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Extracellular vesicles (EVs) were once believed to serve as a means of disposing of cellular waste. However, recent discoveries have identified their crucial roles in intercellular communication between neighboring and distant cells. Almost all cell types have now been identified to produce EVs, which play a vital role in transporting cellular cargo. The functional roles of EVs, along with their implications in (patho)physiology of various diseases, are still being explored. In the last decade, the identification of EV roles in pathophysiology, pharmacology, and diagnostics has gained significant interest, albeit the development of universal methods for the isolation and characterization of EVs has been the limiting factor. A further challenge is ensuring that EVs of various size categories, which are thought to be produced via independent cellular mechanisms and often differ in their cargo and physiological purpose, can be separated and studied in isolation.This protocol provides an efficient and accessible method for isolating and characterizing EV samples from conditioned cell culture media. The combination of differential centrifugation and the use of a commercial EV-precipitation kit allows for the rapid isolation of a highly pure sample of EVs separated by size. A microfluidic resistive pulse sensing (MRPS)-based method is then used to quantify the particles, as well as to assess the size distribution of the EV sample. As a result, this protocol provides a reproducible means to isolate and characterize EVs of a variety of sizes from nearly any cultured cells.
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Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Humanos , Meios de Cultivo Condicionados , Meios de Cultura/química , Fracionamento Celular/métodos , Centrifugação/métodos , Técnicas de Cultura de Células/métodosRESUMO
Cardiac fibrosis is a significant driver of congestive heart failure, a syndrome that continues to affect a growing patient population globally. Cardiac fibrosis results from a constellation of complex processes at the transcription, receptor, and signaling axes levels. Various mediators and signaling cascades, such as the transformation growth factor-beta pathway, have been implicated in the pathophysiology of cardiac tissue fibrosis. Our understanding of these markers and pathways has improved in recent years as more advanced technologies and assays have been developed, allowing for better delineation of the crosstalk between specific factors. There is mounting evidence suggesting that epigenetic modulation plays a pivotal role in the progression of cardiac fibrosis. Transcriptional regulation of key pro- and anti-fibrotic pathways can accentuate or blunt the rate and extent of fibrosis at the tissue level. Exosomes, micro-RNAs, and long non-coding RNAs all belong to factors that can impact the epigenetic signature in cardiac fibrosis. Herein, we comprehensively review the latest literature about exosomes, their contents, and cardiac fibrosis. In doing so, we highlight the specific transcriptional factors with pro- or anti-fibrotic properties. We also assimilate the data supporting these mediators' potential utility as diagnostic or prognostic biomarkers. Finally, we offer insight into where further work can be done to fill existing gaps to translate pre-clinical findings better and improve clinical outcomes.
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The potential of the pericardial space as a therapeutic delivery tool for cardiac fibrosis and heart failure (HF) treatment has yet to be elucidated. Recently, miRNAs and exosomes have been discovered to be present in human pericardial fluid (PF). Novel studies have shown characteristic human PF miRNA compositions associated with cardiac diseases and higher miRNA expressions in PF compared to peripheral blood. Five key studies found differentially expressed miRNAs in HF, angina pectoris, aortic stenosis, ventricular tachycardia, and congenital heart diseases with either atrial fibrillation or sinus rhythm. As miRNA-based therapeutics for cardiac fibrosis and HF showed promising results in several in vivo studies for multiple miRNAs, we hypothesize a potential role of miRNA-based therapeutics delivered through the pericardial cavity. This is underlined by the favorable results of the first phase 1b clinical trial in this emerging field. Presenting the first human miRNA antisense drug trial, inhibition of miR-132 by intravenous administration of a novel antisense oligonucleotide, CDR132L, established efficacy in reducing miR-132 in plasma samples in a dose-dependent manner. We screened the literature, provided an overview of the miRNAs and exosomes present in PF, and drew a connection to those miRNAs previously elucidated in cardiac fibrosis and HF. Further, we speculate about clinical implications and potential delivery methods.
