos , Miócitos Cardíacos/metabolismo , Mutação , Cardiomiopatia Dilatada/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Ferro/metabolismo , Clatrina/genética , Clatrina/metabolismo , Clatrina/farmacologiaRESUMO
AIMS: There is evidence to suggest that the subtype of aortic stenosis (AS), the degree of myocardial fibrosis (MF), and level of aortic valve calcification (AVC) are associated with adverse cardiac outcome in AS. Because little is known about their respective contribution, we sought to investigate their relative importance and interplay as well as their association with adverse cardiac events following transcatheter aortic valve replacement (TAVR). METHODS AND RESULTS: One hundred consecutive patients with severe AS and indication for TAVR were prospectively enrolled between January 2017 and October 2018. Patients underwent transthoracic echocardiography, multidetector computed tomography, and left ventricular endomyocardial biopsies at the time of TAVR. The final study cohort consisted of 92 patients with a completed study protocol, 39 (42.4%) of whom showed a normal ejection fraction (EF) high-gradient (NEFHG) AS, 13 (14.1%) a low EF high-gradient (LEFHG) AS, 25 (27.2%) a low EF low-gradient (LEFLG) AS, and 15 (16.3%) a paradoxical low-flow, low-gradient (PLFLG) AS. The high-gradient phenotypes (NEFHG and LEFHG) showed the largest amount of AVC (807 ± 421 and 813 ± 281 mm3 , respectively) as compared with the low-gradient phenotypes (LEFLG and PLFLG; 503 ± 326 and 555 ± 594 mm3 , respectively, P < 0.05). Conversely, MF was most prevalent in low-output phenotypes (LEFLG > LEFHG > PLFLG > NEFHG, P < 0.05). This was paralleled by a greater cardiovascular (CV) mortality within 600 days after TAVR (LEFLG 28% > PLFLG 26.7% > LEFHG 15.4% > NEFHG 2.5%; P = 0.023). In patients with a high MF burden, a higher AVC was associated with a lower mortality following TAVR (P = 0.045, hazard ratio 0.261, 95% confidence interval 0.07-0.97). CONCLUSIONS: MF is associated with adverse CV outcome following TAVR, which is most prevalent in low EF situations. In the presence of large MF burden, patients with large AVC have better outcome following TAVR. Conversely, worse outcome in large MF and relatively little AVC may be explained by a relative prominence of an underlying cardiomyopathy. The better survival rates in large AVC patients following TAVR indicate TAVR induced relief of severe AS-associated pressure overload with subsequently improved outcome.
Assuntos
Estenose da Valva Aórtica , Cardiomiopatias , Substituição da Valva Aórtica Transcateter , Humanos , Valva Aórtica/cirurgia , Resultado do Tratamento , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/complicações , FibroseRESUMO
BACKGROUND: The prevalence of diabetes mellitus has risen considerably and currently affects more than 422 million people worldwide. Cardiovascular diseases including myocardial infarction and heart failure represent the major cause of death in type 2 diabetes (T2D). Diabetes patients exhibit accelerated aortic stiffening which is an independent predictor of cardiovascular disease and mortality. We recently showed that aortic stiffness precedes hypertension in a mouse model of diabetes (db/db mice), making aortic stiffness an early contributor to cardiovascular disease development. Elucidating how aortic stiffening develops is a pressing need in order to halt the pathophysiological process at an early time point. METHODS: To assess EndMT occurrence, we performed co-immunofluorescence staining of an endothelial marker (CD31) with mesenchymal markers (α-SMA/S100A4) in aortic sections from db/db mice. Moreover, we performed qRT-PCR to analyze mRNA expression of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. To identify the underlying mechanism by which EndMT contributes to aortic stiffening, we used aortas from db/db mice and diabetic patients in combination with high glucose-treated human umbilical vein endothelial cells (HUVECs) as an in vitro model of diabetes-associated EndMT. RESULTS: We demonstrate robust CD31/α-SMA and CD31/S100A4 co-localization in aortic sections of db/db mice which was almost absent in control mice. Moreover, we demonstrate a significant upregulation of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. As underlying regulator, we identified miR-132-3p as the most significantly downregulated miR in the micronome of db/db mice and high glucose-treated HUVECs. Indeed, miR-132-3p was also significantly downregulated in aortic tissue from diabetic patients. We identified Kruppel-like factor 7 (KLF7) as a target of miR-132-3p and show a significant upregulation of KLF7 in aortic sections of db/db mice and diabetic patients as well as in high glucose-treated HUVECs. We further demonstrate that miR-132-3p overexpression and KLF7 downregulation ameliorates EndMT in high glucose-treated HUVECs. CONCLUSIONS: We demonstrate for the first time that EndMT contributes to aortic stiffening in T2D. We identified miR-132-3p and KLF7 as novel EndMT regulators in this context. Altogether, this gives us new insights in the development of aortic stiffening in T2D.
