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1.
Antioxidants (Basel) ; 8(12)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31817022

RESUMO

Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2',7'-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphomaextra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-ß1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.

3.
Mol Biol Rep ; 46(5): 5197-5207, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31309451

RESUMO

Cardiac myofibroblast (CMF) are non-muscle cardiac cells that play a crucial role in wound healing and in pathological remodeling. These cells are mainly derived of cardiac fibroblast (CF) differentiation mediated by TGF-ß1. Evidence suggests that bradykinin (BK) regulates cardiac fibroblast function in the heart. Both B1 and B2 kinin receptors (B1R and B2R, respectively) mediate the biological effects of kinins. We recently showed that both receptors are expressed in CMF and its stimulation decreases collagen secretion. Whether TGF-ß1 regulates B1R and B2R expression, and how these receptors control antifibrotic activity in CMF remains poorly understood. In this work, we sought to study, the regulation of B1R expression in cultured CMF mediated by TGF-ß1, and the molecular mechanisms involved in B1R activation on CMF intracellular collagen type-I levels. Cardiac fibroblast-primary culture was obtained from neonatal rats. Hearts were digested and CFs were attached to dishes and separated from cardiomyoctes. CMF were obtained from CF differentiation with TGF-ß1 5 ng/mL. CF and CMF were treated with B1R and B2R agonists and with TGF-ß1 at different times and concentrations, in the presence or absence of chemical inhibitors, to evaluate signaling pathways involved in B1R expression, collagen type-I and prostacyclin levels. B1R and collagen type-I levels were evaluated by western blot. Prostacyclin levels were quantified by an ELISA kit. TGF-ß1 increased B1R expression via TGFß type I receptor kinase (ALK5) activation and its subsequent signaling pathways involving Smad2, p38, JNK and ERK1/2 activation. Moreover, in CMF, the activation of B1R and B2R by their respective agonists, reduced collagen synthesis. This effect was mediated by the canonical signaling pathway; phospholipase C (PLC), protein kinase C (PKC), phospholipase A2 (PLA2), COX-2 activation and PGI2 secretion and its autocrine effect. TGF-ß1 through ALK5, Smad2, p38, JNK and ERK1/2 increases B1R expression; whereas in CMF, B1R and B2R activation share common signaling pathways for reducing collagen synthesis.

4.
Front Immunol ; 10: 1394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281317

RESUMO

In colorectal cancer (CRC), cancer-associated fibroblasts (CAFs) are the most abundant component from the tumor microenvironment (TM). CAFs facilitate tumor progression by inducing angiogenesis, immune suppression and invasion, thus altering the organization/composition of the extracellular matrix (i.e., desmoplasia) and/or activating epithelial-mesenchymal transition (EMT). Soluble factors from the TM can also contribute to cell invasion through secretion of cytokines and recently, IL-33/ST2 pathway has gained huge interest as a protumor alarmin, promoting progression to metastasis by inducing changes in TM. Hence, we analyzed IL-33 and ST2 content in tumor and healthy tissue lysates and plasma from CRC patients. Tissue localization and distribution of these molecules was evaluated by immunohistochemistry (using localization reference markers α-smooth muscle actin or α-SMA and E-cadherin), and clinical/histopathological information was obtained from CRC patients. In vitro experiments were conducted in primary cultures of CAFs and normal fibroblasts (NFs) isolated from tumor and healthy tissue taken from CRC patients. Additionally, migration and proliferation analysis were performed in HT29 and HCT116 cell lines. It was found that IL-33 content increases in left-sided CRC patients with lymphatic metastasis, with localization in tumor epithelia associated with abundant desmoplasia. Although ST2 content showed similarities between tumor and healthy tissue, a decreased immunoreactivity was observed in left-sided tumor stroma, associated to metastasis related factors (advanced stages, abundant desmoplasia, and presence of tumor budding). A principal component analysis (including stromal and epithelial IL-33/ST2 and α-SMA immunoreactivity with extent of desmoplasia) allowed us to distinguish clusters of low, intermediate and abundant desmoplasia, with potential to develop a diagnostic signature with benefits for further therapeutic targets. IL-33 transcript levels from CAFs directly correlated with CRC cell line migration induced by CAFs conditioned media, with rhIL-33 inducing a mesenchymal phenotype in HT29 cells. These results indicate a role of IL-33/ST2 in tumor microenvironment, specifically in the interaction between CAFs and epithelial tumor cells, thus contributing to invasion and metastasis in left-sided CRC, most likely by activating desmoplasia.