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Cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. The escalating global occurrence of obesity and diabetes mellitus (DM) has led to a significant upsurge in individuals afflicted with CVDs. As the prevalence of CVDs continues to rise, it is becoming increasingly important to identify the underlying cellular and molecular mechanisms that contribute to their development and progression, which will help discover novel therapeutic avenues. Adipose tissue (AT) is a connective tissue that plays a crucial role in maintaining lipid and glucose homeostasis. However, when AT is exposed to diseased conditions, such as DM, this tissue will alter its phenotype to become dysfunctional. AT is now recognized as a critical contributor to CVDs, especially in patients with DM. AT is comprised of a heterogeneous cellular population, which includes adipose-derived stem cells (ADSCs). ADSCs resident in AT are believed to regulate physiological cardiac function and have potential cardioprotective roles. However, recent studies have also shown that ADSCs from various adipose tissue depots become pro-apoptotic, pro-inflammatory, less angiogenic, and lose their ability to differentiate into various cell lineages upon exposure to diabetic conditions. This review aims to summarize the current understanding of the physiological roles of ADSCs, the impact of DM on ADSC phenotypic changes, and how these alterations may contribute to the pathogenesis of CVDs.
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Doenças Cardiovasculares , Diabetes Mellitus , Humanos , Doenças Cardiovasculares/patologia , Células-Tronco/patologia , Tecido Adiposo , FenótipoRESUMO
Abdominal aortic aneurysm (AAA) represents a debilitating vascular disease characterized by aortic dilatation and wall rupture if it remains untreated. We aimed to determine the effects of Ang 1-7 in a murine model of AAA and to investigate the molecular mechanisms involved. Eight- to 10-week-old apolipoprotein E-deficient mice (ApoEKO) were infused with Ang II (1.44 mg/kg/day, s.c.) and treated with Ang 1-7 (0.576 mg/kg/day, i.p.). Echocardiographic and histological analyses showed abdominal aortic dilatation and extracellular matrix remodeling in Ang II-infused mice. Treatment with Ang 1-7 led to suppression of Ang II-induced aortic dilatation in the abdominal aorta. The immunofluorescence imaging exhibited reduced smooth muscle cell (SMC) density in the abdominal aorta. The abdominal aortic SMCs from ApoEKO mice exhibited markedly increased apoptosis in response to Ang II. Ang 1-7 attenuated cell death, as evident by increased SMC density in the aorta and reduced annexin V/propidium iodide-positive cells in flow cytometric analysis. Gene expression analysis for contractile and synthetic phenotypes of abdominal SMCs showed preservation of contractile phenotype by Ang 1-7 treatment. Molecular analyses identified increased mitochondrial fission, elevated cellular and mitochondrial reactive oxygen species (ROS) levels, and apoptosis-associated proteins, including cytochrome c, in Ang II-treated aortic SMCs. Ang 1-7 mitigated Ang II-induced mitochondrial fission, ROS generation, and levels of pro-apoptotic proteins, resulting in decreased cell death of aortic SMCs. These results highlight a critical vasculo-protective role of Ang 1-7 in a degenerative aortic disease; increased Ang 1-7 activity may provide a promising therapeutic strategy against the progression of AAA.
