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1.
J Nippon Med Sch ; 87(5): 268-276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33311008

RESUMO

BACKGROUND: Some cultured neonatal rat cardiomyocytes continue spontaneous beating even in serum-free medium. The present study explored the cause and genes responsible for this phenomenon. METHODS: Ingenuity Pathway Analysis (IPA) software was used to analyze fold changes in gene expression in beating neonatal rat cardiomyocytes, as compared with non-beating cardiomyocytes, which were obtained from DNA microarray data of total RNA extracts of cardiomyocytes. To confirm the involvement of the 8 genes selected by IPA prediction, cellular protein abundances were determined by Western blot. The gene expression of connective tissue growth factor (CTGF) was substantially higher in beating cardiomyocytes than in non-beating cardiomyocytes; thus, CTGF protein content released from cardiomyocytes into the culture medium was examined. RESULTS: IPA showed that the "Apelin Cardiac Fibroblast Signaling Pathway" was significantly inhibited and that microtubule dynamics and cytoskeleton organization were significantly activated. Each fluctuation in the cellular abundances of the 8 proteins in beating cardiomyocytes, as compared with non-beating cardiomyocytes, was primarily in the same direction as that of gene expression. However, the cellular CTGF protein abundance as well as CTGF content released into the medium did not substantially differ between beating and non-beating cardiomyocytes. CONCLUSIONS: The present results suggest that the large increase in CTGF gene expression in beating cardiomyocytes is not a cause but a result of beating, which may provide a putative pathway for controlling beating. Beating is sustained by developed cardiomyofibrils and directly upregulates CTGF gene expression, which is not followed by CTGF protein synthesis.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação da Expressão Gênica , Expressão Gênica , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Regulação para Cima , Animais , Animais Recém-Nascidos , Células Cultivadas , Ratos
2.
Nat Commun ; 11(1): 4467, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948751

RESUMO

Recent studies have reported that upregulation of disulfide-bond A oxidoreductase-like protein (DsbA-L) prevented lipid-induced renal injury in diabetic nephropathy (DN). However, the role and regulation of proximal tubular DsbA-L for renal tubulointerstitial fibrosis (TIF) remains unclear. In current study, we found that a proximal tubules-specific DsbA-L knockout mouse (PT-DsbA-L-KO) attenuated UUO-induced TIF, renal cell apoptosis and inflammation. Mechanistically, the DsbA-L interacted with Hsp90 in mitochondria of BUMPT cells which activated the signaling of Smad3 and p53 to produce connective tissue growth factor (CTGF) and then resulted in accumulation of ECM of BUMPT cells and mouse kidney fibroblasts. In addition, the progression of TIF caused by UUO, ischemic/reperfusion (I/R), aristolochic acid, and repeated acute low-dose cisplatin was also alleviated in PT-DsbA-L-KO mice via the activation of Hsp90 /Smad3 and p53/CTGF axis. Finally, the above molecular changes were verified in the kidney biopsies from patients with obstructive nephropathy (Ob). Together, these results suggest that DsbA-L in proximal tubular cells promotes TIF via activation of the Hsp90 /Smad3 and p53/CTGF axis.


Assuntos
Fibrose/genética , Predisposição Genética para Doença/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Nefropatias/genética , Idoso , Animais , Apoptose , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Nefropatias Diabéticas , Modelos Animais de Doenças , Feminino , Fibrose/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Inflamação , Rim/lesões , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína Supressora de Tumor p53/metabolismo
3.
Life Sci ; 256: 117893, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502539

RESUMO

AIMS: To investigate the effect and underlying mechanism of melittin and tripartite motif (TRIM) family in human embryonic lung fibroblast (HELF). MATERIALS AND METHODS: Lentiviral RNA interference vector and lentiviral overexpression vector were constructed and packaged by transfecting 293T cells; the proliferation of HELF was examined using Cell Counting Kit 8; Western blot and qRT-PCR were performed to examine protein and mRNA expression; the interaction with protein phosphatase magnesium-dependent 1A (PPM1A) was examined by Co-immunoprecipitation. KEY FINDINGS: Compared with the control group, the mRNA expression of the TRIM6, TRIM8 and TRIM47 in the IPF group significantly increased. Melittin inhibited the mRNA expression and protein expression levels of TRIM47, the HELF proliferation, the hydroxyproline levels, and the phosphorylation of Smad2/3; the interference of TRIM47 inhibited the protein expression of Vimentin, α-SMA, CTGF, the phosphorylation of Smad2/3 and the synthesis of hydroxyproline; TRIM47 overexpression elevated the phosphorylation of Smad2/3, induced ubiquitination of PPM1A and decreased the expression level of PPM1A, while TRIM47 RNA interference reversed this result. SIGNIFICANCE: Melittin has anti-fibrotic effect in HELF by directly reducing the phosphorylation of Smad2/3 or indirectly reducing the phosphorylation of Smad2/3 by decreasing the expression levels of TRIM47 whose overexpression induces ubiquitination of PPM1A.