5.
Circulation ; 139(20): 2342-2357, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-30818997

RESUMO

BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

6.
Molecules ; 24(4)2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791388

RESUMO

New histone deacetylases (HDAC) inhibitors with low toxicity to non-cancerous cells, are a prevalent issue at present because these enzymes are actively involved in fibrotic diseases. We designed and synthesized a novel series of thiazolyl-coumarins, substituted at position 6 (R = H, Br, OCH3), linked to classic zinc binding groups, such as hydroxamic and carboxylic acid moieties and alternative zinc binding groups such as disulfide and catechol. Their in vitro inhibitory activities against HDACs were evaluated. Disulfide and hydroxamic acid derivatives were the most potent ones. Assays with neonatal rat cardiac fibroblasts demonstrated low cytotoxic effects for all compounds. Regarding the parameters associated to cardiac fibrosis development, the compounds showed antiproliferative effects, and triggered a strong decrease on the expression levels of both α-SMA and procollagen I. In conclusion, the new thiazolyl-coumarin derivatives inhibit HDAC activity and decrease profibrotic effects on cardiac fibroblasts.


Assuntos
Cumarínicos/síntese química , Cumarínicos/farmacologia , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Cumarínicos/química , Fibrose , Expressão Gênica , Inibidores de Histona Desacetilases/química , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ratos
7.
Front Pharmacol ; 9: 1368, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30555324

RESUMO

Cardiac fibroblasts (CFs) contribute to theinflammatory response to tissue damage, secreting both pro- and anti-inflammatory cytokines and chemokines. Interferon beta (IFN-ß) induces the phosphorylation of signal transducer and activator of transcription (STAT) proteins through the activation of its own receptor, modulating the secretion of cytokines and chemokines which regulate inflammation. However, the role of IFN-ß and STAT proteins in modulating the inflammatory response of CF remains unknown. CF were isolated from adult male rats and subsequently stimulated with IFN-ß to evaluate the participation of STAT proteins in secreting chemokines, cytokines, cell adhesion proteins expression and in their capacity to recruit neutrophils. In addition, in CF in which the TRL4 receptor was pre-activated, the effect of INF-ß on the aforementioned responses was also evaluated. Cardiac fibroblasts stimulation with IFN-ß showed an increase in STAT1, STAT2, and STAT3 phosphorylation. IFN-ß stimulation through STAT1 activation increased proinflammatory chemokines MCP-1 and IP-10 secretion, whereas IFN-ß induced activation of STAT3 increased cytokine secretion of anti-inflammatory IL-10. Moreover, in TLR4-activated CF, IFN-ß through STAT2 and/or STAT3, produced an anti-inflammatory effect, reducing pro-IL-1ß, TNF-α, IL-6, MCP-1, and IP-10 secretion; and decreasing neutrophil recruitment by decreasing ICAM-1 and VCAM-1 expression. Altogether, our results indicate that IFN-ß exerts both pro-inflammatory and anti-inflammatory effects in non-stimulated CF, through differential activation of STAT proteins. When CF were previously treated with an inflammatory agent such as TLR-4 activation, IFN-ß effects were predominantly anti-inflammatory.

