RESUMO
Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.
Assuntos
Adipogenia/genética , Tecido Adiposo Bege/metabolismo , Metabolismo Energético/genética , Quinase 1 de Adesão Focal/metabolismo , Transdução de Sinais/genética , Células-Tronco/metabolismo , Tetraspanina 28/metabolismo , Adipócitos/metabolismo , Tecido Adiposo Bege/citologia , Tecido Adiposo Bege/crescimento & desenvolvimento , Tecido Adiposo Branco/metabolismo , Adulto , Animais , Ataxina-1/metabolismo , Feminino , Fibronectinas/farmacologia , Quinase 1 de Adesão Focal/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Resistência à Insulina/genética , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/metabolismo , RNA-Seq , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Células-Tronco/citologia , Tetraspanina 28/genéticaRESUMO
Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle, resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show in in vitro and in vivo in skeletal muscle in mice that antibody-mediated blockade of the ß5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.
Assuntos
Insulina , Receptor de Insulina , Animais , Humanos , Camundongos , Antígenos de Superfície/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Cadeias beta de Integrinas , Proteínas do Leite/metabolismo , Receptor de Insulina/genética , Camundongos Endogâmicos C57BL , Masculino , Linhagem CelularRESUMO
The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvß5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/ß5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvß5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/ß5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvß5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.
Assuntos
Antígenos de Superfície/genética , Resistência à Insulina/genética , Insulina/genética , Proteínas do Leite/genética , Receptores de Vitronectina/genética , Animais , Antígenos CD/genética , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Glucose/metabolismo , Glicolipídeos/genética , Glicoproteínas/genética , Homeostase/genética , Humanos , Integrina alfaVbeta3/genética , Gotículas Lipídicas , Camundongos , Músculo Esquelético/metabolismo , Receptor de Insulina/genética , Transdução de Sinais/genéticaRESUMO
Asthma affects â¼300 million people worldwide. Despite multiple treatment options, asthma treatment remains unsatisfactory in a subset of patients. Airway obstruction is a hallmark of allergic asthma and is largely due to airway smooth muscle hypercontractility induced by airway inflammation. Identification of molecular pathways that regulate airway smooth muscle hypercontractility is of considerable therapeutic interest. We previously identified roles for milk fat globule epidermal growth factor-like 8 (Mfge8) in opposing the effects of allergic inflammation on increasing airway smooth muscle contractile force. In this study, we delineate the signaling pathway by which Mfge8 mediates these effects. By using genetic and pharmacologic approaches, we show that the α8ß1 integrin and the phosphatase and tensin homolog (PTEN) mediate the effects of Mfge8 on preventing IL-13-induced increases in airway contractility. Tracheal rings from mice with smooth muscle-specific deletion of α8ß1 or PTEN have enhanced contraction in response to treatment with IL-13. Enhanced IL-13-induced tracheal ring contraction in Mfge8-/- mice was abolished by treatment with the PI3K inhibitor. Mechanistically, IL-13 induces ubiquitination and degradation of PTEN protein. Our findings identify a role for the Mfge8-α8ß1-PTEN pathway in regulating the force of airway smooth muscle contraction in the setting of allergic inflammation.-Khalifeh-Soltani, A., Gupta, D., Ha, A., Podolsky, M. J., Datta, R., Atabai, K. The Mfge8-α8ß1-PTEN pathway regulates airway smooth muscle contraction in allergic inflammation.
RESUMO
Pathological hypertrophy and myocardial infarction (MI) are two etiologically different cardiac disorders having differential molecular mechanisms of disease manifestation. However, no study has been conducted so far to analyze and compare the differential status of energy metabolism in these two disease forms. It was shown recently by our group that production of ATP is significantly impaired during MI along with inhibition of pyruvate dehydrogenase E1-ß (PDHE1 B) by pyruvate dehydrogenase kinase 4 (PDK4). However, the ATP levels showed no significant change during pathological hypertrophy compared to control group. To seek a plausible explanation of this phenomenon, the peroxisome proliferator-activated receptor alpha (PPAR) pathway was studied in all the experimental groups which revealed that PGC1α- ERRα axis remains active in MI while the same remained inactive during pathological hypertrophy possibly by NF-κB that plays a significant role in deactivating this pathway during hypertrophy. At the same time, it was observed that reactive oxygen species (ROS) negatively regulates NF-κB activity during MI by oxidation of cysteine residues of p50- the DNA binding subunit of NF-κB. Thus, this study reports for the first time, a possible mechanism for the differential status of energy metabolism during two etiologically different cardiac pathophysiological conditions involving PGC1α-ERRα axis along with p50 subunit of NF-κB.
