RESUMO
Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.
Assuntos
Massagem , Músculo Esquelético , Atrofia Muscular/terapia , Animais , Membro Posterior , Imobilização , Inflamação/terapia , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Atrofia Muscular/etiologia , Condicionamento Físico Animal , PressãoRESUMO
Physical inactivity gives rise to numerous diseases and organismal dysfunctions, particularly those related to aging. Musculoskeletal disorders including muscle atrophy, which can result from a sedentary lifestyle, aggravate locomotive malfunction and evoke a vicious circle leading to severe functional disruptions of vital organs such as the brain and cardiovascular system. Although the significance of physical activity is evident, molecular mechanisms behind its beneficial effects are poorly understood. Here, we show that massage-like mechanical interventions modulate immobilization-induced pro-inflammatory responses of macrophages in situ and alleviate muscle atrophy. Local cyclical compression (LCC) on mouse calves, which generates intramuscular pressure waves with amplitude of 50 mmHg, partially restores the myofiber thickness and contracting forces of calf muscles that are decreased by hindlimb immobilization. LCC tempers the increase in the number of cells expressing pro-inflammatory proteins, tumor necrosis factor-α and monocyte chemoattractant protein-1 (MCP-1), including macrophages in situ The reversing effect of LCC on immobilization-induced thinning of myofibers is almost completely nullified when macrophages recruited from circulating blood are depleted by administration of clodronate liposomes. Furthermore, application of pulsatile fluid shear stress, but not hydrostatic pressure, reduces the expression of MCP-1 in macrophages in vitro Together with the LCC-induced movement of intramuscular interstitial fluid detected by µCT analysis, these results suggest that mechanical modulation of macrophage function is involved in physical inactivity-induced muscle atrophy and inflammation. Our findings uncover the implication of mechanosensory function of macrophages in disuse muscle atrophy, thereby opening a new path to develop a novel therapeutic strategy utilizing mechanical interventions.
Assuntos
Macrófagos/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/fisiopatologia , Estresse Mecânico , Animais , Quimiocina CCL2/metabolismo , Feminino , Elevação dos Membros Posteriores/fisiologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Cardiac cell therapy has been proposed as one of the new strategies against myocardial infarction. Although several reports showed improvement of the function of ischemic heart, the effects of cell therapy vary among the studies and the mechanisms of the beneficial effects are still unknown. Previously, we reported that clonal stem cell antigen-1-positive cardiac progenitor cells exerted a therapeutic effect when transplanted into the ischemic heart. Our aims were to identify the cardiac progenitor-specific paracrine factor and to elucidate the mechanism of its beneficial effect. METHODS AND RESULTS: By using an antibody array, we found that soluble junctional adhesion molecule-A (JAM-A) was abundantly secreted from cardiac progenitor cells. Pretreatment of neutrophils with conditioned medium from cultured cardiac progenitor cells or soluble JAM-A inhibited transendothelial migration and reduced motility of neutrophils. These inhibitory effects were attenuated by anti-JAM-A neutralizing antibody. Injection of cardiac progenitor cells into infarct heart attenuated neutrophil infiltration and expression of inflammatory cytokines. Injection of soluble JAM-A-expressing, but not of JAM-A siRNA-expressing, cardiac progenitor cells into the infarct heart prevented cardiac remodeling and reduced fibrosis area. CONCLUSIONS: Soluble JAM-A secreted from cardiac progenitor cells reduces infiltration of neutrophils after myocardial infarction and ameliorates tissue damage through prevention of excess inflammation. Our finding may lead to a new therapy for cardiovascular disease by using the anti-inflammatory effect of JAM-A.
Assuntos
Anti-Inflamatórios/metabolismo , Moléculas de Adesão Celular/metabolismo , Inflamação/prevenção & controle , Infarto do Miocárdio/cirurgia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Receptores de Superfície Celular/metabolismo , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Interferência de RNA , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Células-Tronco/efeitos dos fármacos , Migração Transendotelial e Transepitelial , Transfecção , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Stem cell antigen-1 (Sca1 or Ly6A/E) is a cell surface marker that is widely expressed in mesenchymal stem cells, including adipose-derived stem cells (ASCs). We hypothesized that the fat depot-specific gene signature of Sca1(high) ASCs may play the major role in defining adipose tissue function and extracellular matrix (ECM) remodeling in a depot-specific manner. Herein we aimed to characterize the unique gene signature and ECM remodeling of Sca1(high) ASCs isolated from subcutaneous (inguinal) and visceral (epididymal) adipose tissues. Sca1(high) ASCs are found in the adventitia and perivascular areas of adipose tissues. Sca1(high) ASCs purified with magnetic-activated cell sorting (MACS) demonstrate dendrite or round shape with the higher expression of cytokines and chemokines (e.g., Il6, Cxcl1) and the lower expression of a glucose transporter (Glut1). Subcutaneous and visceral fat-derived Sca1(high) ASCs particularly differ in the gene expressions of adhesion and ECM molecules. While the expression of the major membrane-type collagenase (MMP14) is comparable between the groups, the expressions of secreted collagenases (MMP8 and MMP13) are higher in visceral Sca1(high) ASCs than in subcutaneous ASCs. Consistently, slow but focal MMP-dependent collagenolysis was observed with subcutaneous adipose tissue-derived vascular stromal cells, whereas rapid and bulk collagenolysis was observed with visceral adipose tissue-derived cells in MMP-dependent and -independent manners. These results suggest that the fat depot-specific gene signatures of ASCs may contribute to the distinct patterns of ECM remodeling and adipose function in different fat depots.
