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Whether in real or simulated microgravity, Humans or animals, the kinetics of cardiovascular adaptation and its regulation by the autonomic nervous system (ANS) remain controversial. In this study, we used hindlimb unloading (HU) in 10 conscious mice. Blood pressure (BP), heart rate (HR), temperature, and locomotor activity were continuously monitored with radio-telemetry, during 3 days of control, 5 days of HU, and 2 days of recovery. Six additional mice were used to assess core temperature. ANS activity was indirectly determined by analyzing both heart rate variability (HRV) and baroreflex sensitivity (BRS). Our study showed that HU induced an initial bradycardia, accompanied by an increase in vagal activity markers of HRV and BRS, together with a decrease in water intake, indicating the early adaptation to fluid redistribution. During HU, BRS was reduced; temperature and BP circadian rhythms were altered, showing a loss in day/night differences, a decrease in cycle amplitude, a drop in core body temperature, and an increase in day BP suggestive of a rise in sympathetic activity. Reloading induced resting tachycardia and a decrease in BP, vagal activity, and BRS. In addition to cardiovascular deconditioning, HU induces disruption in day/night rhythmicity of locomotor activity, temperature, and BP.
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Myocardial infraction (MI) is the principal risk factor for the onset of heart failure (HF). Investigations regarding the physiopathology of MI progression to HF have revealed the concerted engagement of other tissues, such as the autonomic nervous system and the medulla oblongata (MO), giving rise to systemic effects, important in the regulation of heart function. Cardiac sympathetic afferent denervation following application of resiniferatoxin (RTX) attenuates cardiac remodelling and restores cardiac function following MI. While the physiological responses are well documented in numerous species, the underlying molecular responses during the initiation and progression from MI to HF remains unclear. We obtained multi-tissue time course proteomics with a murine model of HF induced by MI in conjunction with RTX application. We isolated tissue sections from the left ventricle (LV), MO, cervical spinal cord and cervical vagal nerves at four time points over a 12-week study. Bioinformatic analyses consistently revealed a high statistical enrichment for metabolic pathways in all tissues and treatments, implicating a central role of mitochondria in the tissue-cellular response to both MI and RTX. In fact, the additional functional pathways found to be enriched in these tissues, involving the cytoskeleton, vesicles and signal transduction, could be downstream of responses initiated by mitochondria due to changes in neuronal pulse frequency after a shock such as MI or the modification of such frequency communication from the heart to the brain after RTX application. Development of future experiments, based on our proteomic results, should enable the dissection of more precise mechanisms whereby metabolic changes in neuronal and cardiac tissues can effectively ameliorate the negative physiological effects of MI via RTX application.
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Insuficiência Cardíaca , Infarto do Miocárdio , Animais , Denervação , Modelos Animais de Doenças , Redes e Vias Metabólicas , Camundongos , Infarto do Miocárdio/metabolismo , Proteômica , Transdução de SinaisRESUMO
The lymphatic network of mammalian heart is an important regulator of interstitial fluid compartment and immune cell trafficking. We observed a remodeling of the cardiac lymphatic vessels and a reduced lymphatic efficiency during heart hypertrophy and failure induced by transverse aortic constriction. The lymphatic endothelial cell number of the failing hearts was positively correlated with cardiac function and with a subset of cardiac macrophages. This macrophage population distinguished by LYVE-1 (Lymphatic vessel endothelial hyaluronic acid receptor-1) and by resident macrophage gene expression signature, appeared not replenished by CCR2 mediated monocyte infiltration during pressure overload. Isolation of macrophage subpopulations showed that the LYVE-1 positive subset sustained in vitro and in vivo lymphangiogenesis through the expression of pro-lymphangiogenic factors. In contrast, the LYVE-1 negative macrophage subset strongly expressed MMP12 and decreased the endothelial LYVE-1 receptors in lymphatic endothelial cells, a feature of cardiac lymphatic remodeling in failing hearts. The treatment of mice with a CCR2 antagonist during pressure overload modified the proportion of macrophage subsets within the pathological heart and preserved lymphatic network from remodeling. This study reports unknown and differential functions of macrophage subpopulations in the regulation of cardiac lymphatic during pathological hypertrophy and may constitute a key mechanism underlying the progression of heart failure.