RESUMO
In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron-specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B-dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.
Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Aprendizagem/fisiologia , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Epigênese Genética , Hipocampo/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Integrases/metabolismo , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/metabolismo , Sítio de Iniciação de Transcrição , Transcriptoma/genéticaRESUMO
Aryl hydrocarbon receptor nuclear translocator (ARNT) mediates anti-fibrotic activity in kidney and liver through induction of ALK3-receptor expression and subsequently increased Smad1/5/8 signaling. While expression of ARNT can be pharmacologically induced by sub-immunosuppressive doses of FK506 or by GPI1046, its anti-fibrotic activity is only realized when ARNT-ARNT homodimers form, as opposed to formation of ARNT-AHR or ARNT-HIF1α heterodimers. Mechanisms underlying ARNTs dimerization decision to specifically form ARNT-ARNT homodimers and possible cues to specifically induce ARNT homodimerization have been previously unknown. Here, we demonstrate that phosphorylation of the Ser77 residue is critical for ARNT-ARNT homodimer formation and stabilization. We further demonstrate that inhibition of PP2A phosphatase activity by LB100 enhances ARNT-ARNT homodimers both in vivo and in vitro (mouse tubular epithelial cells and human embryonic kidney cells). In murine models of kidney fibrosis, and also of liver fibrosis, combinations of FK506 or GPI1046 (to induce ARNT expression) with LB100 (to enhance ARNT homodimerization) elicit additive anti-fibrotic activities. Our study provides additional evidence for the anti-fibrotic activity of ARNT-ARNT homodimers and reveals Ser77 phosphorylation as a novel pharmacological target to realize the therapeutic potential of increased ARNT transactivation activity.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Inibidores Enzimáticos/farmacologia , Multimerização Proteica , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Fibrose , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de SinaisRESUMO
Aberrant promoter hypermethylation leads to gene silencing and is associated with various pathologies including cancer and organ fibrosis. Active DNA demethylation is mediated by TET enzymes: TET1, TET2, and TET3, which convert 5-methylcytosine to 5-hydroxymethylcytosine. Induction of gene-specific hydroxymethylation via CRISPR/Cas9 gene technology provides an opportunity to reactivate a single target gene silenced in pathological conditions. We utilized a spCas9 variant fused with TET3 catalytic domain to mediate gene-specific hydroxymethylation with subsequent gene reactivation which holds promise for gene therapy. Here, we present guidelines for gene-specific hydroxymethylation targeting with a specific focus on designing sgRNA and functional assessments in vitro.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Sistemas CRISPR-Cas , Metilação de DNA , DNA/análise , Edição de Genes , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Cromatina , Biologia Computacional/métodos , DNA/química , DNA/genética , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Oxirredução , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Sulfitos/químicaRESUMO
BACKGROUND: Myocardial fibrosis is a major determinant of outcome in aortic stenosis (AS). Novel fast real-time (RT) cardiovascular magnetic resonance (CMR) mapping techniques allow comprehensive quantification of fibrosis but have not yet been compared against standard techniques and histology. METHODS: Patients with severe AS underwent CMR before (n = 110) and left ventricular (LV) endomyocardial biopsy (n = 46) at transcatheter aortic valve replacement (TAVR). Midventricular short axis (SAX) native, post-contrast T1 and extracellular volume fraction (ECV) maps were generated using commercially available modified Look-Locker Inversion recovery (MOLLI) (native: 5(3)3, post-contrast: 4(1)3(1)2) and RT single-shot inversion recovery Fast Low-Angle Shot (FLASH) with radial undersampling. Focal late gadolinium enhancement was excluded from T1 and ECV regions of interest. ECV and LV mass were used to calculate