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Angiotensina II , Aneurisma da Aorta Abdominal , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Angiotensina II/metabolismo , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/prevenção & controle , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Proteínas Reguladoras de Apoptose/metabolismo , Miócitos de Músculo Liso/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Thoracic aortic aneurysm (TAA) involves extracellular matrix (ECM) remodeling of the aortic wall, leading to reduced biomechanical support with risk of aortic dissection and rupture. Activation of the renin-angiotensin system, and resultant angiotensin (Ang) II synthesis, is critically involved in the onset and progression of TAA. The current study investigated the effects of angiotensin (Ang) 1-7 on a murine model of TAA. Male 8-10-week-old ApoEKO mice were infused with Ang II (1.44 mg/kg/day) and treated with Ang 1-7 (0.576 mg/kg/day). ApoEKO mice developed advanced TAA in response to four weeks of Ang II infusion. Echocardiographic and histological analyses demonstrated increased aortic dilatation, excessive structural remodelling, perivascular fibrosis, and inflammation in the thoracic aorta. Ang 1-7 infusion led to attenuation of pathological phenotypic alterations associated with Ang II-induced TAA. Smooth muscle cells (SMCs) isolated from adult murine thoracic aorta exhibited excessive mitochondrial fission, oxidative stress, and hyperproliferation in response to Ang II. Treatment with Ang 1-7 resulted in inhibition of mitochondrial fragmentation, ROS generation, and hyperproliferation. Gene expression profiling used for characterization of the contractile and synthetic phenotypes of thoracic aortic SMCs revealed preservation of the contractile phenotype with Ang 1-7 treatment. In conclusion, Ang 1-7 prevented Ang II-induced vascular remodeling and the development of TAA. Enhancing Ang 1-7 actions may provide a novel therapeutic strategy to prevent or delay the progression of TAA.
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Aneurisma da Aorta Torácica , Masculino , Animais , Camundongos , Aneurisma da Aorta Torácica/tratamento farmacológico , Aneurisma da Aorta Torácica/prevenção & controle , Aneurisma da Aorta Torácica/genética , Angiotensina I/farmacologia , Angiotensina I/genética , Fenótipo , Angiotensina II/metabolismo , Miócitos de Músculo Liso/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Aortic aneurysm (AA) is a degenerative vascular disease that involves aortic dilatation, and, if untreated, it can lead to rupture. Despite its significant impact on the healthcare system, its multifactorial nature and elusive pathophysiology contribute to limited therapeutic interventions that prevent the progression of AA. Thus, further research into the mechanisms underlying AA is paramount. Adventitial fibroblasts are one of the key constituents of the aortic wall, and they play an essential role in maintaining vessel structure and function. However, adventitial fibroblasts remain understudied when compared with endothelial cells and smooth muscle cells. Adventitial fibroblasts facilitate the production of extracellular matrix (ECM), providing structural integrity. However, during biomechanical stress and/or injury, adventitial fibroblasts can be activated into myofibroblasts, which move to the site of injury and secrete collagen and cytokines, thereby enhancing the inflammatory response. The overactivation or persistence of myofibroblasts has been shown to initiate pathological vascular remodeling. Therefore, understanding the underlying mechanisms involved in the activation of fibroblasts and in regulating myofibroblast activation may provide a potential therapeutic target to prevent or delay the progression of AA. This review discusses mechanistic insights into myofibroblast activation and associated vascular remodeling, thus illustrating the contribution of fibroblasts to the pathogenesis of AA.
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Diabetic cardiomyopathy (DbCM) occurs independently of cardiovascular diseases or hypertension, leading to heart failure and increased risk for death in diabetic patients. To investigate the molecular mechanisms involved in DbCM, we performed a quantitative proteomic profiling analysis in the left ventricle (LV) of type 2 diabetic mice. Six-month-old C57BL/6J-lepr/lepr (db/db) mice exhibited DbCM associated with diastolic dysfunction and cardiac hypertrophy. Using quantitative shotgun proteomic analysis, we identified 53 differentially expressed proteins in the LVs of db/db mice, majorly associated with the regulation of energy metabolism. The subunits of ATP synthase that form the F1 domain, and Cytochrome c1, a catalytic core subunit of the complex III primarily responsible for electron transfer to Cytochrome c, were upregulated in diabetic LVs. Upregulation of these key proteins may represent an adaptive mechanism by diabetic heart, resulting in increased electron transfer and thereby enhancement of mitochondrial ATP production. Conversely, diabetic LVs also showed a decrease in peptide levels of NADH dehydrogenase 1ß subcomplex subunit 11, a subunit of complex I that catalyzes the transfer of electrons to ubiquinone. Moreover, the atypical kinase COQ8A, an essential lipid-soluble electron transporter involved in the biosynthesis of ubiquinone, was also downregulated in diabetic LVs. Our study indicates that despite attempts by hearts from diabetic mice to augment mitochondrial ATP energetics, decreased levels of key components of the electron transport chain may contribute to impaired mitochondrial ATP production. Preserved basal mitochondrial respiration along with the markedly reduced maximal respiratory capacity in the LVs of db/db mice corroborate the association between altered mitochondrial metabolic profile and cardiac dysfunction in DbCM.