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Vasos Linfáticos/metabolismo , Macrófagos/metabolismo , Miocárdio/patologia , Pressão , Animais , Benzoxazinas/farmacologia , Células CHO , Polaridade Celular/efeitos dos fármacos , Cricetulus , Eletrocardiografia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR2/metabolismo , Compostos de Espiro/farmacologia , Transcriptoma , Proteínas de Transporte Vesicular/metabolismoRESUMO
AIM: Impairments in cerebral structure and cognitive performance in chronic heart failure (CHF) are critical components of its comorbidity spectrum. Autonomic afferents that arise from cardiac sensory fibres show enhanced activity with CHF. Desensitization of these fibres by local application of resiniferatoxin (RTX) during myocardial infarction (MI) is known to prevent cardiac hypertrophy, sympathetic hyperactivity and CHF. Whether these afferents mediate cerebral allostasis is unknown. METHODS: CHF was induced by myocardial infarction. To evaluate if cardiac afferents contribute to cerebral allostasis, RTX was acutely applied to the pericardial space in controls (RTX) and in MI treated animals (MI/RTX). Subjects were then evaluated in a series of behavioural tests recapitulating different symptoms of depressive disorders. Proteomics of the frontal cortices (FC) was performed to identify contributing proteins and pathways responsible for behavioural allostasis. RESULTS: Desensitization of cardiac afferents relieves hallmarks of an anxio/depressive-like state in mice. Unique protein signatures and regulatory pathways in FCs isolated from each treatment reveal the degree of complexity inherent in the FC response to stresses originating in the heart. While cortices from the combined treatment (MI/RTX) did not retain protein signatures from the individual treatment groups, all three groups suffer dysregulation in circadian entrainment. CONCLUSION: CHF is comorbid with an anxio/depressive-like state and ablation of cardiac afferents relieves the despair phenotype. The strikingly different proteomic profiles observed in FCs suggest that MI and RTX lead to unique brain-signalling patterns and that the combined treatment, potentially through destructive interference mechanisms, most closely resembles controls.
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Insuficiência Cardíaca , Proteômica , Animais , Cardiomegalia , Coração , Insuficiência Cardíaca/tratamento farmacológico , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
For some years now, gadolinium oxysulfide nanoparticles (NPs) appear as strong candidates for very efficient multimodal in vivo imaging by: 1) Magnetic Resonance (MRI), 2) X-ray Computed Tomography (CT) and 3) photoluminescence imaging. In this paper, we present a selection of results centered on the evaluation of physico-chemical stability, toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ nanoparticles intravenously injected in rats. Two formulations are here tested with a common matrix and different dopants: Gd2O2S:Eu3+5% and Gd2O2S:Yb3+4%/Tm3+0.1%. The NPs appear to be almost insoluble in pure water and human plasma but corrosion/degradation phenomenon appears in acidic conditions classically encountered in cell lysosomes. Whole body in vivo distribution, excretion and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting imaging signal associated with a slow hepatobiliary clearance and very weak urinary excretion. The results show that the majority of the injected product (>60%) has been excreted through the feces after five months. Experiments have evidenced that the NPs mainly accumulate in macrophage-rich organs, that is mainly liver and spleen and to a lesser extent lungs and bones (mainly marrow). No significant amounts have been detected in other organs such as heart, kidneys, brain, intestine and skin. Gd2O2S:Ln3+ NPs appeared to be very well tolerated up to 400 mg/kg when administered intravenously. STATEMENT OF SIGNIFICANCE: Since 2011, we have focused our work on Gd2O2S nanoparticles (NPs) for multimodal bioimaging using fluorescence, Magnetic Resonance Imaging (MRI) and Computed Tomography with very efficient results already published. However, since the European Medicines Agency has concluded its review of gadolinium contrast agents, confirming recommendations to restrict the use of some linear gadolinium agents used in MRI, a particular attention must be paid to any new contrast media containing gadolinium. Therefore, we present in this paper a compilation of studies about toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ NPs intravenously injected into rats. We also present an in vitro kinetic study of NPs degradation in aqueous and biological media to provide some information on chemical and biological stability.