Assuntos
Proteínas de Transporte/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Pulmão/embriologia , Meliteno/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Actinas/metabolismo , Proteínas de Transporte/sangue , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiprolina/metabolismo , Proteínas de Neoplasias/sangue , Proteínas Nucleares/sangue , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2C/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação/efeitos dos fármacos , Vimentina/metabolismo
4.
Poult Sci ; 99(4): 1832-1837, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32241463

RESUMO

Forty broilers maintained under natural hypobaric hypoxia (2,638 m above sea level) and 20 maintained under relative normoxia (460 m above sea level) were selected as pulmonary hypertensive (PHB) and nonpulmonary hypertensive (NPHB), to estimate the degree of the adventitial vascular thickness in lung arterioles and connective tissue growth factor (CTGF) expression in lung. In each group, the adventitial thickness (%AT) of 20 arterioles with 100 to 250 µm of external diameter was measured in lung samples of 24 and 42-day-old broilers. Also, mRNA extraction and real-time reverse transcription-PCR analysis were used to measure lung CTGF expression. The %AT was higher in PHB at 42 D as compared to NPHB at both ages and PHB at 24 D; however, the same differences were not evidenced at 24 D. In the 2 ages evaluated, differences were observed in the %AT between broilers under hypobaric hypoxia (PHB and NPHB) and under relative normoxia (P < 0.01). In broilers subjected to relative normoxia, no significant differences were found at any of the 2 ages. The expression levels of CTGF mRNA were higher in PHB compared to NPHB at the 2 ages. The %AT was higher in PHB with high levels of expression of CTGF mRNA than those NPHB with low expression of CTGF mRNA. This study showed that adventitial thickening is part of the pulmonary hypertension (PH) physiopathology in broilers exposed to hypobaric hypoxia, in which CTGF appears to be a fibrosis enhancer. Although present data suggest that adventitial engrossment could be a time-dependent process, individual susceptibility and the variable time-course of PH pathophysiology have to be considered.


Assuntos
Arteríolas/fisiopatologia , Proteínas Aviárias/genética , Galinhas , Fator de Crescimento do Tecido Conjuntivo/genética , Expressão Gênica , Hipertensão Pulmonar/veterinária , Doenças das Aves Domésticas/fisiopatologia , Animais , Arteríolas/metabolismo , Proteínas Aviárias/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/etiologia , Hipóxia/fisiopatologia , Hipóxia/veterinária , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Doenças das Aves Domésticas/etiologia , Doenças das Aves Domésticas/genética
5.
Mol Cell Biochem ; 468(1-2): 129-142, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32185674

RESUMO

Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Cirrose Hepática/metabolismo , Substâncias Protetoras/farmacologia , Silimarina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Linhagem Celular , Cromatografia Gasosa , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Dimetil Sulfóxido/toxicidade , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Espectrometria de Massas , Triglicerídeos/metabolismo
6.
Sci Rep ; 10(1): 1871, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024900