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Cardiovascular diseases (CVDs) represent a major global health problem, due to their continued high incidences and mortality. The last few decades have witnessed new advances in clinical research which led to increased survival and recovery in CVD patients. Nevertheless, elusive and multifactorial pathophysiological mechanisms of CVD development perplexed researchers in identifying efficacious therapeutic interventions. Search for novel and effective strategies for diagnosis, prevention, and intervention for CVD has shifted research focus on extracellular vesicles (EVs) in recent years. By transporting molecular cargo from donor to recipient cells, EVs modulate gene expression and influence the phenotype of recipient cells, thus EVs prove to be an imperative component of intercellular signaling. Elucidation of the role of EVs in intercellular communications under physiological conditions implied the enormous potential of EVs in monitoring and treatment of CVD. The EVs secreted from the myriad of cells in the cardiovascular system such as cardiomyocytes, cardiac fibroblasts, cardiac progenitor cells, endothelial cells, inflammatory cells may facilitate the communication in physiological and pathological conditions. Understanding EVs-mediated cellular communication may delineate the mechanism of origin and progression of cardiovascular diseases. The current review summarizes exosome-mediated paracrine signaling leading to cardiovascular disease. The mechanistic role of exosomes in cardiovascular disease will provide novel avenues in designing diagnosis and therapeutic interventions.
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Cardiovascular disease is the most prevalent cause of morbidity and mortality in diabetes. Epicardial adipose tissue (EAT) lies in direct contact with the myocardium and coronary arteries and can influence cardiac (patho) physiology through paracrine signaling pathways. This study hypothesized that the proteins released from EAT represent a critical molecular link between the diabetic state and coronary artery endothelial cell dysfunction. To simulate type 2 diabetes-associated metabolic and inflammatory status in an ex vivo tissue culture model, human EAT samples were treated with a cocktail composed of high glucose, high palmitate, and lipopolysaccharide (gplEAT) and were compared with control EAT (conEAT). Compared to conEAT, gplEAT showed a markedly increased gene expression profile of proinflammatory cytokines, corroborating EAT inflammation, a hallmark feature observed in patients with type 2 diabetes. Luminex assay of EAT-secretome identified increased release of various proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interferon-alpha 2 (IFNA2), interleukin 1 beta (IL1B), interleukin 5 (IL5), interleukin 13 (IL13), and CCL5, among others, in response to high glucose, high palmitate, and lipopolysaccharide. Conditioned culture media was used to collect the concentrated proteins (CPs). In response to gplEAT-CPs, human coronary artery endothelial cells (HCAECs) exhibited an inflammatory endothelial cell phenotype, featuring a significantly increased gene expression of proinflammatory cytokines and cell surface expression of VCAM-1. Moreover, gplEAT-CPs severely decreased Akt-eNOS signaling, nitric oxide production, and angiogenic potential of HCAECs, when compared with conEAT-CPs. These findings indicate that EAT inflammation may play a key role in coronary artery endothelial cell dysfunction in type 2 diabetes.
