RESUMO
Cardiac inflammation and fibrosis triggered by left ventricular pressure overload are the major causes of heart dysfunction. Differentiated embryonic chondrocyte gene 1 (Dec1) is a basic helix-loop-helix transcription factor that is comprehensively involved in inflammation and tissue fibrosis, but its role in cardiac hypertrophy remains unclear. This study explored the effects of Dec1 on cardiac fibrosis, inflammation, and apoptosis in hypertrophic conditions. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in wild-type (WT) mice and in Dec1 knock out (KO) mice for 4 weeks. Using the TAC mouse model, prominent differences in cardiac hypertrophy at the morphological, functional, and molecular levels were delineated by Masson's Trichrome and TUNEL staining, immunohistochemistry, RT-PCR and Western Blot. DNA microarray and microRNA (miRNA) array analyses were carried out to identify gene and miRNA expression patterns. Dec1KO mice exhibited a more severe hypertrophic heart, whereas WT mice showed a more pronounced perivascular fibrosis after TAC at 4 weeks. The Dec1 deficiency promoted M2 phenotype macrophages. Dec1KO TAC mice showed fewer apoptotic cells than WT TAC mice. APEX1, WNT16, FGF10 and MMP-10 were differentially expressed according to DNA microarray analysis and expression levels of those genes and the corresponding miRNAs (miR-295, miR-200 b, miR-130a, miR-92a) showed the same trends. Furthermore, luciferase reporter assay confirmed that FGF10 is the direct target gene of miR-130. In conclusion, a Dec1 deficiency protects the heart from perivascular fibrosis, regulates M1/M2 macrophage polarization and reduces cell apoptosis, which may provide a novel insight for the treatment of cardiac hypertrophy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Cardiomegalia/genética , Proteínas de Homeodomínio/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Modelos Animais de Doenças , Expressão Gênica , Proteínas de Homeodomínio/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Miocardite/genética , Miocárdio/citologia , Miocárdio/patologiaRESUMO
Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular , Proliferação de Células , Células Epiteliais/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas Circadianas Period/metabolismo , Proteína Smad3/fisiologia , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Ritmo Circadiano , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Proteínas Circadianas Period/genética , Fatores de Transcrição/genéticaRESUMO
Cardiac fibrosis is a major cause of cardiac dysfunction in hypertrophic hearts. Differentiated embryonic chondrocyte gene 1 (Dec1), a basic helix-loop-helix transcription factor, has circadian expression in the heart; however, its role in cardiac diseases remains unknown. Therefore, using Dec1 knock-out (Dec1KO) and wild-type (WT) mice, we evaluated cardiac function and morphology at one and four weeks after transverse aortic constriction (TAC) or sham surgery. We found that Dec1KO mice retained cardiac function until four weeks after TAC. Dec1KO mice also revealed more severely hypertrophic hearts than WT mice at four weeks after TAC, whereas no significant change was observed at one week. An increase in Dec1 expression was found in myocardial and stromal cells of TAC-treated WT mice. In addition, Dec1 circadian expression was disrupted in the heart of TAC-treated WT mice. Cardiac perivascular fibrosis was suppressed in TAC-treated Dec1KO mice, with positive immunostaining of S100 calcium binding protein A4 (S100A4), alpha smooth muscle actin (αSMA), transforming growth factor beta 1 (TGFß1), phosphorylation of Smad family member 3 (pSmad3), tumor necrosis factor alpha (TNFα), and cyclin-interacting protein 1 (p21). Furthermore, Dec1 expression was increased in myocardial hypertrophy and myocardial infarction of autopsy cases. Taken together, our results indicate that Dec1 deficiency suppresses cardiac fibrosis, preserving cardiac function in hypertrophic hearts. We suggest that Dec1 could be a new therapeutic target in cardiac fibrosis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Obstrução do Fluxo Ventricular Externo/complicações , Animais , Biomarcadores , Cardiomegalia/diagnóstico , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomiopatias/diagnóstico , Modelos Animais de Doenças , Ecocardiografia , Fibrose , Expressão Gênica , Testes de Função Cardíaca , Proteínas de Homeodomínio , Masculino , Camundongos , Camundongos Knockout , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Obstrução do Fluxo Ventricular Externo/diagnóstico , Remodelação VentricularRESUMO