Assuntos
Trifosfato de Adenosina/metabolismo , Cardiomegalia/metabolismo , Infarto do Miocárdio/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Modelos Animais de Doenças , Metabolismo Energético , Humanos , Masculino , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Wistar , Receptores de Estrogênio/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Although extensive work has delineated many of the mechanisms of extracellular matrix (ECM) production, far less is known about pathways that regulate ECM degradation. This is particularly true of cellular internalization and degradation of matrix, which play an underappreciated role in ECM metabolism and lung fibrosis. For example, genetic perturbation of this pathway leads to exacerbated fibrosis in experimental animal models. In this work, we present the results of an unbiased screen of Drosophila phagocytes that yielded multiple genes that, when silenced, led to increased collagen uptake. We further describe the function of cell division cycle 7 kinase (CDC7) as a specific suppressor of collagen uptake. We show that the genetic or pharmacological inhibition of CDC7 results in increased expression of the collagen endocytic receptor Endo180. Chromobox 5 (CBX5) is a putative target of CDC7, and genetic silencing of CBX5 also results in increased Endo180 and collagen uptake. Finally, CRISPR-mediated activation of Endo180 expression results in increased collagen uptake, suggesting that CDC7 regulates collagen internalization through increased Endo180 expression. Targeting the regulatory elements of the collagen degradative machinery may be a useful therapeutic approach in diseases of fibrosis or malignancy.
Assuntos
Colágeno/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Animais , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Colágeno/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose , Regulação Enzimológica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Receptores Mitogênicos/biossíntese , Receptores Mitogênicos/genéticaRESUMO
Cardiomyocyte apoptosis acts as a prime modulator of cardiac hypertrophy leading to heart failure, a major cause of human mortality worldwide. Recent therapeutic interventions have focussed on translational applications of diverse pharmaceutical regimes among which, Curcumin (from Curcuma longa) is known to have an anti-hypertrophic potential but with limited pharmacological efficacies due to low aqueous solubility and poor bioavailability. In this study, Curcumin encapsulated by carboxymethyl chitosan (CMC) nanoparticle conjugated to a myocyte specific homing peptide was successfully delivered in bioactive form to pathological myocardium for effective regression of cardiac hypertrophy in a rat (Rattus norvegicus) model. Targeted nanotization showed higher cardiac bioavailability of Curcumin at a low dose of 5 mg/kg body weight compared to free Curcumin at 35 mg/kg body weight. Moreover, Curcumin/CMC-peptide treatment during hypertrophy significantly improved cardiac function by downregulating expression of hypertrophy marker genes (ANF, ß-MHC), apoptotic mediators (Bax, Cytochrome-c) and activity of apoptotic markers (Caspase 3 and PARP); whereas free Curcumin in much higher dose showed minimal improvement during compromised cardiac function. Targeted Curcumin treatment significantly lowered p53 expression and activation in diseased myocardium via inhibited interaction of p53 with p300-HAT. Thus attenuated acetylation of p53 facilitated p53 ubiquitination and reduced the apoptotic load in hypertrophied cardiomyocytes; thereby limiting cardiomyocytes' need to enter the regeneration cycle during hypertrophy. This study elucidates for the first time an efficient targeted delivery regimen for Curcumin and also attributes towards probable mechanistic insight into its therapeutic potential as a cardio-protective agent for regression of cardiac hypertrophy.
Assuntos
Apoptose/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Curcumina/farmacocinética , Sistemas de Liberação de Medicamentos , Acetilação , Animais , Disponibilidade Biológica , Caspase 3/genética , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quitosana/análogos & derivados , Quitosana/química , Curcumina/administração & dosagem , Citocromos c/genética , Citocromos c/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Nanopartículas/química , Ratos , Ratos Wistar , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress-induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α-crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen-activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20-week-old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress-induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro-survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38ß compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580-treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro-survival role and confers protection of cardiomyocytes, during infarct formation.
Assuntos
Cristalinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/enzimologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Células Cultivadas , Cristalinas/genética , Desmina/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Contração Miocárdica , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Transdução de Sinais , Transfecção , Função Ventricular Esquerda , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.