LV matrix volumes. Variability and agreements were assessed between RT, MOLLI and histology using intraclass correlation coefficients, coefficients of variation and Bland Altman analyses. RESULTS: RT and MOLLI derived ECV were similar for midventricular SAX slice coverage (26.2 vs. 26.5, p = 0.073) and septal region of interest (26.2 vs. 26.5, p = 0.216). MOLLI native T1 time was in median 20 ms longer compared to RT (p < 0.001). Agreement between RT and MOLLI was best for ECV (ICC > 0.91), excellent for post-contrast T1 times (ICC > 0.81) and good for native T1 times (ICC > 0.62). Diffuse collagen volume fraction by biopsies was in median 7.8%. ECV (RT r = 0.345, p = 0.039; MOLLI r = 0.40, p = 0.010) and LV matrix volumes (RT r = 0.45, p = 0.005; MOLLI r = 0.43, p = 0.007) were the only parameters associated with histology. CONCLUSIONS: RT mapping offers fast and sufficient ECV and LV matrix volume calculation in AS patients. ECV and LV matrix volume represent robust and universally comparable parameters with associations to histologically assessed fibrosis and may emerge as potential targets for clinical decision making.
Assuntos
Estenose da Valva Aórtica/diagnóstico por imagem , Valva Aórtica/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética , Miocárdio/patologia , Função Ventricular Esquerda , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/cirurgia , Biópsia , Feminino , Fibrose , Humanos , Masculino , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Substituição da Valva Aórtica Transcateter , Remodelação VentricularRESUMO
Endothelial-to-mesenchymal transition (EndMT) is a cellular transdifferentiation program in which endothelial cells partially lose their endothelial identity and acquire mesenchymal-like features. Renal capillary endothelial cells can undergo EndMT in association with persistent damage of the renal parenchyma. The functional consequence(s) of EndMT in kidney fibrosis remains unexplored. Here, we studied the effect of Twist or Snail deficiency in endothelial cells on EndMT in kidney fibrosis. Conditional deletion of Twist1 (which encodes Twist) or Snai1 (which encodes Snail) in VE-cadherin+ or Tie1+ endothelial cells inhibited the emergence of EndMT and improved kidney fibrosis in two different kidney injury/fibrosis mouse models. Suppression of EndMT limited peritubular vascular leakage, reduced tissue hypoxia, and preserved tubular epithelial health and function. Hypoxia, which was exacerbated by EndMT, resulted in increased Myc abundance in tubular epithelial cells, enhanced glycolysis, and suppression of fatty acid oxidation. Pharmacological suppression or epithelial-specific genetic ablation of Myc in tubular epithelial cells ameliorated fibrosis and restored renal parenchymal function and metabolic homeostasis. Together, these findings demonstrate a functional role for EndMT in the response to kidney capillary endothelial injury and highlight the contribution of endothelial-epithelial cross-talk in the development of kidney fibrosis with a potential for therapeutic intervention.
Assuntos
Reprogramação Celular , Endotélio Vascular/metabolismo , Nefropatias/metabolismo , Túbulos Renais/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Endotélio Vascular/patologia , Fibrose , Rim , Nefropatias/genética , Nefropatias/patologia , Túbulos Renais/patologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleases/metabolismo , Epigenômica/métodos , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/metabolismo , Cromatina/ultraestrutura , DNA/metabolismo , Endonucleases/metabolismo , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Imagem Óptica , RNA Guia de Cinetoplastídeos/genética , Proteínas Recombinantes de Fusão/análise , Especificidade por Substrato , Telômero/ultraestrutura , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Transcrição Gênica , Dedos de ZincoRESUMO
Rationale: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure. Methods: We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. In vitro TGFß1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the Rxfp1 promoter region in MCECs. Results: Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFß-pSMAD2/3 signaling in endothelial cells. Conclusions: This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.