RESUMO

Gallbladder cancer (GBC) is a highly malignant tumor with poorly understood etiology. An insight into phenotypic features of this malignancy may add to the knowledge of its carcinogenesis and pave the way to new therapeutic approaches. We assessed the expression of female sex hormone receptors (ERα, ERß, PR), connective tissue growth factor (CTGF) and HER2 in GBC, and adjacent normal tissue (NT), and determined their prognostic impact. Immunohistochemical (IHC) expression of all biomarkers was performed in formalin-fixed, paraffin-embedded specimens in 60 Caucasian GBC patients (51 women and 9 men). ERß, cytoPR and CTGF expression were found in 89%, 27%, 91% of GBC, and in 63%, 87%, 100% of NT, respectively. No ERα expression was found in GBC and NT. Strong (3+) HER2 expression by IHC or HER2 amplification was seen in five GBC (10.4%). A positive correlation was found between HER2 and CTGF and ERß expression in GBC and matched NT. In the multivariate analysis, patient age >70 years, tumor size and ERß expression in GBC was highly predictive for OS (p = 0.003). The correlation between HER2, CTGF and ERß expression in GBC and NT may indicate the interaction of these pathways in physiological processes and gallbladder pathology.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Feminino , Vesícula Biliar/metabolismo , Vesícula Biliar/patologia , Neoplasias da Vesícula Biliar/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
7.
Am J Physiol Heart Circ Physiol ; 318(4): H764-H777, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32083975

RESUMO

A variant in the PRDM16 locus has been correlated with QRS duration in an electrocardiogram genome-wide association study, and the deletion of PRDM16 has been implicated as a causal factor of the dilated cardiomyopathy that is linked to 1p36 deletion syndrome. We aimed to determine how a null mutation of Prdm16 affects cardiac function and study the underlying mechanism of the resulting phenotype in an appropriate mouse model. We used cardiac-specific Prdm16 conditional knockout mice to examine cardiac function by electrocardiography. QRS duration and QTc interval increased significantly in cardiac-specific Prdm16 knockout animals compared with wild-type mice. Further, we assessed cardiomyopathy-associated features by trichrome staining, densitometry, and hydroxyproline assay. Prdm16-null hearts showed greater fibrosis and cardiomyocyte hypertrophy. By quantitative real-time PCR, Prdm16-null hearts upregulated extracellular matrix-related genes (Ctgf, Timp1) and α-smooth muscle actin (Acta2), a myofibroblast marker. Moreover, TGF-ß signaling was activated in Prdm16-null hearts, as evidenced by increased Tgfb1-3 transcript levels and phosphorylated Smad2. However, the inhibition of TGF-ß receptor did not reverse the aberrations in conduction in cardiac-specific Prdm16 knockout mice. To determine the underlying mechanisms, we performed RNA-seq using mouse left ventricular tissue. By functional analysis, Prdm16-null hearts experienced dysregulated expression of ion channel genes, including Kcne1, Scn5a, Cacna1h, and Cacna2d2. Mice with Prdm16-null hearts develop abnormalities in cardiac conduction and cardiomyopathy-associated phenotypes, including fibrosis and cellular hypertrophy. Further, the RNA-seq findings suggest that impairments in ion homeostasis (Ca2+, K+, and Na+) may at least partially underlie the abnormal conduction in cardiac-specific Prdm16 knockout mice.NEW & NOTEWORTHY This is the first study that describes aberrant cardiac function and cardiomyopathy-associated phenotypes in an appropriate murine genetic model with cardiomyocyte-specific Prdm16-null mutation. It is noteworthy that the correlation of PRDM16 with QRS duration is replicated in a murine animal model and the potential underlying mechanism may be the impairment of ion homeostasis.


Assuntos
Cardiomiopatias/genética , Proteínas de Ligação a DNA/genética , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , Fenótipo , Fatores de Transcrição/genética , Actinas/genética , Actinas/metabolismo , Animais , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroencefalografia , Fibrose , Canais Iônicos/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
8.
Oxid Med Cell Longev ; 2020: 4910280, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104533

RESUMO

Reactive species play a pivotal role in orchestrating wound healing responses. They act as secondary messengers and drive redox-signalling pathways that are involved in the homeostatic, inflammatory, proliferative, and remodelling phases of wound healing. The application of Cold Atmospheric Plasma (CAP) to the wound site produces a profusion of short- and long-lived reactive species that have been demonstrated to be effective in promoting wound healing; however, knowledge of the mechanisms underlying CAP-mediated wound healing remains scarce. To address this, an in vitro coculture model was used to study the effects of CAP on wound healing and on paracrine crosstalk between dermal keratinocytes and fibroblasts. Using this coculture model, we observed a stimulatory effect on the migration ability of HaCaT cells that were cocultured with dermal fibroblasts. Additionally, CAP treatment resulted in an upregulation of the HIPPO transcription factor YAP in HaCaTs and fibroblasts. Downstream effectors of the HIPPO signalling pathway (CTGF and Cyr61) were also upregulated in dermal fibroblasts, and the administration of antioxidants could inhibit CAP-mediated wound healing and abrogate the gene expression of the HIPPO downstream effectors. Interestingly, we observed that HaCaT cells exhibited an improved cell migration rate when incubated with CAP-treated fibroblast-conditioned media compared to that observed after incubation with untreated media. An induction of CTGF and Cyr61 secretion was also observed upon CAP treatment in the fibroblast-conditioned media. Finally, exposure to recombinant CTGF and Cyr61 could also significantly improve HaCaT cell migration. In summary, our results validated that CAP activates a regenerative signalling pathway at the onset of wound healing. Additionally, CAP also stimulated a reciprocal communication between dermal fibroblasts and keratinocytes, resulting in improved keratinocyte wound healing in coculture.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Comunicação Parácrina , Gases em Plasma/farmacologia , Fatores de Transcrição/metabolismo , Cicatrização/efeitos dos fármacos , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Queratinócitos/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes , Fatores de Transcrição/genética
9.
Am J Sports Med ; 48(5): 1141-1150, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32074471

RESUMO

BACKGROUND: The pathogenesis of patellar tendon fibrosis caused by overuse remains unclear. In an effort to further investigate effective treatments for patellar tendon fibrosis attributed to overuse, it is necessary to construct a reliable animal model. PURPOSE: A rabbit patellar tendon fibrosis model was developed with the use of electrical stimulation to induce jumping. The pathogenesis and development of patellar tendon fibrosis were subsequently investigated with this model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 32 New Zealand White rabbits were randomly divided into a jumping group and a control group. Rabbits in the control group did not receive any treatment, while those in the jumping group jumped 150 times daily, 5 days per week. At 2, 4, 6, and 8 weeks after the initiation of treatment, the patellar tendons of 4 rabbits from each group were harvested and subjected to hematoxylin and eosin staining, immunohistochemical staining, and real-time polymerase chain reaction. The influence of jumping training on the expressions of histology- and fibrosis-related factors in the patellar tendon was assessed. RESULTS: The histological changes of patellar tendon fibrosis in the jumping group were most pronounced at 4 weeks. When compared with the control group at corresponding time points, the mRNA and protein expressions of TGF-ß1, CTGF, COL-I, and COL-III were upregulated significantly in the patellar tendon after jumping training for 4 weeks (P < .05). Intragroup comparison at different time points indicated that the mRNA and protein expressions of TGF-ß1, COL-I, and COL-III were the highest at 4 weeks in the jumping group (P < .01). CONCLUSION: It was found that patellar tendon fibrosis occurred because of overuse and the peak changes occurred at 4 weeks. Jumping load increased the secretions of TGF-ß1 and Smad3 in the patellar tendon, with CTGF upregulation and higher synthesis of COL-I and COL-III, which were considered the pathogenesis of fibrosis. CLINICAL RELEVANCE: This study simulated the effects of jumping load on tendon fibrosis at different time points. Moreover, the time course relationship between jumping training and patellar tendon fibrosis in the rabbit model was determined, which provided a new animal model for the study of patellar tendon fibrosis.


Assuntos
Transtornos Traumáticos Cumulativos/patologia , Ligamento Patelar/patologia , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Modelos Animais de Doenças , Fibrose , Coelhos , Distribuição Aleatória , Fator de Crescimento Transformador beta1/metabolismo
10.
Sci Rep ; 10(1): 3043, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080296

RESUMO

Altered pulmonary angiogenesis contributes to disrupted alveolarization, which is the main characteristic of bronchopulmonary dysplasia (BPD). Transforming growth factor ß (TGFß) plays an important role during lung vascular development, and recent studies have demonstrated that endoglin is engaged in the modulation of TGFß downstream signalling. Although there are two different isoforms of endoglin, L- and S-endoglin, little is known about the effect of S-endoglin in developing lungs. We analysed the expression of both L- and S-endoglin in the lung vasculature and its contribution to TGFß-activin-like kinase (ALK)-Smad signalling with respect to BPD development. Hyperoxia impaired pulmonary angiogenesis accompanied by alveolar simplification in neonatal mouse lungs. S-endoglin, phosphorylated Smad2/3 and connective tissue growth factor levels were significantly increased in hyperoxia-exposed mice, while L-endoglin, phosphor-Smad1/5 and platelet-endothelial cell adhesion molecule-1 levels were significantly decreased. Hyperoxia suppressed the tubular growth of human pulmonary microvascular endothelial cells (ECs), and the selective inhibition of ALK5 signalling restored tubular growth. These results indicate that hyperoxia alters the balance in two isoforms of endoglin towards increased S-endoglin and that S-endoglin attenuates TGFß-ALK1-Smad1/5 signalling but stimulates TGFß-ALK5-Smad2/3 signalling in pulmonary ECs, which may lead to impaired pulmonary angiogenesis in developing lungs.


Assuntos
Displasia Broncopulmonar/metabolismo , Endoglina/metabolismo , Hiperóxia/metabolismo , Pulmão/irrigação sanguínea , Neovascularização Patológica/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/complicações , Displasia Broncopulmonar/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Endoteliais/metabolismo , Humanos , Hiperóxia/complicações , Hiperóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Pessoa de Meia-Idade , Fosforilação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteínas Smad/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Mayo Clin Proc ; 95(4): 688-697, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31954524

RESUMO

OBJECTIVE: To assess gene expression in cardiomyocytes isolated from patients with aortic stenosis, hypothesizing that maladaptive remodeling and inflammation-related genes are higher in male vs female patients. PATIENTS AND METHODS: In this study, 34 patients with aortic stenosis undergoing aortic valve replacement from March 20, 2016, through May 24, 2017, at the German Heart Centre in Berlin, Germany, were included. Isolated cardiomyocytes from interventricular septum samples were used for gene expression analysis. Clinical and echocardiographic data were collected preoperatively. RESULTS: Age, body mass index, systolic and diastolic blood pressure, comorbidities, and medication were similar between the 17 male and 17 female patients. The mean ± SD left ventricular end-diastolic diameter (52±9 vs 45±4 mm; P=.007) and posterior wall thickness (14.2±2.5 vs 12.1±1.6 mm; P=.03) were higher in male vs female patients, while ejection fraction was lower in male patients (49%±14% vs 59%±5%; P=.01). Focusing on structural genes involved in the development of cardiac hypertrophy and remodeling, we found that most were expressed higher in male vs female patients. Our modeling analysis revealed that 2 inflammation-related genes, CCN2 and NFKB1, were negatively related to ejection fraction, with this effect being male specific (P=.03 and P=.02, respectively). CONCLUSION: These findings provide novel insight into cardiomyocyte-specific molecular changes related to sex differences in pressure overload and a significant male-specific association between cardiac function and inflammation-related genes. Considering these sex differences may contribute toward a more accurate design of research and the development of more appropriate therapeutic approaches for both male and female patients.


Assuntos
Estenose da Valva Aórtica/metabolismo , Regulação da Expressão Gênica , Pressão Ventricular/fisiologia , Idoso , Estenose da Valva Aórtica/fisiopatologia , Pressão Sanguínea/fisiologia , Índice de Massa Corporal , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Feminino , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Subunidade p50 de NF-kappa B/metabolismo , Fatores Sexuais , Volume Sistólico/fisiologia
12.
Mol Med Rep ; 21(3): 1390-1398, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31922209

RESUMO

Carbon tetrachloride (CCl4) is widely used to induce hepatic fibrosis. Therapeutic agents alleviate hepatic fibrosis by inhibiting signal transducer and activator of transcription 3 (STAT3) activation. To understand the direct effects of CCl4 on STAT3 expression in the liver, the present study incubated cultured hepatocytes expressing connective tissue growth factor (CTGF) with CCl4. Rats exposed to CCl4 for 8 weeks exhibited hepatic fibrosis, which was confirmed through the assessment of plasma biomarkers. Isolated liver samples were used to determine the protein levels of CTGF and STAT3 using western blotting. In addition, STAT3 expression was silenced in α mouse liver 12 (AML­12) cells using small interfering RNA transfection. In addition, a pharmacological inhibitor, stattic, was used to inhibit STAT3 expression. The incubation of AML­12 cells with CCl4 induced a dose­dependent increase in CTGF expression and STAT3 activation. Notably, silymarin, an extract from milk thistle, inhibited these changes in AML­12 cells and the antioxidant tiron produced similar effects. Silencing of STAT3 reduced the CTGF expression promoted by CCl4 in the hepatocytes. Additionally, similar to tiron, stattic inhibited CTGF expression induced by CCl4. In conclusion, CCl4 may activate STAT3 through oxidative stress to promote CTGF expression, which is one of the main factors contributing to the risk of hepatic fibrosis.


Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Tetracloreto de Carbono/toxicidade , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Intoxicação por Tetracloreto de Carbono/patologia , Linhagem Celular , Hepatócitos/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Ratos , Ratos Sprague-Dawley
13.
Elife ; 92020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31913124

RESUMO

The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tamanho Celular , Fatores de Transcrição/metabolismo , Apoptose , Contagem de Células , Divisão Celular , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Biomater Sci ; 8(6): 1658-1668, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-31971526

RESUMO

An abnormal tumor growth induces solid stress in tumors, thus reducing blood perfusion. As a result, the impaired blood perfusion, with dense interstitial matrix in tumors significantly reduces the penetration and efficacy of nanotherapeutics. In this study, we have developed a losartan-loaded polydopamine nanoparticle (PLST) for the enhanced delivery of nanoparticles to tumors and improved photothermal cancer therapy. Losartan, an angiotensin inhibitor, is also able to alleviate the solid stress in tumors. It was laden on polydopamine nanoparticles via π-π stacking and was released upon tumor extracellular acidity. PLST reduced collagen production in vitro along with the lowered expression of profibrotic factors of TGF-ß1, CCN2, and TIMP-1. The in vivo studies reveal that PLST reduced solid stress in tumors, and the amount of PLST accumulated in tumors was enhanced. The efficiency of the photothermal ablation of tumors was significantly enhanced by using PLST.


Assuntos
Angiotensinas/antagonistas & inibidores , Neoplasias da Mama/terapia , Indóis/química , Losartan/administração & dosagem , Fototerapia/métodos , Polímeros/química , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hipertermia Induzida/métodos , Losartan/química , Losartan/farmacologia , Melaninas/química , Camundongos , Nanopartículas , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
15.
Cells ; 9(2)2020 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991791

RESUMO

: During chronic liver injury, hepatic stellate cells (HSC) undergo activation and are the principal cellular source of collagenous scar. In this study, we found that activation of mouse HSC (mHSC) was associated with a 4.5-fold increase in extracellular vesicle (EV) production and that fibrogenic gene expression (CCN2, Col1a1) was suppressed in Passage 1 (P1; activated) mHSC exposed to EVs from Day 4 (D4; relatively quiescent) mHSC but not to EVs from P1 mHSC. Conversely, gene expression (CCN2, Col1a1, αSMA) in D4 mHSC was stimulated by EVs from P1 mHSC but not by EVs from D4 mHSC. EVs from Day 4 mHSC contained only 46 proteins in which histones and keratins predominated, while EVs from P1 mHSC contained 337 proteins and these were principally associated with extracellular spaces or matrix, proteasome, collagens, vesicular transport, metabolic enzymes, ribosomes and chaperones. EVs from the activated LX-2 human HSC (hHSC) line also promoted fibrogenic gene expression in D4 mHSC in vitro and contained 524 proteins, many of which shared identity or had functional overlap with those in P1 mHSC EVs. The activation-associated changes in production, function and protein content of EVs from HSC likely contribute to the regulation of HSC function in vivo and to the fine-tuning of fibrogenic pathways in the liver.


Assuntos
Vesículas Extracelulares/metabolismo , Regulação da Expressão Gênica/genética , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Proteoma/metabolismo , Transdução de Sinais/genética , Animais , Linhagem Celular , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/metabolismo , Vesículas Extracelulares/genética , Ontologia Genética , Células Estreladas do Fígado/enzimologia , Histonas/genética , Histonas/metabolismo , Humanos , Queratinas/genética , Queratinas/metabolismo , Cirrose Hepática/genética , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteômica , Ribossomos/metabolismo , Espectrometria de Massas em Tandem , Fatores de Tempo
16.
Mol Med Rep ; 21(2): 795-805, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974601

RESUMO

The aim of the present study was to investigate the involvement of B cell­activating factor (BAFF) in the pathogenesis of IgA nephropathy by activating the tumor necrosis factor receptor­associated factor 6 (TRAF6)/NF­κB signaling pathway in glomerular mesangial cells. For the clinical analysis, blood, urine and kidney tissue samples were collected from 58 patients diagnosed with primary IgA nephropathy by renal biopsy. For the in vitro study, glomerular mesangial cells were divided into five groups: Control (con)­short hairpin RNA (shRNA) (control group); con­shRNA + BAFF (20 ng/ml); con­shRNA + BAFF + BAFF­RFc chimera protein (500 µg/ml); TRAF6­shRNA; and TRAF6­shRNA + BAFF (20 ng/ml). For the in vivo experiments, 60 Sprague­Dawley rats were randomly divided into four groups: Con­small interfering RNA (siRNA) (control group); con­siRNA + IgA (IgA nephropathy group), BAFF­RFc chimera protein (2 µg/ml) + IgA, and TRAF6­siRNA (0.2 µM) + IgA. Reverse transcription­quantitative PCR was performed to evaluate the mRNA expression levels of TRAF6, connective tissue growth factor (CTGF), fibronectin (FN) and NF­κBP65. Western blot analysis was used to detect the protein expression levels of TRAF6, FN, CTGF and phosphorylated­NF­κBP65 in glomerular mesangial cells and kidney tissues. The results revealed that plasma BAFF levels were positively correlated with the severity of pathological damage in patients with IgA nephropathy. In vitro, BAFF induced the mRNA and protein expression of TRAF6, CTGF, FN and NF­κBP65 in glomerular mesangial cells. After the BAFF­RFc chimera protein was added to inhibit the binding of BAFF and BAFF­receptor (­R), this effect was reduced. In vivo, inhibition of the effects of BAFF via injection with the BAFF­R Fc chimera protein reduced kidney damage in rats suffering from IgA nephropathy. The effect on the expression of signaling pathway­associated proteins was also alleviated. In conclusion, BAFF enhanced the expression of fibroblast factors in the kidneys by activating the TRAF6/NF­κB signaling pathway.


Assuntos
Fator Ativador de Células B/metabolismo , Glomerulonefrite por IGA/metabolismo , Glomérulos Renais/patologia , Células Mesangiais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Fator Ativador de Células B/sangue , Receptor do Fator Ativador de Células B/metabolismo , Biomarcadores/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Creatinina/sangue , Feminino , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Glomerulonefrite por IGA/sangue , Humanos , Masculino , Células Mesangiais/patologia , Pessoa de Meia-Idade , Proteinúria/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição RelA/metabolismo
17.
Exp Mol Pathol ; 113: 104373, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31917285

RESUMO

Asthma is a chronic inflammatory airway disease. Icariside II has been reported to exert anti-inflammatory effect in multiple human diseases. The present study aimed to investigate the effects and mechanisms of Icariside II on airway inflammation and remodeling in asthma. We established an asthma mouse model with ovalbumin (OVA) immunization. Histological analysis using H&E, PAS and Masson staining showed that administration of Icariside II attenuated OVA-induced airway inflammation and remodeling. Icariside II reduced the numbers of total white blood cells and eosinophils in bronchoalveolar lavage fluid (BALF). The levels of interleukin (IL)-4, IL-5, IL-13 and transforming growth factor (TGF)-ß1 in peripheral blood and the expression of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), eotaxin-1, CC-chemokine receptor-3 (CCR-3), Toll-like receptor (TLR)-2 and TLR-4 were significantly down-regulated in lung tissues of OVA-induced mouse model. These results suggested that Icariside II inhibited eosinophil activation and thus decreased eosinophils-induced airway inflammation and remodeling in asthma. Moreover, Icariside II suppressed TGF-ß1-induced cell proliferation, migration, and CTGF expression in airway smooth muscle cells (ASMCs). In both OVA-induced mouse model of asthma and TGF-ß1-induced ASMCs, Icariside II decreased IκBα degradation, nuclear translocation of NF-κB p65 and STAT3 phophorylation, indicating an inactivation of NF-κB and STAT3 in the presence of Icariside II. Therefore, we demonstrate that Icariside II attenuates eosinophils-induced airway inflammation and remodeling in asthmatic mice and inhibits TGF-ß1-induced cell proliferation and migration in ASMCs via suppressing NF-κB and STAT3 signalings.


Assuntos
Remodelação das Vias Aéreas , Asma/tratamento farmacológico , Eosinófilos/patologia , Flavonoides/uso terapêutico , Inflamação/patologia , Pulmão/patologia , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Asma/metabolismo , Asma/patologia , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Flavonoides/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
18.
J Cell Physiol ; 235(2): 979-992, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267533

RESUMO

This study is carried out to investigate the role of microRNA-26a (miR-26a) in cartilage injury and chondrocyte proliferation and apoptosis in rats with rheumatoid arthritis (RA) by regulating expression of CTGF. A rat model of RA induced by type II collagen was established. The rats were assigned into normal, RA, RA + mimics negative control (NC), and RA + miR-26a mimics groups, and the cells were classified into blank, mimics NC, and miR-26a mimics groups. The degree of secondary joint swelling and arthritis index, expression of miR-26a, pathological changes, proliferation and apoptosis of chondrocytes, and expression of CTGF, interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α, Bax, and Bcl-2 were also determined through a series of experiments. The targeting relationship between miR-26a and CTGF was verified. Initially, downregulated miR-26a was found in cartilage tissues and inflammatory articular chondrocytes of RA rats. In addition, CTGF was determined as a direct target gene of miR-26a, and upregulation of miR-26a inhibited CTGF expression in cartilage tissues of RA rats. Furthermore, upregulation of miR-26a reduced swelling and inflammation of joints, inhibited cartilage damage, apoptosis of chondrocytes, inflammatory injury, promotes proliferation, and inhibited apoptosis of inflammatory articular chondrocytes, which may be correlated with the targeting inhibition of CTGF expression. Collectively, the results demonstrate that upregulating the expression of miR-26a could attenuate cartilage injury, stimulate the proliferation, and inhibit apoptosis of chondrocytes in RA rats.


Assuntos
Artrite Reumatoide/induzido quimicamente , Cartilagem/lesões , Proliferação de Células/fisiologia , Condrócitos/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , MicroRNAs/metabolismo , Animais , Apoptose/fisiologia , Cartilagem/metabolismo , Colágeno Tipo II/toxicidade , Fator de Crescimento do Tecido Conjuntivo/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley
19.
Am J Pathol ; 190(1): 206-221, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610176

RESUMO

Tumor stroma resembles a fibrotic microenvironment, being characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs). In wild-type mice injected with melanoma cells, we show that the stem cell transcription factor Sox2 is expressed by tumor cells and induced in CAFs derived from synthetic fibroblasts. These fibroblasts were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase under the control of a fibroblast-specific promoter/enhancer. Conversely, fibroblast activation was impaired in mice with a fibroblast-specific deletion of cellular communication network 2 (Ccn2), associated with reduced expression of α-smooth muscle actin and Sox2. Multipotent Sox2-expressing skin-derived precursor (SKP) spheroids were cultured from murine back skin. Using lineage tracing and flow cytometry, approximately 40% of SKPs were found to be derived from type I collagen-lineage cells and acquired multipotency in culture. Inhibition of mechanotransduction pathways prevented myofibroblast differentiation of SKPs and expression of Ccn2. In SKPs deleted for Ccn2, differentiation into a myofibroblast, but not an adipocyte or neuronal phenotype, was also impaired. In human melanoma, CCN2 expression was associated with a profibrotic integrin alpha (ITGA) 11-expressing subset of CAFs that negatively associated with survival. These results suggest that synthetic dermal fibroblasts are plastic, and that CCN2 is required for the differentiation of dermal progenitor cells into a myofibroblast/CAF phenotype and is, therefore, a therapeutic target in melanoma.


Assuntos
Fibroblastos Associados a Câncer/patologia , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Fibroblastos/patologia , Fibrose/patologia , Melanoma Experimental/patologia , Pele/patologia , Células-Tronco/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Diferenciação Celular , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Fibrose/metabolismo , Humanos , Mecanotransdução Celular , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Knockout , Prognóstico , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Pele/metabolismo , Células-Tronco/metabolismo , Taxa de Sobrevida , Microambiente Tumoral
20.
Biochim Biophys Acta Mol Basis Dis ; 1866(1): 165560, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31648019

RESUMO

Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1ß and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.


Assuntos
Humor Aquoso/metabolismo , Glaucoma/metabolismo , Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Drenagem/métodos , Feminino , Fibronectinas/metabolismo , Humanos , Pressão Intraocular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Hipertensão Ocular/metabolismo , Proteína Smad3/metabolismo , Malha Trabecular/metabolismo
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