Assuntos
Colágeno , Matriz Extracelular , Animais , Humanos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Proteólise , Pulmão/patologia , Mamíferos/metabolismo , Proteínas/metabolismoRESUMO
The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show that ß5 blockade inhibits and MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in reduced or increased insulin-stimulated myotube glucose uptake respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wild type but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.
RESUMO
Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, we executed unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discovered a previously unappreciated mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism is dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of SEL1L. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus cell-autonomous, rheostatic collagen clearance is a previously unidentified pathway of tissue homeostasis.
RESUMO
Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvß5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.
Assuntos
Gorduras na Dieta , Enterócitos , Animais , Camundongos , Antígenos de Superfície/metabolismo , Gorduras na Dieta/metabolismo , Enterócitos/metabolismo , Ácidos Graxos/metabolismo , Hidrolases/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Proteínas do Leite/metabolismo , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismoRESUMO
IKKγ prototypically promotes NFκBp65 activity by regulating the assembly of the IKK holocomplex. In hypertrophied cardiomyocytes, the p65-p300 complex-induced regenerative efforts are neutralized by the p53-p300 complex-mediated apoptotic load resulting in compromised cardiac function. The present study reports that nitrosative stress leads to S-Nitrosylation of IKKγ in hypertrophied cardiomyocytes in a pre-clinical model. Using a cardiomyocyte-targeted nanoconjugate, IKKγ S-Nitrosylation-resistant mutant plasmids were delivered to the pathologically hypertrophied heart that resulted in improved cardiac function by amelioration of cardiomyocyte apoptosis and simultaneous induction of their cell cycle re-entry machinery. Mechanistically, in IKKγ S-Nitrosyl mutant-transfected hypertrophied cells, increased IKKγ-p300 binding downregulated the binding of p53 and p65 with p300. This shifted the binding preference of p65 from p300 to HDAC1 resulting in upregulated expression of cyclin D1 and CDK2 via the p27/pRb pathway. This approach has therapeutic advantage over mainstream anti-hypertrophic remedies which concomitantly reduce the regenerative prowess of resident cardiomyocytes during hypertrophy upon downregulation of myocyte apoptosis. Therefore, cardiomyocyte-targeted delivery of IKKγ S-Nitrosyl mutants during hypertrophy can be exploited as a novel strategy to re-muscularize the diseased heart.
Assuntos
Quinase I-kappa B , Miócitos Cardíacos , Cardiomegalia/patologia , Humanos , Quinase I-kappa B/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Nitrosativo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although aging represents the most important epidemiologic risk factor for fibrotic disease, the reasons for this are incompletely understood. Excess collagen deposition in tissues is the sine qua non of tissue fibrosis and can be viewed as an imbalance between collagen production and collagen degradation. Yet we still lack a detailed understanding of the changes that take place during development, maturation, and aging in extracellular matrix (ECM) dynamics. Resolution of fibrosis is impaired in aging, and this impairment may explain why age is the most important risk factor for fibrotic diseases, such as idiopathic pulmonary fibrosis. However, ECM dynamics and impaired resolution of fibrosis in aging remain understudied. Here we show that cell-mediated collagen uptake and degradation are diminished in aged animals and this finding correlates with downregulation of the collagen endocytic receptor mannose receptor, C-type 2 (Mrc2). We identify myeloid zinc finger-1 as a potentially novel transcriptional regulator of Mrc2, and both this transcription factor and Mrc2 are downregulated in multiple tissues and organisms in an age-dependent manner. Thus, cell-mediated degradation of collagen is an essential process that promotes resolution of fibrosis, and impairment in this process contributes to age-related fibrosis.
Assuntos
Envelhecimento/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Proteólise , Envelhecimento/genética , Animais , Colágeno/genética , Matriz Extracelular/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transcrição GênicaRESUMO
AIMS: Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. RESULTS: Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3ß (GSK3ß), which upregulated inactive phospho-GSK3ß (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. INNOVATION: Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. CONCLUSION: PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac function, thereby, opening up a potential avenue for cardiac tissue engineering during hypertrophic cardiac pathophysiology.