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Gadolínio , Nanopartículas , Animais , Meios de Contraste/toxicidade , Gadolínio/toxicidade , Imageamento por Ressonância Magnética , Nanopartículas/toxicidade , Ratos , Distribuição TecidualRESUMO
Restricted and controlled drug delivery to the heart remains a challenge giving frequent off-target effects as well as limited retention of drugs in the heart. There is a need to develop and optimize tools to allow for improved design of drug candidates for treatment of heart diseases. Over the last decade, novel drug platforms and nanomaterials were designed to confine bioactive materials to the heart. Yet, the research remains in its infancy, not only in the development of tools but also in the understanding of effects of these materials on cardiac function and tissue integrity. Upconverting nanoparticles are nanomaterials that recently accelerated interest in theranostic nanomedicine technologies. Their unique photophysical properties allow for sensitive in vivo imaging that can be combined with spatio-temporal control for targeted release of encapsulated drugs. Here we synthesized upconverting NaYF4:Yb,Tm nanoparticles and show for the first time their innocuity in the heart, when injected in the myocardium or in the pericardial space in mice. Nanoparticle retention and upconversion in the cardiac region did not alter heart rate variability, nor cardiac function as determined over a 15-day time course ensuing the sole injection. Altogether, our nanoparticles show innocuity primarily in the pericardial region and can be safely used for controlled spatiotemporal drug delivery. Our results support the use of upconverting nanoparticles as potential theranostics tools overcoming some of the key limitations associated with conventional experimental cardiology.
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Cardiopatias/diagnóstico , Cardiopatias/terapia , Nanopartículas/uso terapêutico , Nanomedicina Teranóstica , Animais , Peso Corporal , Cardiopatias/fisiopatologia , Testes de Função Cardíaca , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestruturaRESUMO
Alarmins and damage-associated molecular patterns (DAMPs) are powerful inflammatory mediators, capable of initiating and maintaining sterile inflammation during acute or chronic tissue injury. Recent evidence suggests that alarmins/DAMPs may also trigger tissue regeneration and repair, suggesting a potential contribution to tissue fibrogenesis. High mobility group B1 (HMGB1), a bona fide alarmin/DAMP, may be released passively by necrotic cells or actively secreted by innate immune cells. Macrophages can release large amounts of HMGB1 and play a key role in wound healing and regeneration processes. Here, we hypothesized that macrophages may be a key source of HMGB1 and thereby contribute to wound healing and fibrogenesis. Surprisingly, cell-specific deletion approaches, demonstrated that macrophage-derived HMGB1 is not involved in tissue fibrogenesis in multiple organs with different underlying pathologies. Compared to control HMGB1Flox mice, mice with macrophage-specific HMGB1 deletion (HMGB1ΔMac) do not display any modification of fibrogenesis in the liver after CCL4 or thioacetamide treatment and bile duct ligation; in the kidney following unilateral ureter obstruction; and in the heart after transverse aortic constriction. Of note, even under thermoneutral housing, known to exacerbate inflammation and fibrosis features, HMGB1ΔMac mice do not show impairment of fibrogenesis. In conclusion, our study clearly establishes that macrophage-derived HMGB1 does not contribute to tissue repair and fibrogenesis.
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Aims: Tenascin-C (TNC) is an endogenous danger signal molecule strongly associated with inflammatory diseases and with poor outcome in patients with cardiomyopathies. Its function within pathological cardiac tissue during pressure overload remains poorly understood. Methods and results: We showed that TNC accumulates after 1 week of transverse aortic constriction (TAC) in the heart of 12-week-old male mice. By cross bone marrow transplantation experiments, we determined that TNC deposition relied on cardiac cells and not on haematopoietic cells. The expression of TNC induced by TAC, or by administration of a recombinant lentivector coding for TNC, triggered a pro-inflammatory cardiac microenvironment, monocyte/macrophage (MO/MΦ) accumulation, and systolic dysfunction. TNC modified macrophage polarization towards the pro-inflammatory phenotype and stimulated RhoA/Rho-associated protein kinase (ROCK) pathways to promote mesenchymal to amoeboid transition that enhanced macrophage migration into fibrillar collagen matrices. The amplification of inflammation and MO/MΦ recruitment by TNC was abrogated by genetic invalidation of TNC in knockout mice. These mice showed less ventricular remodelling and an improved cardiac function after TAC as compared with wild-type mice. Conclusions: By promoting a pro-inflammatory microenvironment and macrophage migration, TNC appears to be a key factor to enable the MO/MΦ accumulation within fibrotic hearts leading to cardiac dysfunction. As TNC is highly expressed during inflammation and sparsely during the steady state, its inhibition could be a promising therapeutic strategy to control inflammation and immune cell infiltration in heart disease.
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Movimento Celular , Hipertrofia Ventricular Esquerda/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Miocárdio/metabolismo , Tenascina/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Microambiente Celular , Quimiocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Fenótipo , Transdução de Sinais , Tenascina/genética , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.