Intramyocardial hydrogel injection is a promising therapy to prevent negative remodeling following myocardial infarction (MI). In this study, we report a mechanism for in-situ gel formation without external stimulation, resulting in an injectable and tissue-retainable hydrogel for MI treatment, and investigate its therapeutic outcomes. A liquid-like polymeric solution comprising poly(3-acrylamidophenylboronic acid-co-acrylamide) (BAAm), polyvinyl alcohol (PVA), and sorbitol (S) increases the viscous modulus by reducing the pre-added sorbitol concentration is developed. This solution achieves a sol-gel transition in-vitro in heart tissue by spontaneously diffusing the sorbitol. After intramyocardial injection, the BAAm/PVA/S with lower initial viscous modulus widely spreads in the myocardium and gelate compared to a viscoelastic alginate (ALG) hydrogel and is retained longer than the BAAm/S solution. Serial echocardiogram analyses prove that injecting the BAAm/PVA/S into the hearts of subacute MI rats significantly increases the fraction shortening and ejection shortening and attenuates the expansion of systolic LV diameter for up to 21 d after injection compared to the saline injection as a control, but the ALG injection does not. In addition, histological evaluation shows that only the BAAm/PVA/S decreases the infarct size and increases the wall thickness 21 d after injection. The BAAm/PVA/S intramyocardial injection is better at restraining systolic ventricular dilatation and cardiac failure in the rat MI model than in the control groups. Our findings highlight an effective injectable hydrogel therapy for MI by optimizing injectability-dependent distribution and retention of injected material. STATEMENT OF SIGNIFICANCE: In-situ gelling material is a promising strategy for intramyocardial hydrogel injection therapy for myocardial infarction (MI). Since the sol-gel transition of reported materials is driven by external stimulation such as temperature, pH, or ultraviolet, their application in vivo remains challenging. In this study, we first reported a synthetic in-situ gelling material (BAAm/PVA/S) whose gelation is stimulated by spontaneously reducing pre-added sorbitol after contacting the heart tissue. The BAAm/PVA/S solution spreads evenly, and is retained for at least 21 d in the heart tissue. Our study demonstrated that intramyocardial injection of the BAAm/PVA/S with more extensive distribution and longer retention had better effects on preventing LV dilation and improving cardiac function after MI than that of viscoelastic ALG and saline solution. We expect that these findings provide fundamental information for the optimum design of injectable biomaterials for treating MI.
Assuntos
Alprenolol/análogos & derivados , Hidrogel de Polietilenoglicol-Dimetacrilato , Infarto do Miocárdio , Ratos , Animais , Hidrogel de Polietilenoglicol-Dimetacrilato/uso terapêutico , Hidrogéis/farmacologia , Hidrogéis/uso terapêutico , Infarto do Miocárdio/patologia , Sorbitol/farmacologia , Sorbitol/uso terapêuticoRESUMO
Decellularized vascular tissue has high potential as a tissue-engineered vascular graft because of its similarity to native vessels in terms of mechanical strength. However, exposed collagen on the tissue induces blood coagulation, and low hemocompatibility is a major obstacle to its vascular application. Here we report that freeze-drying and ethanol treatment effectively modify collagen fiber structure and drastically reduce blood coagulation on the graft surface without exogenous chemical modification. Decellularized carotid artery of ostrich was treated with freeze-drying and ethanol solution at concentrations ranging between 5 and 99.5 %. Collagen fiber distance in the graft was narrowed by freeze-drying, and the non-helical region increased by ethanol treatment. Although in vitro blood coagulation pattern was similar on the grafts, platelet adhesion on the grafts was largely suppressed by freeze-drying and ethanol treatments. Ex vivo blood circulation tests also indicated that the adsorption of platelets and Von Willebrand Factor was largely reduced to approximately 80 % by ethanol treatment. These results indicate that structural modification of collagen fibers in decellularized tissue reduces blood coagulation on the surface by inhibiting platelet adhesion.