Assuntos
Adipogenia/genética , Antígenos Ly/biossíntese , Gordura Intra-Abdominal/metabolismo , Proteínas de Membrana/biossíntese , Gordura Subcutânea/metabolismo , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Mesenquimais/metabolismo , CamundongosRESUMO
Thrombospondin 1 (THBS1 or TSP-1) is a circulating glycoprotein highly expressed in hypertrophic visceral adipose tissues of humans and mice. High-fat diet (HFD) feeding induces the robust increase of circulating THBS1 in the early stages of HFD challenge. The loss of Thbs1 protects male mice from diet-induced weight gain and adipocyte hypertrophy. Hyperinsulinemic euglycemic clamp study has demonstrated that Thbs1-null mice are protected from HFD-induced insulin resistance. Tissue-specific glucose uptake study has revealed that the insulin-sensitive phenotype of Thbs1-null mice is mostly mediated by skeletal muscles. Further assessments of the muscle phenotype using RNA sequencing, quantitative PCR, and histological studies have demonstrated that Thbs1-null skeletal muscles are protected from the HFD-dependent induction of Col3a1 and Col6a1, coupled with a new collagen deposition. At the same time, the Thbs1-null mice display a better circadian rhythm and higher amplitude of energy expenditure with a browning phenotype in sc adipose tissues. These results suggest that THBS1, which circulates in response to a HFD, may induce insulin resistance and fibrotic tissue damage in skeletal muscles as well as the de-browning of sc adipose tissues in the early stages of a HFD challenge. Our study may shed new light on the pathogenic role played by a circulating extracellular matrix protein in the cross talk between adipose tissues and skeletal muscles during obesity progression.
Assuntos
Gorduras na Dieta/efeitos adversos , Fibrose/induzido quimicamente , Resistência à Insulina/fisiologia , Doenças Musculares/etiologia , Trombospondina 1/metabolismo , Células 3T3-L1 , Tecido Adiposo Branco/metabolismo , Animais , Gorduras na Dieta/administração & dosagem , Relação Dose-Resposta a Droga , Epididimo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnica Clamp de Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Obesidade , Trombospondina 1/genética , TranscriptomaRESUMO
Implantation of various types of cells into the heart has been reported to be effective for heart failure, however, it is unknown what kinds of cells are most suitable for myocardial repair. To examine which types of cells are most effective, we injected cell-Puramatrix™ (PM) complex into the border area and overlaid the cell-PM patch on the myocardial infarction (MI) area. We compared cardiac morphology and function at 2 weeks after transplantation. Among clonal stem cell antigen-1 positive cardiac progenitors with PM (cSca-1/PM), bone marrow mononuclear cells with PM (BM/PM), skeletal myoblasts with PM (SM/PM), adipose tissue-derived mesenchymal cells with PM (AMC/PM), PM alone (PM), and non-treated MI group (MI), the infarct area of cSca-1/PM was smaller than that of BM/PM, SM/PM, PM and MI. cSca-1/PM and AMC/PM attenuated ventricular enlargement and restored cardiac function in comparison with MI. Capillary density in the infarct area of cSca-1/PM was higher than that of other five groups. The percentage of TUNEL positive cardiomyocytes in the infarct area of cSca-1/PM was lower than that of MI and PM. cSca-1 secreted VEGF and some of them differentiated into cardiomyocytes and vascular smooth muscle cells. These results suggest that transplantation of cSca-1/PM most effectively prevents cardiac remodeling and dysfunction through angiogenesis, inhibition of apoptosis and myocardial regeneration.
Assuntos
Testes de Função Cardíaca/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Miocárdio/citologia , Peptídeos/farmacologia , Transplante de Células-Tronco , Células-Tronco/citologia , Actinas/metabolismo , Indutores da Angiogênese/metabolismo , Animais , Antígenos Ly/metabolismo , Apoptose/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/patologia , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Células-Tronco/efeitos dos fármacos , Sístole/efeitos dos fármacos , Ultrassonografia , Remodelação Ventricular/efeitos dos fármacos , Fator de von Willebrand/metabolismoRESUMO
Cardiac progenitor cells are a potential source of cell therapy for heart failure. Although recent studies have shown that transplantation of cardiac stem/progenitor cells improves function of infarcted hearts, the precise mechanisms of the improvement in function remain poorly understood. The present study demonstrates that transplantation of sheets of clonally expanded stem cell antigen 1-positive (Sca-1-positive) cells (CPCs) ameliorates cardiac dysfunction after myocardial infarction in mice. CPC efficiently differentiated into cardiomyocytes and secreted various cytokines, including soluble VCAM-1 (sVCAM-1). Secreted sVCAM-1 induced migration of endothelial cells and CPCs and prevented cardiomyocyte death from oxidative stress through activation of Akt, ERK, and p38 MAPK. Treatment with antibodies specific for very late antigen-4 (VLA-4), a receptor of sVCAM-1, abolished the effects of CPC-derived conditioned medium on cardiomyocytes and CPCs in vitro and inhibited angiogenesis, CPC migration, and survival in vivo, which led to attenuation of improved cardiac function following transplantation of CPC sheets. These results suggest that CPC transplantation improves cardiac function after myocardial infarction through cardiomyocyte differentiation and paracrine mechanisms mediated via the sVCAM-1/VLA-4 signaling pathway.