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Tecido Adiposo/patologia , Doença da Artéria Coronariana/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Células Endoteliais/patologia , Inflamação/patologia , Pericárdio/patologia , Tecido Adiposo/metabolismo , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/metabolismo , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Pericárdio/metabolismo , Mapas de Interação de ProteínasRESUMO
A simple, accurate, precise and economical Q- Absorption Ratio spectrophotometric method was developed and validated for estimation of Anagliptin and Metformin HCl in synthetic mixture. Anagliptin and Metformin HCl showed an iso-absorptive point at 238 nm in distilled water. The second wavelength used was 233 nm which is λmax of Metformin HCl in distilled water. The concentration of the drugs was determined by using ratio of absorbance at iso-absorptive point (λ1 = 238 nm) and at the λmax of Metformin HCl (λ2 = 233 nm). This method is linear for both drugs; in range of 2-12 µg/mL at λ1 (R2 = 0.999) and at λ2 (R2 = 0.9998) for Anagliptin, and in the range of 5-30 µg/mL for Metformin HCl found at λ1 (R2 = 0.9995) and at λ2 (R2 = 0.9997). The % Recovery was 100.42-101.83 % of Anagliptin and 99.94-101.63 % of Metformin HCl by standard addition method. The LOD was found to be 0.201 µg/mL and 0.262 µg/mL for Anagliptin at λ1 and λ2 respectively. The LOD was found to be 0.320 µg/mL and 0.167 µg/mL for Metformin HCl at λ1 and λ2 respectively. The LOQ was found to be 0.610 µg/mL and 0.794 µg/mL for Anagliptin at λ1 and λ2 respectively. The LOQ was found to be 0.972 µg/mL and 0.506 µg/mL for Metformin HCl at λ1 and λ2 respectively. The method was found to be precise as % RSD was less than 2.00 in Repeatability, Interday and Intraday precision for Anagliptin and Metformin HCl. The % assay of analyte drugs in synthetic mixture was found to be 100.601% of Anagliptin and 100.206 % of Metformin HCl which showed good applicability of the developed method.
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Discovered in the late 1980s as an extracellular vesicle of endosomal origin secreted from reticulocytes, exosomes recently gained scientific attention due to its role in intercellular communication. Exosomes have now been identified to carry cell-specific cargo of nucleic acids, proteins, lipids, and other biologically active molecules. Exosomes can be selectively taken up by neighboring or distant cells, which has shown to result in structural and functional responses in the recipient cells. Recent advances indicate the regulation of exosomes at various steps, including their biogenesis, selection of their cargo, as well as cell-specific uptake. This review will shed light on the differences between the type of extracellular vesicles. In this review, we discuss the recent progress in our understanding of the regulation of exosome biogenesis, secretion, and uptake.
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Exossomos/metabolismo , Reticulócitos/citologia , Transporte Biológico , Comunicação Celular , HumanosRESUMO
Humans have a lower risk of death from myocardial infarction (MI) living at low elevations (<2500 m), which are not high enough to induce hypoxia. Both chronic hypoxia pre-MI, achieved by altitude simulation >5000 m, and intermittent hypobaric hypoxia post-MI can reduce MI size in rodents, and it is believed that hypoxia is the key stimulus. To explore mechanisms beyond hypoxia we studied whether altitude simulation <2500 m would also be associated with reduced infarct size. We performed left-anterior descending artery ligation on C57BL6 mice. Control mice (n = 12) recovered at 754 mmHg (atmospheric pressure, control), and treatment group mice (n = 13) were placed in a hypobaric chamber to recover 3-hours daily at 714 mmHg for 1 week. Echocardiographic evaluation of left ventricular function was performed on Day 0, Day 1 and Day 8. Intermittent hypobaric treatment was associated with a 14.2±5.3% improvement in ejection fraction for treatment group mice (p<0.01 vs. Day 1), with no change observed in control mice. Cardiac output, stroke volume, and infarct size were also improved in treated mice, but no changes were observed in HIF-1 activation or neovascularization. Next, we studied the acute hemodynamic effects of low altitude stimulation in intact mice breathing 100% oxygen using left ventricular catheterization and recording of pressure-volume loops. Acute reductions in barometric pressure from 754 mmHg to 714 mmHg and 674 mmHg were associated with reduced systemic vascular resistance, increased stroke volume and cardiac output, and no change in blood pressure or heart rate. Ex-vivo vascular function was studied using murine mesenteric artery segments. Acute reductions in barometric pressure were associated with greater vascular distensibility. We conclude that intermittent hypobaric treatment using simulated altitudes <2500 m reduces infarct size and increases ventricular function post-MI, and that these changes are related to altered arterial function and not hypoxia-associated neovascularization.