Assuntos
Cardiomegalia/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Mitocôndrias/patologia , Miocárdio/metabolismo , Nanopartículas/metabolismo , PPAR alfa/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Nanopartículas/química , Estresse Oxidativo , PPAR alfa/química , PPAR alfa/genéticaRESUMO
At the simplest level, obesity is the manifestation of an imbalance between caloric intake and expenditure; however, the pathophysiological mechanisms that govern the development of obesity and associated complications are enormously complex. Fibrosis within the adipose tissue compartment is one such factor that may influence the development of obesity and/or obesity-related comorbidities. Furthermore, the functional consequences of adipose tissue fibrosis are a matter of considerable debate, with evidence that fibrosis serves both adaptive and maladaptive roles. Tissue fibrosis itself is incompletely understood, and multiple cellular and molecular pathways are involved in the development, maintenance, and resolution of the fibrotic state. Within the context of obesity, fibrosis influences molecular and cellular events that relate to adipocytes, inflammatory cells, inflammatory mediators, and supporting adipose stromal tissue. In this Review, we explore what is known about the interplay between the development of adipose tissue fibrosis and obesity, with a view toward future investigative and therapeutic avenues.
Assuntos
Tecido Adiposo/patologia , Obesidade/etiologia , Adipócitos/efeitos dos fármacos , Adipócitos/imunologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Animais , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêutico , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Fibrose , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Obesidade/tratamento farmacológico , Obesidade/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Células Estromais/metabolismoRESUMO
Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy.
Assuntos
Cardiomegalia/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Miócitos Cardíacos/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , Animais , Benzoquinonas/farmacologia , Cardiomegalia/patologia , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Fibroblastos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Interleucina-6/metabolismo , Lactamas Macrocíclicas/farmacologia , Masculino , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Ratos Wistar , Fator de Transcrição RelA/metabolismo , Ubiquitina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Cardiac hypertrophy and myocardial infarction (MI) are two etiologically different disease forms with varied pathological characteristics. However, the precise molecular mechanisms and specific causal proteins associated with these diseases are obscure to date. In this study, a comparative cardiac proteome profiling was performed in Wistar rat models for diseased and control (sham) groups using two-dimensional difference gel electrophoresis followed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified using Protein Pilot™ software (version 4.0) and were subjected to stringent statistical analysis. Alteration of key proteins was validated by Western blot analysis. The differentially expressed protein sets identified in this study were associated with different functional groups, involving various metabolic pathways, stress responses, cytoskeletal organization, apoptotic signaling and other miscellaneous functions. It was further deciphered that altered energy metabolism during hypertrophy in comparison to MI may be predominantly attributed to induced glucose oxidation level, via reduced phosphorylation of pyruvate dehydrogenase E1 component subunit ß (PDHE1-B) protein during hypertrophy. This study reports for the first time the global changes in rat cardiac proteome during two etiologically different cardiac diseases and identifies key signaling regulators modulating ontogeny of these two diseases culminating in heart failure. This study also pointed toward differential activation of PDHE1-B that accounts for upregulation of glucose oxidation during hypertrophy. Downstream analysis of altered proteome and the associated modulators would enhance our present knowledge regarding altered pathophysiology of these two etiologically different cardiac disease forms.
Assuntos
Cardiomegalia/metabolismo , Glucose/metabolismo , Infarto do Miocárdio/metabolismo , Piruvato Desidrogenase (Lipoamida)/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Metabolismo Energético , Ativação Enzimática , Masculino , Oxirredução , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cardiac hypertrophy is accompanied by excessive collagen deposition in the heart. Despite painstaking research on this fatal disease, the precise role of molecular chaperones in myocardial fibrosis has not yet been elucidated. In this study, we have analyzed the mechanism by which Heat shock protein 90 (Hsp90)/Cell division cycle 37 (Cdc37) assembly modulates cardiac hypertrophy associated fibrosis. For the in vitro hypertrophy model, Angiotensin II (AngII) treated cultured adult cardiac fibroblasts were used, whereas the in vivo hypertrophy model was generated by renal artery ligation in adult male Wistar rats (Rattus norvegicus). Pretreatment with the Hsp90 inhibitor or the blocking of Hsp90-Cdc37 interactions during pressure overload hypertrophy resulted in ubiquitin-mediated proteasomal degradation of TGFß receptor-II (TßR-II) leading to termination of TGFß mediated signaling. In both cases significant reduction in collagen synthesis was observed revealing the Hsp90/Cdc37 complex as an integral profibrotic component of TGFß signaling during cardiac hypertrophy.