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Apelina/genética , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica , Redes Reguladoras de Genes , Isquemia Miocárdica/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Cardiomegalia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Fibrose , Ordem dos Genes , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Lentivirus/genética , Camundongos , Isquemia Miocárdica/patologia , Isquemia Miocárdica/terapia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Transcriptoma , Transdução GenéticaRESUMO
Lymphatic endothelium serves as a barrier to control fluid balance and immune cell trafficking to maintain tissue homeostasis. Long-term alteration of lymphatic vasculature promotes edema and fibrosis, which is an aggravating factor in the onset of cardiovascular diseases such as myocardial infarction. Apelin is a bioactive peptide that plays a central role in angiogenesis and cardiac contractility. Despite an established role of apelin in lymphangiogenesis, little is known about its function in the cardiac lymphatic endothelium. Here, we show that apelin and its receptor APJ were exclusively expressed on newly formed lymphatic vasculature in a pathological model of myocardial infarction. Using an apelin-knockout mouse model, we identified morphological and functional defects in lymphatic vasculature associated with a proinflammatory status. Surprisingly, apelin deficiency increased the expression of lymphangiogenic growth factors VEGF-C and VEGF-D and exacerbated lymphangiogenesis after myocardial infarction. Conversely, the overexpression of apelin in ischemic heart was sufficient to restore a functional lymphatic vasculature and to reduce matrix remodeling and inflammation. In vitro, the expression of apelin prevented the alteration of cellular junctions in lymphatic endothelial cells induced by hypoxia. In addition, we demonstrated that apelin controls the secretion of the lipid mediator sphingosine-1-phosphate in lymphatic endothelial cells by regulating the level of expression of sphingosine kinase 2 and the transporter SPNS2. Taken together, our results show that apelin plays a key role in lymphatic vessel maturation and stability in pathological settings. Thus, apelin may represent a novel candidate to prevent pathological lymphatic remodeling in diseases.
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BACKGROUND: Although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. RESULTS: We have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. CONCLUSIONS: Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.
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MicroRNAs/genética , Terapia de Alvo Molecular , Pelve , Biologia de Sistemas , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/genética , Animais , Estudos de Casos e Controles , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND AND PURPOSE: Apelin-13, an endogenous ligand for the apelin (APJ) receptor, behaves as a potent modulator of metabolic and cardiovascular disorders. Here, we examined the effects of apelin-13 on myocardial injury in a mouse model combining ischaemia/reperfusion (I/R) and obesity and explored their underlying mechanisms. EXPERIMENTAL APPROACH: Adult male C57BL/6J mice were fed a normal diet (ND) or high-fat diet (HFD) for 6 months and then subjected to cardiac I/R. The effects of apelin-13 post-treatment on myocardial injury were evaluated in HFD-fed mice after 24 h I/R. Changes in protein abundance, phosphorylation, subcellular localization and mRNA expression were determined in cardiomyoblast cell line H9C2, primary cardiomyocytes and cardiac tissue from ND- and HFD-fed mice. Apoptosis was evaluated by TUNEL staining and caspase-3 activity. Mitochondrial ultrastructure was analysed by electron microscopy. KEY RESULTS: In HFD-fed mice subjected to cardiac I/R, i.v. administration of apelin-13 significantly reduced infarct size, myocardial apoptosis and mitochondrial damage compared with vehicle-treated animals. In H9C2 cells and primary cardiomyocytes, apelin-13 induced FoxO1 phosphorylation and nuclear exclusion. FoxO1 silencing by siRNA abolished the protective effects of apelin-13 against hypoxia-induced apoptosis and mitochondrial ROS generation. Finally, apelin deficiency in mice fed a HFD resulted in reduced myocardial FoxO1 expression and impaired FoxO1 distribution. CONCLUSIONS AND IMPLICATIONS: These data reveal apelin as a novel regulator of FoxO1 in cardiac cells and provide evidence for the potential of apelin-13 in prevention of apoptosis and mitochondrial damage in conditions combining I/R injury and obesity.