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Altitude , Infarto do Miocárdio/fisiopatologia , Função Ventricular Esquerda , Animais , Regulação da Expressão Gênica , Hemodinâmica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Volume SistólicoRESUMO
Background Cancer therapies inhibiting PI 3Kα (phosphoinositide 3-kinase-α)-dependent growth factor signaling, including trastuzumab inhibition of HER 2 (Human Epidermal Growth Factor Receptor 2), can cause adverse effects on the heart. Direct inhibition of PI 3Kα is now in clinical trials, but the effects of PI 3Kα pathway inhibition on heart atrophy, remodeling, and function in the context of cancer therapy are not well understood. Method and Results Pharmacological PI 3Kα inhibition and heart-specific genetic deletion of p110α, the catalytic subunit of PI 3Kα, was characterized in conjunction with anthracycline (doxorubicin) treatment in female murine models. Biventricular changes in heart morphological characteristics and function were analyzed, with molecular characterization of signaling pathways. Both PI 3Kα inhibition and anthracycline therapy promoted heart atrophy and a combined effect of distinct right ventricular dilation, dysfunction, and cardiomyocyte remodeling in the absence of pulmonary arterial hypertension. Congruent findings of right ventricular dilation and dysfunction were seen with pharmacological and genetic suppression of PI 3Kα signaling when combined with doxorubicin treatment. Increased p38 mitogen-activated protein kinase activation was mechanistically linked to heart atrophy and correlated with right ventricular dysfunction in explanted failing human hearts. Conclusions PI 3Kα pathway inhibition promotes heart atrophy in mice. The right ventricle is specifically at risk for dilation and dysfunction in the setting of PI 3K inhibition in conjunction with chemotherapy. Inhibition of p38 mitogen-activated protein kinase is a proposed therapeutic target to minimize this mode of cardiotoxicity.
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Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Coração/efeitos dos fármacos , Miocárdio/patologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Tiazóis/farmacologia , Disfunção Ventricular Direita/fisiopatologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Atrofia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Coração/fisiopatologia , Camundongos , Disfunção Ventricular Direita/induzido quimicamente , Disfunção Ventricular Direita/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Biomechanical stress and cytoskeletal remodeling are key determinants of cellular homeostasis and tissue responses to mechanical stimuli and injury. Here we document the increased activity of gelsolin, an actin filament severing and capping protein, in failing human hearts. Deletion of gelsolin prevents biomechanical stress-induced adverse cytoskeletal remodeling and heart failure in mice. We show that phosphatidylinositol (3,4,5)-triphosphate (PIP3) lipid suppresses gelsolin actin-severing and capping activities. Accordingly, loss of PI3Kα, the key PIP3-producing enzyme in the heart, increases gelsolin-mediated actin-severing activities in the myocardium in vivo, resulting in dilated cardiomyopathy in response to pressure-overload. Mechanical stretching of adult PI3Kα-deficient cardiomyocytes disrupts the actin cytoskeleton, which is prevented by reconstituting cells with PIP3. The actin severing and capping activities of recombinant gelsolin are effectively suppressed by PIP3. Our data identify the role of gelsolin-driven cytoskeletal remodeling in heart failure in which PI3Kα/PIP3 act as negative regulators of gelsolin activity.