9.
Toxicol Appl Pharmacol ; 351: 46-56, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29775649

RESUMO

Cardiac fibroblasts (CF) are key cells for maintaining extracellular matrix (ECM) protein homeostasis in the heart, and for cardiac repair through CF-to-cardiac myofibroblast (CMF) differentiation. Additionally, CF play an important role in the inflammatory process after cardiac injury, and they express Toll like receptor 4 (TLR4), B1 and B2 bradykinin receptors (B1R and B2R) which are important in the inflammatory response. B1R and B2R are induced by proinflammatory cytokines and their activation by bradykinin (BK: B2R agonist) or des-arg-kallidin (DAKD: B1R agonist), induces NO and PGI2 production which is key for reducing collagen I levels. However, whether TLR4 activation regulates bradykinin receptor expression remains unknown. CF were isolated from human, neonatal rat and adult mouse heart. B1R mRNA expression was evaluated by qRT-PCR, whereas B1R, collagen, COX-2 and iNOS protein levels were evaluated by Western Blot. NO and PGI2 were evaluated by commercial kits. We report here that in CF, TLR4 activation increased B1R mRNA and protein levels, as well as COX-2 and iNOS levels. B1R mRNA levels were also induced by interleukin-1α via its cognate receptor IL-1R1. In LPS-pretreated CF the DAKD treatment induced higher responses with respect to those observed in non LPS-pretreated CF, increasing PGI2 secretion and NO production; and reducing collagen I protein levels in CF. In conclusion, no significant response to DAKD was observed (due to very low expression of B1R in CF) - but pre-activation of TLR4 in CF, conditions that significantly enhanced B1R expression, led to an additional response of DAKD.


Assuntos
Fibroblastos/metabolismo , Miócitos Cardíacos/metabolismo , Receptor B1 da Bradicinina/biossíntese , Receptor 4 Toll-Like/biossíntese , Animais , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Expressão Gênica , Humanos , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética
10.
Biochim Biophys Acta Mol Basis Dis ; 1864(3): 831-842, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29222072

RESUMO

Cardiac fibroblasts (CF) act as sentinel cells responding to chemokines, cytokines and growth factors released in cardiac tissue in cardiac injury events, such as myocardial infarction (MI). Cardiac injury involves the release of various damage-associated molecular patterns (DAMPs) including heparan sulfate (HS), a constituent of the extracellular matrix (ECM), through the TLR4 receptor activation triggering a strong inflammatory response, inducing leukocytes recruitment. This latter cells are responsible of clearing cell debris and releasing cytokines that promote CF differentiation to myofibroblast (CMF), thus initiating scar formation. CF were isolated from adult male rats and subsequently stimulated with HS or LPS, in the presence or absence of chemical inhibitors, to evaluate signaling pathways involved in ICAM-1 and VCAM-1 expression. siRNA against ICAM-1 and VCAM-1 were used to evaluate participation of these adhesion molecules on leukocytes recruitment. HS through TLR4, PI3K/AKT and NF-ΚB increased ICAM-1 and VCAM-1 expression, which favored the adhesion of spleen mononuclear cells (SMC) and bone marrow granulocytes (PMN) to CF. These effects were prevented by siRNA against ICAM-1 and VCAM-1. Co-culture of CF with SMC increased α-SMA expression, skewing CF towards a pro-fibrotic phenotype, while CF pretreatment with HS partially reverted this effect. CONCLUSION: These data show the dual role of HS during the initial stages of wound healing. Initially, HS enhance the pro-inflammatory role of CF increasing cytokines secretion; and later, by increasing protein adhesion molecules allows the adhesion of SMC on CF, which trigger CF-to-CMF differentiation.


Assuntos
Adesão Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Miocárdio/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Células Cultivadas , Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Leucócitos/fisiologia , Masculino , Miocárdio/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Molécula 1 de Adesão de Célula Vascular/genética
11.
Biosci Trends ; 11(2): 154-162, 2017 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-28239053

RESUMO

Fibroblasts play several homeostatic roles, including electrical coupling, paracrine signaling and tissue repair after injury. Fibroblasts have low secretory activity. However, in response to injury, they differentiate to myofibroblasts. These cells have an increased extracellular matrix synthesis and secretion, including collagen fibers, providing stiffness to the tissue. In pathological conditions myofibroblasts became resistant to apoptosis, remaining in the tissue, causing excessive extracellular matrix secretion and deposition, which contributes to the progressive tissue remodeling. Therefore, increased myofibroblast content within damaged tissue is a characteristic hallmark of heart, lung, kidney and liver fibrosis. Recently, it was described that cardiac fibroblast to myofibroblast differentiation is triggered by the transforming growth factor ß1 (TGF-ß1) through a Smad-independent activation of Forkhead box O (FoxO). FoxO proteins are a transcription factor family that includes FoxO1, FoxO3, FoxO4 and FoxO6. In several cells types, they play an important role in cell cycle arrest, oxidative stress resistance, cell survival, energy metabolism, and cell death. Here, we review the role of FoxO family members on the regulation of cardiac fibroblast proliferation and differentiation.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Miofibroblastos/metabolismo
12.
Cardiovasc Toxicol ; 17(4): 458-470, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28220374

RESUMO

Bacterial lipopolysaccharide (LPS) is a known ligand of Toll-like receptor 4 (TLR4) which is expressed in cardiac fibroblasts (CF). Differentiation of CF to cardiac myofibroblasts (CMF) is induced by transforming growth factor-ß1 (TGF-ß1), increasing alpha-smooth muscle actin (α-SMA) expression. In endothelial cells, an antagonist effect between LPS-induced signaling and canonical TGF-ß1 signaling was described; however, it has not been studied whether in CF and CMF the expression of α-SMA induced by TGF-ß1 is antagonized by LPS and the mechanism involved. In adult rat CF and CMF, α-SMA, ERK1/2, Akt, NF-κß, Smad3, and Smad7 protein levels were determined by western blot, TGF-ß isoforms by ELISA, and α-SMA stress fibers by immunocytochemistry. CF and CMF secrete the three TGF-ß isoforms, and the secretion levels of TGF-ß2 was affected by LPS treatment. In CF, LPS treatment decreased the protein levels of α-SMA, and this effect was prevented by TAK-242 (TLR4 inhibitor) and LY294002 (Akt inhibitor), but not by BAY 11-7082 (NF-κß inhibitor) and PD98059 (ERK1/2 inhibitor). TGF-ß1 increased α-SMA protein levels in CF, and LPS prevented partially this effect. In addition, in CMF α-SMA protein levels were decreased by LPS treatment, which was abolished by TAK-242. Finally, in CF LPS decreased the p-Smad3 phosphorylation and increased the Smad7 protein levels. LPS treatment prevents the CF-to-CMF differentiation and reverses the CMF phenotype induced by TGF-ß1, through decreasing p-Smad3 and increasing Smad7 protein levels.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Masculino , Miócitos Cardíacos/fisiologia , Miofibroblastos/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor 4 Toll-Like/agonistas
13.
J Mol Cell Cardiol ; 2016 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-27983968

RESUMO

Macrophage polarization plays an essential role in cardiac remodeling after injury, evolving from an initial accumulation of proinflammatory M1 macrophages to a greater balance of anti-inflammatory M2 macrophages. Whether cardiac fibroblasts themselves influence this process remains an intriguing question. In this work, we present evidence for a role of cardiac fibroblasts (CF) as regulators of macrophage recruitment and skewing. Adult rat CF, were treated with lipopolysaccharide (LPS) or TGF-ß1, to evaluate ICAM-1 and VCAM-1 expression using Western blot and proinflammatory/profibrotic cytokine secretion using LUMINEX. We performed in vitro migration and adhesion assays of rat spleen monocytes to layers of TGF-ß1- or LPS-pretreated CF. Finally, TGF-ß1- or LPS-pretreated CF were co-cultured with monocyte, to evaluate their effects on macrophage polarization, using flow cytometry and cytokine secretion. There was a significant increase in monocyte adhesion to LPS- or TGF-ß1-stimulated CF, associated with increased CF expression of ICAM-1 and VCAM-1. siRNA silencing of either ICAM-1 or VCAM-1 inhibited monocyte adhesion to LPS-pretreated CF; however, monocyte adhesion to TGF-ß1-treated CF was dependent on only VCAM-1 expression. Pretreatment of CF with LPS or TGF-ß1 increased monocyte migration to CF, and this effect was completely abolished with an MCP-1 antibody blockade. LPS-treated CF secreted elevated levels of TNF-α and MCP-1, and when co-cultured with monocyte, LPS-treated CF stimulated increased macrophage M1 polarization and secretion of proinflammatory cytokines (TNF-α, IL-12 and MCP-1). On the other hand, CF stimulated with TGF-ß1 produced an anti-inflammatory cytokine profile (high IL-10 and IL-5, low TNF-α). When co-cultured with monocytes, the TGF-ß1 stimulated fibroblasts skewed monocyte differentiation towards M2 macrophages accompanied by increased IL-10 and decreased IL-12 levels. Taken together, our results show for the first time that CF can recruit monocytes (via MCP-1-mediated chemotaxis and adhesion to ICAM-1/VCAM-1) and induce their differentiation to M1 or M2 macrophages (through the CF cytokine profile induced by proinflammatory or profibrotic stimuli).

14.
Mol Immunol ; 74: 96-105, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27174187

RESUMO

UNLABELLED: Cardiac inflammation can be produced by pathogen-associated molecular patterns (PAMPs), from parasitic, bacterial or viral origin; or by danger-associated molecular patterns (DAMPs), released from dead cells after cardiac tissue damage, for example by cardiac infarction. Both, PAMPS and DAMPS activate TLR4 on resident immune cells and heart tissue cells, triggering an inflammatory process necessary to begin the wound healing process. Cardiac fibroblasts (CF) are the most abundant cells in the heart and are critical to wound healing, along with cardiac myofibroblasts (CMF), which are differentiated from CF through a TGF-ß1-mediated process. While TLR4 and the inflammasome complex are known to play important roles in CF function, the effects of TGF-ß1 on TLR4 and inflammasome expression and activity remain unknown. To elucidate this important point, we evaluated the effect of TGF-ß1 on TLR4, and the inflammasome protein expression and activity through activation by LPS, mimicking a myocarditis condition by bacterial origin. We found that TGF-ß1 increased TLR4 expression in CF and that the process was mediated by the TGFßRI and p38 signaling pathways. In both CF and CMF, LPS triggered ERK1/2, PI3K-Akt, and p65-NF-κB phosphorylation. All of these effects were blocked by TAK-242, a TLR4 signaling pathway inhibitor. LPS increased pro-IL-1ß levels, which were dependent on the ERK1/2, PI3K-Akt, and NF-κB signaling pathways, and levels were higher in CF than CMF. NLRP3 and ASC levels were similar in CF and CMF, while pro-caspase-1 levels and caspase-1 activity were higher in CMF. LPS+ATP treatment induced inflammasome complex assembly and activation, triggering the release of IL-1ß in both CMF and CF. Finally, the unsecreted pro-IL-1ß in the CF was degraded by autophagy. CONCLUSION: TGF-ß1 increases TLR4 expression in CF. Despite different pro-IL-1ß and caspase-1 activity levels in CF versus CMF, the two cell types secreted similar levels of IL-1ß after LPS+ATP treatment. These findings suggest that both cell types are active participants in inflammation.


Assuntos
Fibroblastos/imunologia , Inflamassomos/imunologia , Interleucina-1beta/biossíntese , Miocárdio/imunologia , Miofibroblastos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Masculino , Miocárdio/citologia , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley
15.
Biochim Biophys Acta ; 1863(1): 128-38, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26518453

RESUMO

Cardiac fibroblast differentiation to myofibroblast is a crucial process in the development of cardiac fibrosis and is tightly dependent on transforming growth factor beta-1 (TGF-ß1). The transcription factor forkhead box O1 (FoxO1) regulates many cell functions, including cell death by apoptosis, proliferation, and differentiation. However, several aspects of this process remain unclear, including the role of FoxO1 in cardiac fibroblast differentiation and the regulation of FoxO1 by TGF-ß1. Here, we report that TGF-ß1 stimulates FoxO1 expression, promoting its dephosphorylation, nuclear localization and transcriptional activity in cultured cardiac fibroblasts. TGF-ß1 also increases differentiation markers such as α-smooth muscle actin, connective tissue growth factor, and pro-collagen I, whereas it decreases cardiac fibroblast proliferation triggered by fetal bovine serum. TGF-ß1 also increases levels of p21waf/cip-cycle inhibiting factor protein, a cytostatic factor promoting cell cycle arrest and cardiac fibroblast differentiation. In addition, TGF-ß1 increases cardiac fibroblast contractile capacity as assessed by collagen gel contraction assay. The effect of TGF-ß1 on cardiac fibroblast differentiation was prevented by FoxO1 down-regulation and enhanced by FoxO1 overexpression. Thus, our findings reveal that FoxO1 is regulated by TGF-ß1 and plays a critical role in cardiac fibroblast differentiation. We propose that FoxO1 is an attractive new target for anti-fibrotic therapy.


Assuntos
Diferenciação Celular , Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Bovinos , Núcleo Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição Forkhead/genética , Miocárdio/citologia , Miofibroblastos/citologia , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/genética
16.
Toxicol Appl Pharmacol ; 279(1): 53-62, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24844443

RESUMO

RATIONALE: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca(2+) channels and their renowned antioxidant properties. METHODS: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca(2+) channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca(2+) channel-blocking activity and antioxidant properties. The Ca(2+) channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. RESULTS: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca(2+) channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca(2+) channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. CONCLUSIONS: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Di-Hidropiridinas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Sítios de Ligação , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Cardiotônicos/farmacologia , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Di-Hidropiridinas/química , Frequência Cardíaca/efeitos dos fármacos , Hidroxilação , Masculino , Modelos Moleculares , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
17.
J Hypertens ; 32(4): 771-83, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24463937

RESUMO

BACKGROUND: Little is known about the biological effects of angiotensin-(1-9), but available evidence shows that angiotensin-(1-9) has beneficial effects in preventing/ameliorating cardiovascular remodeling. OBJECTIVE: In this study, we evaluated whether angiotensin-(1-9) decreases hypertension and reverses experimental cardiovascular damage in the rat. METHODS AND RESULTS: Angiotensin-(1-9) (600  ng/kg per min for 2 weeks) reduced already-established hypertension in rats with early high blood pressure induced by angiotensin II infusion or renal artery clipping. Angiotensin-(1-9) also improved cardiac (assessed by echocardiography) and endothelial function in small-diameter mesenteric arteries, cardiac and aortic wall hypertrophy, fibrosis, oxidative stress, collagen and transforming growth factor type ß - 1 protein expression (assessed by western blot). The beneficial effect of angiotensin-(1-9) was blunted by coadministration of the angiotensin type 2(AT2) receptor blocker PD123319 (36  ng/kg per min) but not by coadministration of the Mas receptor blocker A779 (100  ng/kg per min). Angiotensin-(1-9) treatment also decreased circulating levels of Ang II, angiotensin-converting enzyme activity and oxidative stress in aorta and left ventricle. Whereas, Ang-(1-9) increased endothelial nitric oxide synthase mRNA levels in aorta as well as plasma nitrate levels. CONCLUSION: Angiotensin-(1-9) reduces hypertension, ameliorates structural alterations (hypertrophy and fibrosis), oxidative stress in the heart and aorta and improves cardiac and endothelial function in hypertensive rats. These effects were mediated by the AT2 receptor but not by the angiotensin-(1-7)/Mas receptor axis.


Assuntos
Angiotensina II/química , Angiotensina I/química , Inibidores da Enzima Conversora de Angiotensina/química , Doenças Cardiovasculares/tratamento farmacológico , Hipertensão/tratamento farmacológico , Fragmentos de Peptídeos/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Animais , Aorta/patologia , Pressão Sanguínea/efeitos dos fármacos , Doenças Cardiovasculares/prevenção & controle , Modelos Animais de Doenças , Ecocardiografia , Endotélio Vascular/patologia , Ventrículos do Coração , Hemodinâmica , Hipertensão/fisiopatologia , Imidazóis/química , Masculino , Estresse Oxidativo , Piridinas/química , Ratos , Ratos Sprague-Dawley
18.
Toxicol Appl Pharmacol ; 272(2): 414-22, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23845590

RESUMO

UNLABELLED: In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor ß1 (TGF-ß1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction. METHOD: Rat neonatal CF and CMF were treated with TGF-ß1 at different times and concentrations. EPAC-1 protein levels and Rap1 activation were measured by western blot and pull down assay respectively. EPAC cellular functions were determined by adhesion, migration and collagen gel contraction assay; and collagen expression was determined by western blot. RESULTS: TGF-ß1 through Smad and JNK significantly reduced EPAC-1 expression in CF, while in CMF this cytokine increased EPAC-1 expression through ERK1/2, JNK, p38, AKT and Smad3. EPAC activation was able to induce higher Rap1-GTP levels in CMF than in CF. EPAC and PKA, both cAMP effectors, promoted CF and CMF adhesion on fibronectin, as well as CF migration; however, this effect was not observed in CMF. EPAC but not PKA activation mediated collagen gel contraction in CF, while in CMF both PKA and EPAC mediated collagen gel contraction. Finally, the EPAC and PKA activation reduced collagen synthesis in CF and CMF. CONCLUSION: TGF-ß1 differentially regulates the expression of EPAC in CF and CMF; and EPAC regulates differentially CF and CMF functions associated with cardiac remodeling.


Assuntos
Fibroblastos/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Ventrículos do Coração/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Animais , Animais Recém-Nascidos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Colágeno/biossíntese , Fibroblastos/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Ratos , Ratos Sprague-Dawley , Remodelação Ventricular/fisiologia
19.
IUBMB Life ; 65(7): 593-601, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23671040

RESUMO

Insulin-like growth factor-1 (IGF-1) signaling is a key pathway in the control of cell growth and survival. Three critical nodes in the IGF-1 signaling pathway have been described in cardiomyocytes: protein kinase Akt/mammalian target of rapamycin (mTOR), Ras/Raf/extracellular signal-regulated kinase (ERK), and phospholipase C (PLC)/inositol 1,4,5-triphosphate (InsP3 )/Ca(2+) . The Akt/mTOR and Ras/Raf/ERK signaling arms govern survival in the settings of cardiac stress and hypertrophic growth. By contrast, PLC/InsP3 /Ca(2+) functions to regulate metabolic adaptability and gene transcription. Autophagy is a catabolic process involved in protein degradation, organelle turnover, and nonselective breakdown of cytoplasmic components during nutrient starvation or stress. In the heart, autophagy is observed in a variety of human pathologies, where it can be either adaptive or maladaptive, depending on the context. We proposed the hypothesis that IGF-1 protects the heart by rescuing the mitochondrial metabolism and the energetics state, reducing cell death and controls the potentially exacerbate autophagic response to nutritional stress. In light of the importance of IGF-1 and autophagy in the heart, we review here IGF-1 signaling and autophagy regulation in the context of cardiomyocyte nutritional stress.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Fisiológico , Autofagia , Proliferação de Células , Humanos , Mitocôndrias/metabolismo , Miócitos Cardíacos/fisiologia , Transdução de Sinais
20.
Biochim Biophys Acta ; 1832(6): 754-62, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23416528

RESUMO

Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-ß1 has shown cardioprotective effects in cardiac damage; however, if TGF-ß1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-ß1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-ß1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-ß1 during reperfusion. TGF-ß1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-ß1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-ß1 prevents cardiac fibroblast apoptosis induced by simulated ischemia-reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.


Assuntos
Apoptose , Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Fibroblastos/patologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
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