Assuntos
Células Endoteliais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Cardiopatias/tratamento farmacológico , Coração/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Relaxina/farmacologia , Animais , Células Cultivadas , Doença Crônica/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Fibrose/tratamento farmacológico , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Cardiovascular fibrosis is a major contributor to cardiovascular disease, the primary cause of death in patients with chronic kidney disease (CKD). We previously reported expression of endogenous Klotho in human arteries, and that CKD is a state of Klotho deficiency, resulting in vascular calcification, but myocardial expression of Klotho is poorly understood. This study aimed to further clarify endogenous Klotho's functional roles in cardiac fibrosis in patients with underlying CKD. METHODS AND RESULTS: Human atrial appendage specimens were collected during cardiac surgery from individuals with or without CKD. Cardiac fibrosis was quantified using trichrome staining. For endogenous Klotho functional studies, primary human cardiomyocytes (HCMs) were treated with uremic serum from CKD patients or recombinant human TGF-ß1. The effects of endogenous Klotho in HCMs were studied using Klotho-siRNA and Klotho-plasmid transfection. Both gene and protein expression of endogenous Klotho are found in human heart, but decreased Klotho expression is clearly associated with the degree of cardiac fibrosis in CKD patients. Moreover, we show that endogenous Klotho is expressed by HCMs and cardiac fibroblasts (HCFs) but that HCM expression is suppressed by uremic serum or TGF-ß1. Klotho knockdown or overexpression aggravates or mitigates TGF-ß1-induced fibrosis and canonical Wnt signaling in HCMs, respectively. Furthermore, co-culture of HCMs with HCFs increases TGF-ß1-induced fibrogenic proteins in HCFs, but overexpression of endogenous Klotho in HCMs mitigates this effect, suggesting functional crosstalk between HCMs and HCFs. CONCLUSIONS: Our data from analysis of human hearts as well as functional in vitro studies strongly suggests that the loss of cardiac endogenous Klotho in CKD patients, specifically in cardiomyocytes, facilitates intensified TGF-ß1 signaling which enables more vigorous cardiac fibrosis through upregulated Wnt signaling. Upregulation of endogenous Klotho inhibits pathogenic Wnt/ß-catenin signaling and may offer a novel strategy for prevention and treatment of cardiac fibrosis in CKD patients.
Assuntos
Glucuronidase/metabolismo , Miocárdio/patologia , Insuficiência Renal Crônica/complicações , Fator de Crescimento Transformador beta1/metabolismo , Via de Sinalização Wnt , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Fibrose , Glucuronidase/genética , Humanos , Proteínas Klotho , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Insuficiência Renal Crônica/metabolismoRESUMO
Endothelial cells can acquire a mesenchymal phenotype upon irritation [endothelial-to-mesenchymal transition (EndMT)]. Macrophages accumulate in the atherosclerotic plaque. This study addressed whether macrophages modulate EndMT and delineated a reciprocal effect of EndMT on macrophage functions in atherosclerosis. In atherosclerotic murine and human aortas, endothelial cells with mesenchymal markers were elevated by confocal microscopy and flow cytometric analysis. Increased EndMT master transcription factor Snai1 expression and extracellular matrix are consistent with enhanced EndMT in this condition. Hypoxia was detected in individual aortic EndMT cells in vivo and rapidly induced a similar EndMT phenotype in vitro. As a novel inducer of EndMT, macrophages, which are abundant in the atherosclerotic lesions, enhance mesothelial marker expression during coculture in vitro. In the reverse relationship, EndMT altered endothelial colony-stimulating factor expression. Functionally, EndMT cell-conditioned media attenuated macrophage proliferation, antigen-presenting cell marker expression, and TNF-α production in response to oxidized LDL but increased oxidized LDL uptake and scavenger receptor expression. These experiments demonstrate that macrophages promote partial EndMT. In turn, EndMT cells modulate macrophage phenotype and lipid uptake. Our data suggest that EndMT shapes macrophage and endothelial cell phenotypes, thus affecting internal atherosclerotic plaque in addition to surface structure.-Helmke, A., Casper, J., Nordlohne, J., David, S., Haller, H., Zeisberg, E. M., von Vietinghoff, S. Endothelial-to-mesenchymal transition shapes the atherosclerotic plaque and modulates macrophage function.