Assuntos
Coagulação Sanguínea , Colágeno , Adesividade Plaquetária , Animais , Adesividade Plaquetária/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacos , Colágeno/química , Engenharia Tecidual/métodos , Teste de Materiais , Liofilização , Prótese Vascular , Alicerces Teciduais/química , Plaquetas/metabolismo , Plaquetas/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Artérias Carótidas/efeitos dos fármacos , Humanos , Etanol/químicaRESUMO
Abnormal proliferation of vascular smooth muscle cells (VSMCs) induces graft anastomotic stenosis, resulting in graft failure. Herein, we developed a drug-loaded tissue-adhesive hydrogel as artificial perivascular tissue to suppress VSMCs proliferation. Rapamycin (RPM), an anti-stenosis drug, is selected as the drug model. The hydrogel was composed of poly (3-acrylamidophenylboronic acid-co-acrylamide) (BAAm) and polyvinyl alcohol. Since phenylboronic acid reportedly binds to sialic acid of glycoproteins which is distributed on the tissues, the hydrogel is expected to be adherent to the vascular adventitia. Two hydrogels containing 25 or 50 mg/mL of BAAm (BAVA25 and BAVA50, respectively) were prepared. A decellularized vascular graft with a diameter of <2.5 mm was selected as a graft model. Lap-shear test indicates that both hydrogels adhered to the graft adventitia. In vitro release test indicated that 83 and 73 % of RPM in BAVA25 and BAVA50 hydrogels was released after 24 h, respectively. When VSMCs were cultured with RPM-loaded BAVA hydrogels, their proliferation was suppressed at an earlier stage in RPM-loaded BAVA25 hydrogels compared to RPM-loaded BAVA50 hydrogels. An in vivo preliminary test reveals that the graft coated with RPM-loaded BAVA25 hydrogel shows better graft patency for at least 180 d than the graft coated with RPM-loaded BAVA50 hydrogel or without hydrogel. Our results suggest that RPM-loaded BAVA25 hydrogel with tissue adhesive characteristics has potential to improve decellularized vascular graft patency.
Assuntos
Sirolimo , Enxerto Vascular , Sirolimo/farmacologia , Hidrogéis , Prótese VascularRESUMO
Although the clinical application of cell-free tissue-engineered vascular grafts (TEVGs) has been proposed, vascular tissue regeneration mechanisms have not been fully clarified. Here, we report that monocyte subpopulations reconstruct vascular-like tissues through integrin signaling. An Arg-Glu-Asp-Val peptide-modified acellular long-bypass graft was used as the TEVG, and tissue regeneration in the graft was evaluated using a cardiopulmonary pump system and porcine transplantation model. In 1 day, the luminal surface of the graft was covered with cells that expressed CD163, CD14, and CD16, which represented the monocyte subpopulation, and they exhibited proliferative and migratory abilities. RNA sequencing showed that captured cells had an immune-related phenotype similar to that of monocytes and strongly expressed cell adhesion-related genes. In vitro angiogenesis assay showed that tube formation of the captured cells occurred via integrin signal activation. After medium- and long-term graft transplantation, the captured cells infiltrated the tunica media layer and constructed vascular with a CD31/CD105-positive layer and an αSMA-positive structure after 3 months. This finding, including multiple early-time observations provides clear evidence that blood-circulating monocytes are directly involved in vascular remodeling.
RESUMO
Mood disorders such as depression, anxiety, and bipolar disorder are highly associated with disrupted daily rhythms of activity, which are often observed in shift work and sleep disturbance in humans. Recent studies have proposed the REV-ERBα protein as a key circadian nuclear receptor that links behavioural rhythms to mood regulation. However, how the Rev-erbα gene participates in the regulation of mood remains poorly understood. Here, we show that the regulation of the serotonergic (5-HTergic) system, which plays a central role in stress-induced mood behaviours, is markedly disrupted in Rev-erbα-/- mice. Rev-erbα-/- mice exhibit both negative and positive behavioural phenotypes, including anxiety-like and mania-like behaviours, when subjected to a stressful environment. Importantly, Rev-erbα-/- mice show a significant decrease in the expression of a gene that encodes the rate-limiting enzyme of serotonin (5-HT) synthesis in the raphe nuclei (RN). In addition, 5-HT levels in Rev-erbα-/- mice are significantly reduced in the prefrontal cortex, which receives strong inputs from the RN and controls stress-related behaviours. Our findings indicate that Rev-erbα plays an important role in controlling the 5-HTergic system and thus regulates mood and behaviour.
Assuntos
Relógios Circadianos , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Humanos , Camundongos , Camundongos Knockout , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , SerotoninaRESUMO
The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.