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Citoesqueleto de Actina/metabolismo , Gelsolina/metabolismo , Insuficiência Cardíaca/etiologia , Mecanotransdução Celular , Miocárdio/metabolismo , Animais , Cães , Feminino , Gelsolina/genética , Humanos , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Modelos Cardiovasculares , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Remodelação VentricularRESUMO
Chronic iron overload results in heart and liver diseases and is a common cause of morbidity and mortality in patients with genetic hemochromatosis and secondary iron overload. We investigated the role of tissue inhibitor of metalloproteinase 3 (TIMP3) in iron overload-mediated tissue injury by subjecting male mice lacking Timp3 ( Timp3-/-) and wild-type (WT) mice to 12 wk of chronic iron overload. Whereas WT mice with iron overload developed diastolic dysfunction, iron-overloaded Timp3-/- mice showed worsened cardiac dysfunction coupled with systolic dysfunction. In the heart, loss of Timp3 was associated with increased myocardial fibrosis, greater Timp1, matrix metalloproteinase ( Mmp) 2, and Mmp9 expression, increased active MMP-2 levels, and gelatinase activity. Iron overload in Timp3-/- mice showed twofold higher iron accumulation in the liver compared with WT mice because of constituently lower levels of ferroportin. Loss of Timp3 enhanced the hepatic inflammatory response to iron overload, leading to greater neutrophil and macrophage infiltration and increased hepatic fibrosis. Expression of inflammation-related MMPs (MMP-12 and MMP-13) and inflammatory cytokines (IL-1ß and monocyte chemoattractant protein-1) was elevated to a greater extent in iron-overloaded Timp3-/- livers. Gelatin zymography demonstrated equivalent increases in MMP-2 and MMP-9 levels in WT and Timp3-/- iron-overloaded livers. Loss of Timp3 enhanced the susceptibility to iron overload-mediated heart and liver injury, suggesting that Timp3 is a key protective molecule against iron-mediated pathology. NEW & NOTEWORTHY In mice, loss of tissue inhibitor of metalloproteinase 3 ( Timp3) was associated with systolic and diastolic dysfunctions, twofold higher hepatic iron accumulation (attributable to constituently lower levels of ferroportin), and increased hepatic inflammation. Loss of Timp3 enhanced the susceptibility to iron overload-mediated injury, suggesting that Timp3 plays a key protective role against iron-mediated pathology.
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Cardiomiopatias/metabolismo , Sobrecarga de Ferro/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Miocárdio/metabolismo , Inibidor Tecidual de Metaloproteinase-3/deficiência , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proteínas de Transporte de Cátions/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Mediadores da Inflamação/metabolismo , Sobrecarga de Ferro/genética , Fígado/patologia , Hepatopatias/genética , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Inibidor Tecidual de Metaloproteinase-3/genética , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Iron-overload cardiomyopathy is prevalent on a worldwide basis and is a major comorbidity in patients with genetic hemochromatosis and secondary iron overload. Therapies are limited in part due to lack of a valid preclinical model, which recapitulates advanced iron-overload cardiomyopathy. Male hemojuvelin (HJV) knockout (HJVKO) mice, which lack HJV, a bone morphogenetic co-receptor protein required for hepcidin expression and systemic iron homeostasis, were fed a high-iron diet starting at 4 weeks of age for a duration of 1 year. Aged HJVKO mice in response to iron overload showed increased myocardial iron deposition and mortality coupled with oxidative stress and myocardial fibrosis culminating in advanced iron-overload cardiomyopathy. In a parallel group, iron-overloaded HJVKO mice received resveratrol (240 mg/day) at 9 months of age until 1 year of age. Echocardiography and invasive pressure-volume (PV) loop analyses revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. In addition, myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCa2a) levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCa2a levels and suppressed up-regulation of the sodium-calcium exchanger (NCX1). Further, iron-mediated oxidative stress and myocardial fibrosis were suppressed by resveratrol treatment with concomitant activation of the p-Akt and p-AMP-activated protein kinase (AMPK) signaling pathways. A combination of ageing and high-iron diet in male HJVKO mice results in a valid preclinical model that recapitulates iron-overload cardiomyopathy in humans. Resveratrol therapy resulted in normalization of cardiac function demonstrating that resveratrol represents a feasible therapeutic intervention to reduce the burden of iron-overload cardiomyopathy.