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Apoptose/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Proteína Forkhead Box O1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Obesidade/tratamento farmacológico , Animais , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Obesidade/metabolismo , Obesidade/patologiaRESUMO
The increasing incidence of obesity accentuates the importance of identifying mechanisms and optimal therapeutic strategies for patients with heart failure (HF) in relation to obesity status. Here, we investigated the association between plasma level of apelin, an adipocyte-derived factor, and clinicopathological features of obese and non-obese patients with HF. We further explored potential regulatory mechanisms of cardiac cell fate responses in conditions combining myocardial injury and obesity. In a prospective, cross-sectional study involving patients with HF we show that obese patients (BMI ≥ 30 kg/m(2)) have higher left ventricular ejection fraction (LVEF) and greater levels of plasma apelin (p < 0.005) than non-obese patients (< 30 kg/m(2)), independently of ischemic etiology. In a mouse model combining ischemia-reperfusion (I/R) injury and high-fat diet (HFD)-induced obesity, we identify apelin as a novel regulator of FoxO3 trafficking in cardiomyocytes. Confocal microscopy analysis of cardiac cells revealed that apelin prevents nuclear translocation of FoxO3 in response to oxygen deprivation through a PI3K pathway. These findings uncover apelin as a novel regulator of FoxO3 nucleocytoplasmic trafficking in cardiac cells in response to stress and provide insight into its potential clinical relevance in obese patients with HF.
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Cardiotônicos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Obesidade/metabolismo , Adipócitos/metabolismo , Idoso , Animais , Células Cultivadas , Estudos Transversais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Estudos Prospectivos , Ratos , Função Ventricular Esquerda/fisiologiaRESUMO
BACKGROUND: Renal tubulointerstitial fibrosis is the pathological hallmark of chronic kidney disease (CKD). Currently, inhibitors of the renin-angiotensin system (RAS) remain the sole therapy in human displaying antifibrotic properties. Further antifibrotic molecules are needed. We have recently reported that the delayed blockade of the bradykinin B1 receptor (B1R) reduced the development of fibrosis in two animal models of renal fibrosis. The usefulness of new drugs also resides in outperforming the gold standards and eventually being additive or complementary to existing therapies. METHODS: In this study we compared the efficacy of a B1R antagonist (B1Ra) with that of an angiotensin type 1 receptor antagonist (AT1a) in the unilateral ureteral obstruction (UUO) model of renal fibrosis and determined whether bi-therapy presented higher efficacy than any of the drugs alone. RESULTS: B1R antagonism was as efficient as the gold-standard AT1a treatment. However, bitherapy did not improve the antifibrotic effects at the protein level. We sought for the reason of the absence of this additive effect by studying the expression of a panel of genes involved in the fibrotic process. Interestingly, at the molecular level the different drugs targeted different players of fibrosis that, however, in this severe model did not result in improved reduction of fibrosis at the protein level. CONCLUSIONS: As the B1R is induced specifically in the diseased organ and thus potentially displays low side effects it might be an interesting alternative in cases of poor tolerability to RAS inhibitors.
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Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.
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Neointima/enzimologia , Neointima/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Animais , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Inibidores Enzimáticos/farmacologia , Artéria Femoral/enzimologia , Artéria Femoral/imunologia , Artéria Femoral/lesões , Marcação de Genes , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neointima/tratamento farmacológico , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Tiazolidinedionas/farmacologiaRESUMO
BACKGROUND: The mechanisms by which the heart adapts to chronic pressure overload, producing compensated hypertrophy and eventually heart failure (HF), are still not well defined. We aimed to investigate the involvement of T cells in the progression to HF using a transverse aortic constriction (TAC) model. METHODS AND RESULTS: Chronic HF was associated with accumulation of T lymphocytes and activated/effector CD4(+) T cells within cardiac tissue. After TAC, enlarged heart mediastinal draining lymph nodes showed a high density of both CD4(+) and CD8(+) T-cell subsets. To investigate the role of T cells in HF, TAC was performed on mice deficient for recombination activating gene 2 expression (RAG2KO) lacking B and T lymphocytes. Compared with wild-type TAC mice, RAG2KO mice did not develop cardiac dilation and showed improved contractile function and blunted adverse remodeling. Reconstitution of the T-cell compartment into RAG2KO mice before TAC enhanced contractile dysfunction, fibrosis, collagen accumulation, and cross-linking. To determine the involvement of a specific T-cell subset, we performed TAC on mice lacking CD4(+) (MHCIIKO) and CD8(+) T-cell subsets (CD8KO). In contrast to CD8KO mice, MHCIIKO mice did not develop ventricular dilation and dysfunction. MHCIIKO mice also displayed very low fibrosis, collagen accumulation, and cross-linking within cardiac tissue. Interestingly, mice with transgenic CD4(+) T-cell receptor specific for ovalbumin failed to develop HF and adverse remodeling. CONCLUSIONS: These results demonstrate for the first time a crucial role of CD4(+) T cells and specific antigen recognition in the progression from compensated cardiac hypertrophy to HF.