Assuntos
Endotélio/patologia , Macrófagos/citologia , Mesoderma/patologia , Placa Aterosclerótica/patologia , Animais , Antígenos/imunologia , Aorta/metabolismo , Biomarcadores/metabolismo , Antígenos CD36/metabolismo , Hipóxia Celular , Proliferação de Células , Técnicas de Cocultura , Meios de Cultivo Condicionados , Endotélio/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Lipoproteínas LDL/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/imunologia , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Cardiovascular disease and heart failure are the primary cause of morbidity and mortality in patients with chronic kidney disease. Because impairment of kidney function correlates with heart failure and cardiac fibrosis, a kidney-heart axis is suspected. Although our understanding of the underlying mechanisms still is evolving, the possibility that kidney-heart messengers could be intercepted offers ample reason to focus on this clinically highly relevant problem. Here, we review the current knowledge of how kidney injury causes heart failure and fibrosis.
Assuntos
Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Células Endoteliais/patologia , Miocárdio/patologia , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/fisiopatologia , Animais , Células Endoteliais/fisiologia , Transição Epitelial-Mesenquimal , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Fibrose , Glucuronidase/sangue , Humanos , Inflamação/complicações , Inflamação/fisiopatologia , Proteínas Klotho , MicroRNAs , Estresse Oxidativo , Fosfatos/sangue , Sistema Renina-AngiotensinaRESUMO
Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose their properties and transform into fibroblast-like cells. This transition process contributes to cardiac fibrosis, a common feature of patients with chronic heart failure. To date, no specific therapies to halt or reverse cardiac fibrosis are available, so knowledge of the underlying mechanisms of cardiac fibrosis is urgently needed. In addition, EndMT contributes to other cardiovascular pathologies such as atherosclerosis and pulmonary hypertension, but also to cancer and organ fibrosis. Remarkably, the molecular mechanisms driving EndMT are largely unknown. Epigenetics play an important role in regulating gene transcription and translation and have been implicated in the EndMT process. Therefore, epigenetics might be the missing link in unraveling the underlying mechanisms of EndMT. Here, we review the involvement of epigenetic regulators during EndMT in the context of cardiac fibrosis. The role of DNA methylation, histone modifications (acetylation and methylation), and noncoding RNAs (microRNAs, long noncoding RNAs, and circular RNAs) in the facilitation and inhibition of EndMT are discussed, and potential therapeutic epigenetic targets will be highlighted.
Assuntos
Epigênese Genética , Transição Epitelial-Mesenquimal , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Acetilação , Doença Crônica , Metilação de DNA , Fibrose , Insuficiência Cardíaca/patologia , Histonas/metabolismo , Humanos , Metilação , RNA não Traduzido/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca2+ leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36- versus placebo-treated mice. Reduction of SR Ca2+ leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca2+ leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca2+ leak-independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca2+ waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca2+ transient, SR Ca2+ load, SR Ca2+ adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca2+ leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.
Assuntos
Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Progressão da Doença , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Retículo Sarcoplasmático/metabolismo , Animais , Aorta/patologia , Arritmias Cardíacas/fisiopatologia , Constrição Patológica , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/patologia , Homeostase , Humanos , Camundongos , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Fenótipo , Análise de Sobrevida , Remodelação VentricularRESUMO
While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.