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Cardiomiopatias/tratamento farmacológico , Coração/efeitos dos fármacos , Sobrecarga de Ferro/tratamento farmacológico , Proteínas de Membrana/genética , Miocárdio/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Modelos Animais de Doenças , Proteínas Ligadas por GPI , Coração/fisiopatologia , Proteína da Hemocromatose , Hepcidinas/genética , Humanos , Ferro/metabolismo , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Camundongos , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases/genética , Resveratrol , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Trocador de Sódio e Cálcio/genética , Estilbenos/administração & dosagemRESUMO
Obesity and diabetes are strongly associated with metabolic and cardiovascular disorders including dyslipidemia, coronary artery disease, hypertension, and heart failure. Adipose tissue is identified as a complex endocrine organ, which by exerting a wide array of regulatory functions at the cellular, tissue and systemic levels can have profound effects on the cardiovascular system. Different terms including "epicardial," "pericardial," and "paracardial" have been used to describe adipose tissue deposits surrounding the heart. Epicardial adipose tissue (EAT) is a unique and multifaceted fat depot with local and systemic effects. The functional and anatomic proximity of EAT to the myocardium enables endocrine, paracrine, and vasocrine effects on the heart. EAT displays a large secretosome, which regulates physiological and pathophysiological processes in the heart. Perivascular adipose tissue (PVAT) secretes adipose-derived relaxing factor, which is a "cocktail" of cytokines, adipokines, microRNAs, and cellular mediators, with a potent effect on paracrine regulation of vascular tone, vascular smooth muscle cell proliferation, migration, atherosclerosis-susceptibility, and restenosis. Although there are various physiological functions of the EAT and PVAT, a phenotypic transformation can lead to a major pathogenic role in various cardiovascular diseases. The equilibrium between the physiological and pathophysiological properties of EAT is very delicate and susceptible to the influences of intrinsic and extrinsic factors. Various adipokines secreted from EAT and PVAT have a profound effect on the myocardium and coronary arteries; targeting these adipokines could be an important therapeutic approach to counteract cardiovascular disease.
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Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Doença da Artéria Coronariana/metabolismo , Citocinas/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Miocárdio/metabolismo , PericárdioRESUMO
Myocardial fibrosis is excess accumulation of the extracellular matrix fibrillar collagens. Fibrosis is a key feature of various cardiomyopathies and compromises cardiac systolic and diastolic performance. TIMP1 (tissue inhibitor of metalloproteinase-1) is consistently upregulated in myocardial fibrosis and is used as a marker of fibrosis. However, it remains to be determined whether TIMP1 promotes tissue fibrosis by inhibiting extracellular matrix degradation by matrix metalloproteinases or via an matrix metalloproteinase-independent pathway. We examined the function of TIMP1 in myocardial fibrosis using Timp1-deficient mice and 2 in vivo models of myocardial fibrosis (angiotensin II infusion and cardiac pressure overload), in vitro analysis of adult cardiac fibroblasts, and fibrotic myocardium from patients with dilated cardiomyopathy (DCM). Timp1 deficiency significantly reduced myocardial fibrosis in both in vivo models of cardiomyopathy. We identified a novel mechanism for TIMP1 action whereby, independent from its matrix metalloproteinase-inhibitory function, it mediates an association between CD63 (cell surface receptor for TIMP1) and integrin ß1 on cardiac fibroblasts, initiates activation and nuclear translocation of Smad2/3 and ß-catenin, leading to de novo collagen synthesis. This mechanism was consistently observed in vivo, in cultured cardiac fibroblasts, and in human fibrotic myocardium. In addition, after long-term pressure overload, Timp1 deficiency persistently reduced myocardial fibrosis and ameliorated diastolic dysfunction. This study defines a novel matrix metalloproteinase-independent function of TIMP1 in promoting myocardial fibrosis. As such targeting TIMP1 could prove to be a valuable approach in developing antifibrosis therapies.