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Linfócitos T CD4-Positivos/patologia , Cardiomegalia/patologia , Progressão da Doença , Insuficiência Cardíaca/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Cardiomegalia/imunologia , Insuficiência Cardíaca/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Three-dimensional (3D) scaffolds hold great potential for stem cell-based therapies. Indeed, recent results have shown that biomimetic scaffolds may enhance cell survival and promote an increase in the concentration of therapeutic cells at the injury site. The aim of this work was to engineer an original polymeric scaffold based on the respective beneficial effects of alginate and chitosan. Formulations were made from various alginate/chitosan ratios to form opposite-charge polyelectrolyte complexes (PECs). After freeze-drying, the resultant matrices presented a highly interconnected porous microstructure and mechanical properties suitable for cell culture. In vitro evaluation demonstrated their compatibility with mesenchymal stell cell (MSC) proliferation and their ability to maintain paracrine activity. Finally, the in vivo performance of seeded 3D PEC scaffolds with a polymeric ratio of 40/60 was evaluated after an acute myocardial infarction provoked in a rat model. Evaluation of cardiac function showed a significant increase in the ejection fraction, improved neovascularization, attenuated fibrosis as well as less left ventricular dilatation as compared to an animal control group. These results provide evidence that 3D PEC scaffolds prepared from alginate and chitosan offer an efficient environment for 3D culturing of MSCs and represent an innovative solution for tissue engineering.
Assuntos
Alginatos/química , Quitosana/química , Eletrólitos/química , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Isquemia Miocárdica/terapia , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Feminino , Fibrose , Testes de Função Cardíaca , Humanos , Fenômenos Mecânicos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Isquemia Miocárdica/fisiopatologia , Comunicação Parácrina/efeitos dos fármacos , Próteses e Implantes , Ratos , Ratos Endogâmicos LewRESUMO
Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.
Assuntos
Axônios/metabolismo , Fibroblastos/metabolismo , Miocárdio/metabolismo , Fator de Crescimento Neural/metabolismo , Sistema Nervoso Simpático/metabolismo , Sinapses/metabolismo , Animais , Técnicas de Cocultura , Fibroblastos/citologia , Miocárdio/citologia , Células PC12 , Ratos , Ratos Endogâmicos Lew , Sistema Nervoso Simpático/citologiaRESUMO
Mice with genetic alterations are used in heart research for the extrapolation of human diseases. Echocardiography is an essential tool for evaluating cardiac and hemodynamic functions in small animals. The purpose of this study was to compare the effect of different anesthetic regimens and the conscious state on the evaluation of cardiac function by echocardiography. Mice were examined in the conscious state after three days of training, and then for a 7 min period after a single intraperitoneal injection of ketamine at 100 mg/kg, etomidate at 10, 20 or 30 mg/kg, or after inhalation of isoflurane at 1.5% with or without a short period of induction with isoflurane 3%. Intra- and inter-observer variabilities were assessed. The operator's comfort was also assessed. Heart rate, left ventricular end diastolic diameter, fraction shortening and cardiac output were measured using echocardiography. Ketamine at 5 and 7 min after induction and isoflurane at 3, 5 and 7 min after induction provided good anesthetic conditions and a quick awakening time, and did not influence cardiac performance, whereas the conscious state was associated with a non-physiological sympathetic activation and other anesthetic drugs induced a significant decrease in heart rate. Etomidate 10 mg/kg and 20 mg/kg were not enough to provide adequate anesthesia. Etomidate 30 mg/kg induced a good anesthetic condition but influenced cardiac performance and had a long awakening time. Our results indicate that ketamine and isoflurane with a short induction period are better anesthetic drugs than isoflurane without induction or etomidate for evaluating cardiac function in healthy mice.
Assuntos
Anestésicos/administração & dosagem , Ecocardiografia/veterinária , Etomidato/administração & dosagem , Coração/efeitos dos fármacos , Isoflurano/administração & dosagem , Ketamina/administração & dosagem , Anestésicos/farmacologia , Animais , Débito Cardíaco/efeitos dos fármacos , Etomidato/farmacologia , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Isoflurano/farmacologia , Ketamina/farmacologia , Camundongos , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis - TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0-8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0-3 days) decrease of inflammatory cell infiltration followed (3-8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10-14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages.