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1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445337

RESUMO

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFß2 and IL1ß. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.


Assuntos
Transdiferenciação Celular/genética , Rim/patologia , MicroRNAs/fisiologia , Miocárdio/patologia , Animais , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Fibrose/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Rim/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia
2.
Biomolecules ; 11(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34356613

RESUMO

Renal fibrosis is a hallmark of chronic kidney disease (CKD) and a common manifestation of end-stage renal disease that is associated with multiple types of renal insults and functional loss of the kidney. Unresolved renal inflammation triggers fibrotic processes by promoting the activation and expansion of extracellular matrix-producing fibroblasts and myofibroblasts. Growing evidence now indicates that diverse T cells and macrophage subpopulations play central roles in the inflammatory microenvironment and fibrotic process. The present review aims to elucidate the role of CD8+ T cells in renal fibrosis, and identify its possible mechanisms in the inflammatory microenvironment.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Rim/imunologia , Miofibroblastos/imunologia , Insuficiência Renal Crônica/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Fibrose , Humanos , Inflamação/imunologia , Inflamação/patologia , Rim/patologia , Miofibroblastos/patologia , Insuficiência Renal Crônica/patologia
3.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200497

RESUMO

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Insuficiência Cardíaca/patologia , Miofibroblastos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Miofibroblastos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Ativação Transcricional
4.
J Pediatr Hematol Oncol ; 43(5): e718-e722, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34157012

RESUMO

Most inflammatory myofibroblastic tumors (IMTs) harbor ALK fusions but oncogene fusions involving ROS1, RET, NTRK, and PDGFR also occur. The recognition that most IMTs harbor receptor tyrosine kinase fusions has provided a rationale for the use of tyrosine kinase inhibitors to target these oncogenic drivers in advanced IMTs. Crizotinib has been effective in ALK and ROS1-positive IMTs but resistance eventually develops. Here we report the successful use of lorlatinib in a patient with heavily pretreated ROS1-positive IMT of the chest wall with acquired crizotinib-resistance and metastasis to the brain.


Assuntos
Aminopiridinas/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/secundário , Lactamas/uso terapêutico , Neoplasias de Tecido Muscular/tratamento farmacológico , Neoplasias de Tecido Muscular/patologia , Pirazóis/uso terapêutico , Neoplasias Torácicas/tratamento farmacológico , Neoplasias Torácicas/patologia , Adolescente , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Humanos , Masculino , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Neoplasias de Tecido Muscular/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/uso terapêutico , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Torácicas/genética
5.
BMC Cardiovasc Disord ; 21(1): 308, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34154526

RESUMO

BACKGROUND: Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. METHODS: In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). RESULTS: In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. CONCLUSIONS: Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.


Assuntos
Potenciais de Ação , Comunicação Celular , Fibroblastos/metabolismo , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , RNA Circular/metabolismo , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Transdiferenciação Celular , Técnicas de Cocultura , Fibroblastos/patologia , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , RNA Circular/genética , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
6.
Am J Physiol Renal Physiol ; 321(2): F170-F178, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34180718

RESUMO

Pericytes play an important role in the recovery process after ischemic injury of many tissues. Brain pericytes in the peri-infarct area express macrophage markers in response to injury stimuli and are involved in neovascularization. In the kidney, nerve/glial antigen 2 (NG2)+ pericytes have been found to accumulate after renal injury. These accumulated NG2+ cells are not involved in scar formation. However, the role of accumulated NG2+ cells in injured kidneys remains unknown. Here, using a reversible ischemia-reperfusion (I/R) model, we found that renal NG2+ cells were increased in injured kidneys and expressed macrophage markers (CD11b or F4/80) on day 3 after reperfusion. Isolated NG2+ cells from I/R kidneys also had phagocytic activity and expressed anti-inflammatory cytokine genes, including mannose receptor and IL-10. These macrophage-like NG2+ cells did not likely differentiate into myofibroblasts because they did not increase α-smooth muscle actin expression. Intravenous transfusion of renal NG2+ cells isolated from donor mice on day 3 after reperfusion into recipient mice on day 1 after I/R surgery revealed that NG2+ cell-injected mice had lower plasma blood urea nitrogen, reduced kidney injury molecule-1 mRNA expression, ameliorated renal damage, and reduced cellular debris accumulation compared with PBS-injected mice on day 5 after reperfusion. In conclusion, these data suggest that renal NG2+ cells have an M2 macrophage-like ability and play a novel role in facilitating the recovery process after renal I/R injury.NEW & NOTEWORTHY Brain pericytes have macrophage-like activities after injury. However, such properties of pericytes in peripheral tissues have not been investigated. Here, we provide evidence that nerve/glial antigen 2-positive cells increase after renal injury. The population of nerve/glial antigen 2-positive cells, which does not increase expression of myofibroblast-associated gene, express macrophage markers and anti-inflammatory cytokine genes, have phagocytic activity, and play a role in renal recovery after kidney injury.


Assuntos
Antígenos/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Proteoglicanas/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Isquemia/patologia , Rim/patologia , Macrófagos/patologia , Masculino , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fagocitose/fisiologia , Fenótipo , Traumatismo por Reperfusão/patologia
7.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068694

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening lung disease characterized by the proliferation of myofibroblasts and deposition of extracellular matrix that results in irreversible distortion of the lung structure and the formation of focal fibrosis. The molecular mechanism of IPF is not fully understood, and there is no satisfactory treatment. However, most studies suggest that abnormal activation of transforming growth factor-ß1 (TGF-ß1) can promote fibroblast activation and epithelial to mesenchymal transition (EMT) to induce pulmonary fibrosis. Deglycosylated azithromycin (Deg-AZM) is a compound we previously obtained by removing glycosyls from azithromycin; it was demonstrated to exert little or no antibacterial effects. Here, we discovered a new function of Deg-AZM in pulmonary fibrosis. In vivo experiments showed that Deg-AZM could significantly reduce bleomycin-induced pulmonary fibrosis and restore respiratory function. Further study revealed the anti-inflammatory and antioxidant effects of Deg-AZM in vivo. In vitro experiments showed that Deg-AZM inhibited TGF-ß1 signaling, weakened the activation and differentiation of lung fibroblasts, and inhibited TGF-ß1-induced EMT in alveolar epithelial cells. In conclusion, our findings show that Deg-AZM exerts antifibrotic effects by inhibiting TGF-ß1-induced myofibroblast activation and EMT.


Assuntos
Azitromicina/uso terapêutico , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Transdução de Sinais , Animais , Azitromicina/química , Azitromicina/farmacologia , Bleomicina , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Inflamação/patologia , Pulmão/patologia , Camundongos , Modelos Biológicos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Células NIH 3T3 , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo
8.
Exp Eye Res ; 208: 108611, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33992624

RESUMO

PURPOSE: Corneal fibroblast can be transformed into corneal myofibroblasts by TGF-ß1. Enhancer of zeste homolog 2 (EZH2) upregulation has been observed in the occurrence of other fibrotic disorders. We investigated the role of EZH2 in the progression of corneal fibrosis and the antifibrotic effect of EZH2 inhibition in corneal fibroblasts (CFs). METHODS: Primary CFs were isolated from corneal limbi and the CFs were treated with TGF-ß1 to induce fibrosis. EPZ-6438 and EZH2 siRNA were used to inhibit EZH2 expression. Myofibroblast activation and extracellular matrix (ECM) protein synthesis was detected by quantitative real-time PCR, western blotting, and immunofluorescence staining assay. The functions of myofibroblast were evaluated by cell migration and collagen gel contraction assays. Molecular mechanisms involved in EZH2 inhibition were investigated by RNA sequencing. RESULTS: TGF-ß1 activated EZH2 expression in CFs. Treatment with EPZ-6438 (5 µM) and EZH2 siRNA considerably suppressed corneal myofibroblast activation and ECM protein synthesis in CFs induced by TGF-ß1 when compared to the control group. EPZ-6438 (5 µM) suppressed cell migration and gel contraction in CFs. RNA sequencing results revealed that antifibrotic genes were activated after EZH2 inhibition to suppress corneal myofibroblast activation. CONCLUSION: Inhibition of EZH2 suppresses corneal myofibroblast activation and ECM protein synthesis, and could serve as a novel therapeutic target for preventing corneal scarring.


Assuntos
Córnea/metabolismo , Doenças da Córnea/terapia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação da Expressão Gênica , Miofibroblastos/metabolismo , RNA/genética , Animais , Movimento Celular , Células Cultivadas , Córnea/patologia , Doenças da Córnea/genética , Doenças da Córnea/patologia , Modelos Animais de Doenças , Proteína Potenciadora do Homólogo 2 de Zeste/biossíntese , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/patologia
9.
Commun Biol ; 4(1): 546, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972658

RESUMO

Disturbed shear stress is thought to be the driving factor of neointimal hyperplasia in blood vessels and grafts, for example in hemodialysis conduits. Despite the common occurrence of neointimal hyperplasia, however, the mechanistic role of shear stress is unclear. This is especially problematic in the context of in situ scaffold-guided vascular regeneration, a process strongly driven by the scaffold mechanical environment. To address this issue, we herein introduce an integrated numerical-experimental approach to reconstruct the graft-host response and interrogate the mechanoregulation in dialysis grafts. Starting from patient data, we numerically analyze the biomechanics at the vein-graft anastomosis of a hemodialysis conduit. Using this biomechanical data, we show in an in vitro vascular growth model that oscillatory shear stress, in the presence of cyclic strain, favors neotissue development by reducing the secretion of remodeling markers by vascular cells and promoting the formation of a dense and disorganized collagen network. These findings identify scaffold-based shielding of cells from oscillatory shear stress as a potential handle to inhibit neointimal hyperplasia in grafts.


Assuntos
Simulação por Computador , Oclusão de Enxerto Vascular/patologia , Hiperplasia/patologia , Monócitos/patologia , Miofibroblastos/patologia , Estresse Mecânico , Células Cultivadas , Humanos , Técnicas In Vitro
10.
Biochem Biophys Res Commun ; 561: 180-186, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34023784

RESUMO

Fibrosis is a condition characterized by the overproduction of extracellular matrix (ECM) components (e.g., collagen) in the myofibroblasts, causing tissue hardening and eventual organ dysfunction. Currently, the molecular mechanisms that regulate ECM production in the myofibroblasts are still obscure. In this study, we investigated the function of GPRC5B in the cardiac and lung myofibroblasts using real-time RT-PCR and siRNA-mediated knockdown. We discovered a significantly high expression of Gprc5b in the tissues of the fibrosis mice models and confirmed that Gprc5b was consistently expressed in the myofibroblasts of fibrotic hearts and lungs. We also found that Gprc5b expression was associated and may be dependent on the actin-MRTF-SRF signaling pathway. Notably, we observed that Gprc5b knockdown reduced the expression of collagen genes in the cardiac and lung myofibroblasts. Therefore, our findings reveal that GPRC5B enhances collagen production in the myofibroblasts, which directly promotes fibrosis in the tissues.


Assuntos
Colágeno/metabolismo , Fibrose/patologia , Coração/fisiopatologia , Fígado/metabolismo , Pulmão/metabolismo , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose/metabolismo , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Miofibroblastos/patologia , Transdução de Sinais
11.
Toxicol Appl Pharmacol ; 422: 115559, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961903

RESUMO

The occurrence and development of silicosis is related to the interaction of multiple cells through signal transmission caused by silica dust. Including inflammatory changes reduced by macrophages and phenotypic transdifferentiation reduced by lung fibroblasts. As a communication medium between cells, exosomes have become a hot research topic. To explore the role of exosomal proteins in the occurrence and development of silicosis and the possible intervention targets, this study conducted proteomic analysis of macrophage-derived exosomes induced by silica, to identify specific proteins for intervention. In this study, we used proteomic analysis to screen exosomal protein profiles from the RAW264.7 macrophages exposed to silica. A total of 291 proteins were differentially expressed, of which 178 were upregulated and 113 were downregulated. By performing functional annotation and analysis of the differentially expressed proteins, we identified proteins SPP1, HMGB3, and HNRNPAB, which were consistent with the proteomics analysis. The involvement of SPP1 protein in fibrosis was studied further. Knocking down the expression of SPP1 in exosomes resulted in a decrease in fibrosis-related indicators. These results help to understand that exosomal protein can mediate cell communication and play a key role in the transition from fibroblasts to myofibroblasts. Further, this study also provided strategies and scientific basis for future studies on the intervention of silicosis.


Assuntos
Comunicação Celular , Transdiferenciação Celular , Exossomos/efeitos dos fármacos , Fibroblastos/metabolismo , Macrófagos/efeitos dos fármacos , Osteopontina/metabolismo , Dióxido de Silício/toxicidade , Silicose/metabolismo , Animais , Proliferação de Células , Exossomos/genética , Exossomos/metabolismo , Fibroblastos/patologia , Macrófagos/metabolismo , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células NIH 3T3 , Osteopontina/genética , Proteoma , Proteômica , Células RAW 264.7 , Silicose/genética , Silicose/patologia
12.
Medicine (Baltimore) ; 100(20): e25972, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34011083

RESUMO

RATIONALE: Inflammatory myofibroblastic tumor (IMT) is a rare mesenchymal tumor that is prevalent among children and adolescents. Surgery is the most important therapeutic approach for IMT and complete resection is recommended. Although 50% of IMTs show anaplastic lymphoma kinase (ALK) rearrangements, crizotinib has proven an effective therapeutic approach. However, the genetic landscape of this tumor is still not fully understood and treatment options are limited, especially in the majority of ALK-negative tumors. PATIENT CONCERNS: We describe the clinical case of a healthy 18-year-old female in whom a pulmonary nodule was incidentally detected. DIAGNOSES: Following a small increase in the size of the nodule, the patient underwent both 18FDG-PET/CT and 68Ga-PET/CT, resulting in a suspicion of bronchial hamartoma. INTERVENTIONS: The patient underwent surgery and a salivary gland-like lung tumor was diagnosed. OUTCOMES: After surgery, the patient was referred to our cancer center, where a review of the histology slides gave a final diagnosis of ALK-negative lung IMT. Given the histology, it was decided not to administer adjuvant therapy and the patient was placed in a 3-monthly follow-up program. The patient is still disease-free 2 years post-surgery. LESSONS: Although there is no standard of care for the treatment of IMT, identifying genomic alterations could help to redefine the management of patients with negative-ALK disease. Our review of the literature on IMT and other kinase fusions revealed, in addition to ALK rearrangements, the potential association of ROS1, NTRK, RET, or PDGFR beta alterations with the tumor.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias de Tecido Muscular/diagnóstico , Adolescente , Quinase do Linfoma Anaplásico/genética , Broncopatias/diagnóstico , Diagnóstico Diferencial , Feminino , Hamartoma/diagnóstico , Humanos , Achados Incidentais , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/cirurgia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/cirurgia , Miofibroblastos/patologia , Neoplasias de Tecido Muscular/genética , Neoplasias de Tecido Muscular/imunologia , Neoplasias de Tecido Muscular/cirurgia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada
13.
Methods Mol Biol ; 2299: 237-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028748

RESUMO

Aberrant deposition of the extracellular matrix (ECM) causes fibrosis and leads to ECM stiffening. This fibrotic ECM provides biological and biophysical stimulations to alter cell activity and drive progression of fibrosis. As an emerging discipline, mechanobiology aims to access the impact of both these cues on cell behavior and relates the reciprocity of mechanical and biological interactions; it incorporates concepts from different fields, like biology and physics, to help study the mechanical and biological facets of fibrosis extensively. A useful experimental platform in mechanobiology is decellularized ECM (dECM), which mimics the native microenvironment more accurately than standard 2D culture techniques as its composition includes similar ECM protein components and stiffness. dECM, therefore, generates more reliable results that better recapitulate in vivo fibrosis.


Assuntos
Técnicas de Cultura de Células/métodos , Matriz Extracelular/patologia , Miofibroblastos/patologia , Animais , Fenômenos Biomecânicos , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Humanos , Espectrometria de Massas , Microscopia de Força Atômica , Modelos Biológicos , Miofibroblastos/metabolismo
14.
Methods Mol Biol ; 2299: 277-290, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028750

RESUMO

Reductionist cell culture systems are not only convenient but essential to understand molecular mechanisms of myofibroblast activation and action in carefully controlled conditions. However, tissue myofibroblasts do not act in isolation and the complexity of tissue repair and fibrosis in humans cannot be captured even by the most elaborate culture models. Over the past five decades, numerous animal models have been developed to study different aspects of myofibroblast biology and interactions with other cells and extracellular matrix. The underlying principles can be broadly classified into: (1) organ injury by trauma such as prototypical full thickness skin wounds or burns; (2) mechanical challenges, such as pressure overload of the heart by ligature of the aorta or the pulmonary vein; (3) toxic injury, such as administration of bleomycin to lungs and carbon tetrachloride to the liver; (4) organ infection with viruses, bacteria, and parasites, such as nematode infections of liver; (5) cytokine and inflammatory models, including local delivery or viral overexpression of active transforming growth factor beta; (6) "lifestyle" and metabolic models such as high-fat diet; and (7) various genetic models. We will briefly summarize the most widely used mouse models used to study myofibroblasts in tissue repair and fibrosis as well as genetic tools for manipulating myofibroblast repair functions in vivo.


Assuntos
Técnicas de Cultura de Células/métodos , Modelos Animais de Doenças , Miofibroblastos/citologia , Animais , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Fibrose , Humanos , Miofibroblastos/patologia , Cicatrização
15.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800912

RESUMO

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.


Assuntos
Displasia Arritmogênica Ventricular Direita/fisiopatologia , Endocárdio/patologia , Células-Tronco Mesenquimais/patologia , Miofibroblastos/patologia , Fator de Crescimento Transformador beta1/fisiologia , Adulto , Displasia Arritmogênica Ventricular Direita/sangue , Displasia Arritmogênica Ventricular Direita/patologia , Diferenciação Celular , Endocárdio/metabolismo , Feminino , Fibrose , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Transdução de Sinais/fisiologia , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Fator de Crescimento Transformador beta1/sangue
16.
Mar Drugs ; 19(5)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922418

RESUMO

Scleroderma is an autoimmune disease caused by the abnormal regulation of extracellular matrix synthesis and is activated by non-regulated inflammatory cells and cytokines. Echinochrome A (EchA), a natural pigment isolated from sea urchins, has been demonstrated to have antioxidant activities and beneficial effects in various disease models. The present study demonstrates for the first time that EchA treatment alleviates bleomycin-induced scleroderma by normalizing dermal thickness and suppressing collagen deposition in vivo. EchA treatment reduces the number of activated myofibroblasts expressing α-SMA, vimentin, and phosphorylated Smad3 in bleomycin-induced scleroderma. In addition, it decreased the number of macrophages, including M1 and M2 types in the affected skin, suggesting the induction of an anti-inflammatory effect. Furthermore, EchA treatment markedly attenuated serum levels of inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ, in a murine scleroderma model. Taken together, these results suggest that EchA is highly useful for the treatment of scleroderma, exerting anti-fibrosis and anti-inflammatory effects.


Assuntos
Anti-Inflamatórios/farmacologia , Macrófagos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Naftoquinonas/farmacologia , Escleroderma Sistêmico/prevenção & controle , Pele/efeitos dos fármacos , Actinas/metabolismo , Animais , Bleomicina , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/imunologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fosforilação , Células RAW 264.7 , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/patologia , Proteína Smad3/metabolismo , Vimentina/metabolismo
17.
J Proteome Res ; 20(5): 2867-2881, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33789425

RESUMO

Heart failure (HF) is associated with pathological remodeling of the myocardium, including the initiation of fibrosis and scar formation by activated cardiac fibroblasts (CFs). Although early CF-dependent scar formation helps prevent cardiac rupture by maintaining the heart's structural integrity, ongoing deposition of the extracellular matrix in the remote and infarct regions can reduce tissue compliance, impair cardiac function, and accelerate progression to HF. In our study, we conducted mass spectrometry (MS) analysis to identify differentially altered proteins and signaling pathways between CFs isolated from 7 day sham and infarcted murine hearts. Surprisingly, CFs from both the remote and infarct regions of injured hearts had a wide number of similarly altered proteins and signaling pathways that were consistent with fibrosis and activation into pathological myofibroblasts. Specifically, proteins enriched in CFs isolated from MI hearts were involved in pathways pertaining to cell-cell and cell-matrix adhesion, chaperone-mediated protein folding, and collagen fibril organization. These results, together with principal component analyses, provided evidence of global CF activation postinjury. Interestingly, however, direct comparisons between CFs from the remote and infarct regions of injured hearts identified 15 differentially expressed proteins between MI remote and MI infarct CFs. Eleven of these proteins (Gpc1, Cthrc1, Vmac, Nexn, Znf185, Sprr1a, Specc1, Emb, Limd2, Pawr, and Mcam) were higher in MI infarct CFs, whereas four proteins (Gstt1, Gstm1, Tceal3, and Inmt) were higher in MI remote CFs. Collectively, our study shows that MI injury induced global changes to the CF proteome, with the magnitude of change reflecting their relative proximity to the site of injury.


Assuntos
Infarto do Miocárdio , Remodelação Ventricular , Animais , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Proteínas com Domínio LIM , Camundongos , Proteínas dos Microfilamentos , Infarto do Miocárdio/genética , Miocárdio/patologia , Miofibroblastos/patologia
18.
Front Immunol ; 12: 641973, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777037

RESUMO

Cardiac allograft vasculopathy (CAV) charactered with aberrant remodeling and fibrosis usually leads to the loss of graft after heart transplantation. Our previous work has reported that extracellular high-mobility group box 1 (HMGB1) participated in the CAV progression via promoting inflammatory cells infiltration and immune damage. The aim of this study was to investigate the involvement of HMGB1 in the pathogenesis of CAV/fibrosis and potential mechanisms using a chronic cardiac rejection model in mice. We found high levels of transforming growth factor (TGF)-ß1 in cardiac allografts after transplantation. Treatment with HMGB1 neutralizing antibody markedly prolonged the allograft survival accompanied by attenuated fibrosis of cardiac allograft, decreased fibroblasts-to-myofibroblasts conversion, and reduced synthesis and release of TGF-ß1. In addition, recombinant HMGB1 stimulation promoted release of active TGF-ß1 from cardiac fibroblasts and macrophages in vitro, and subsequent phosphorylation of Smad2 and Smad3 which were downstream of TGF-ß1 signaling. These data indicate that HMGB1 contributes to the CAV/fibrosis via promoting the activation of TGF-ß1/Smad signaling. Targeting HMGB1 might become a new therapeutic strategy for inhibiting cardiac allograft fibrosis and dysfunction.


Assuntos
Rejeição de Enxerto/metabolismo , Proteína HMGB1/metabolismo , Transplante de Coração/efeitos adversos , Miocárdio/patologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Feminino , Fibrose/metabolismo , Fibrose/patologia , Rejeição de Enxerto/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Transdução de Sinais/fisiologia
19.
PLoS One ; 16(3): e0248196, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33667270

RESUMO

INTRODUCTION: Regulating excessive activation of fibroblasts may be a promising target to optimize extracellular matrix deposition and myocardial stiffness. Fibroblast activation protein alpha (FAP) is upregulated in activated fibroblasts after myocardial infarction (MI), and alters fibroblast migration in vitro. We hypothesized that FAP depletion may have a protective effect on left ventricular (LV) remodeling after MI. MATERIALS AND METHODS: We used the model of chronic MI in homozygous FAP deficient mice (FAP-KO, n = 51) and wild type mice (WT, n = 55) to analyze wound healing by monocyte and myofibroblast infiltration. Heart function and remodeling was studied by echocardiography, morphometric analyses including capillary density and myocyte size, collagen content and in vivo cell-proliferation. In non-operated healthy mice up to 6 months of age, morphometric analyses and collagen content was assessed (WT n = 10, FAP-KO n = 19). RESULTS: Healthy FAP-deficient mice did not show changes in LV structure or differences in collagen content or cardiac morphology. Infarct size, survival and cardiac function were not different between FAP-KO and wildtype mice. FAP-KO animals showed less LV-dilation and a thicker scar, accompanied by a trend towards lower collagen content. Wound healing, assessed by infiltration with inflammatory cells and myofibroblasts were not different between groups. CONCLUSION: We show that genetic ablation of FAP does not impair cardiac wound healing, and attenuates LV dilation after MI in mice. FAP seems dispensable for normal cardiac function and homeostasis.


Assuntos
Endopeptidases/deficiência , Ventrículos do Coração/metabolismo , Proteínas de Membrana/deficiência , Monócitos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Animais , Dilatação Patológica , Endopeptidases/metabolismo , Feminino , Ventrículos do Coração/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Monócitos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miofibroblastos/patologia
20.
Mol Cell Biochem ; 476(7): 2685-2701, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33666830

RESUMO

Cardiac fibrosis is an important pathological change after myocardial infarction (MI). Its progression is essential for post-MI infarct healing, during which transforming growth factor beta1 (TGF-ß1) plays a critical role. Limb-bud and Heart (LBH), a newly discovered target gene of TGF-ß1, was shown to promote normal cardiogenesis. αB-crystallin (CRYAB), an LBH-interacting protein, was demonstrated to be involved in TGF-ß1-induced fibrosis. The roles and molecular mechanisms of LBH and CRYAB during cardiac fibrosis remain largely unexplored. In this study, we investigated the alterations of LBH and CRYAB expression in mouse cardiac tissue after MI. LBH and CRYAB were upregulated in activated cardiac fibroblasts (CFs), while in vitro TGF-ß1 stimulation induced the upregulation of LBH, CRYAB, and fibrogenic genes in primary CFs of neonatal rats. The results of the ectopic expression of LBH proved that LBH accelerated CF proliferation under hypoxia, mediated the expression of CRYAB and fibrogenic genes, and promoted epithelial-mesenchymal transition (EMT)-like processes in rat CFs, while subsequent CRYAB silencing reversed the effects induced by elevated LBH expression. We also verified the protein-protein interaction (PPI) between LBH and CRYAB in fibroblasts. In summary, our work demonstrated that LBH promotes the proliferation of CFs, mediates TGF-ß1-induced fibroblast-to-myofibroblast transition and EMT-like processes through CRYAB upregulation, jointly functioning in post-MI infarct healing. These findings suggest that LBH could be a promising potential target for the study of cardiac repair and cardiac fibrosis.


Assuntos
Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fibrose , Infarto do Miocárdio/patologia , Miofibroblastos/patologia , Ratos , Ratos Sprague-Dawley
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