RESUMO
Phenotypic transformation from adventitial fibroblasts (AFs) to myofibroblasts (MFs) is critical for vascular remodeling. Septin 2 was found to be downregulated during the differentiation of AFs to MFs induced by angiotensin II (Ang II); however, the role of septin 2 in this process is still unknown. In this study, we investigate whether septin 2 contributes to the adventitial MF phenotypic modulation caused by Ang II. The decreased level of septin 2 and the increased expression of α-smooth muscle actin (α-SMA), a marker of MFs, were readily observed in Ang II-stimulated MF differentiation. After gene transfer of septin 2, the expression of α-SMA was markedly decreased and the MF migration response to Ang II was inhibited. Furthermore, the inhibition of RhoA, another molecule involved in MF phenotypic modulation, decreased the motility of MFs and the expression of septin 2 triggered in Ang II. Finally, transfection of septin 2 rescued the level of acetyl-α-tubulin in MFs. These findings demonstrate that, as a downstream molecule of RhoA, septin 2 blunted the responses of AFs to Ang II by protecting α-tubulin acetylation, which suggests that septin 2 may serve as a potential therapeutic target for vascular injury.
Assuntos
Actinas/metabolismo , Adenoviridae/genética , Túnica Adventícia/efeitos dos fármacos , Angiotensina II/farmacologia , Aorta Torácica/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Vetores Genéticos , Miofibroblastos/efeitos dos fármacos , Septinas/metabolismo , Transfecção/métodos , Acetilação , Actinas/genética , Túnica Adventícia/metabolismo , Animais , Aorta Torácica/metabolismo , Células Cultivadas , Masculino , Miofibroblastos/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional , Ratos Sprague-Dawley , Septinas/genética , Transdução de Sinais/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Regulação para Cima , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Apelin has been proved to be a critical mediator of vascular function and homeostasis. Here, we investigated roles of Apelin in aortic remodeling and fibrosis in rats with transverse aortic constriction (TAC). Male Sprague-Dawley rats were subjected to TAC and then randomized to daily deliver Apelin-13 (50µg/kg) or angiotensin type 1 receptor (AT1) blocker Irbesartan (50mg/kg) for 4 weeks. Pressure overload resulted in myocardial hypertrophy, systolic dysfunction, aortic remodeling and adventitial fibrosis with reduced levels of Apelin in ascending aortas of rat after TAC compared with sham-operated group. These changes were associated with marked increases in levels of miRNA-122, TGFß1, CTGF, NFAT5, LGR4, and ß-catenin. More importantly, Apelin and Irbesartan treatment strikingly prevented TAC-mediated aortic remodeling and adventitial fibrosis in pressure overloaded rats by blocking AT1 receptor and miRNA-122 levels and repressing activation of the CTGF-NFAT5 and LGR4-ß-catenin signaling. In cultured primary rat adventitial fibroblasts, exposure to angiotensin II (100nmolL-1) led to significant increases in cellular migration and levels of TGFß1, CTGF, NFAT5, LGR4 and ß-catenin, which were effectively reversed by pre-treatment with Apelin (100nmolL-1) and miRNA-122 inhibitor (50nmolL-1). In conclusion, Apelin counterregulated against TAC-mediated ascending aortic remodeling and angiotensin II-induced promotion of cellular migration by blocking AT1 receptor and miRNA-122 levels and preventing activation of the TGFß1-CTGF-NFAT5 and LGR4-ß-catenin signaling, ultimately contributing to attenuation of aortic adventitial fibrosis. Our data point to Apelin as an important regulator of aortic remodeling and adventitial fibrosis and a promising target for vasoprotective therapies.
Assuntos
Túnica Adventícia/patologia , Aorta/patologia , Aneurisma da Aorta Torácica/patologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Animais , Aneurisma da Aorta Torácica/metabolismo , Cardiomegalia/metabolismo , Células Cultivadas , Fibrose , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Remodelação Vascular , Remodelação Ventricular , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
AIM: To investigate whether apocynin, a NADPH oxidase inhibitor, produced cardioproteictive effects in Ang II-induced hypertensive mice, and to elucidate the underlying mechanisms. METHODS: C57BL/6 mice were subcutaneously infused Ang II for 4 weeks to mimic cardiac remodeling and fibrosis. Concomitantly the mice were administered apocynin (100 mg·kg(-1)·d(-1)) or/and the aldosterone receptor blocker eplerenone (200 mg·kg(-1)·d(-1)) via gavage for 4 weeks. Systolic blood pressure (SBP) and heart rate were measured, and transthoracic echocardiography was performed. For in vitro study, cardiac fibroblasts were treated with Ang II (10(-7) mol/L) in the presence of apocynin (10(-5) mol/L) or/and eplerenone (10(-5) mol/L). Immunohistochemistry and Western blotting were used to quantify the expression levels of NADPH oxidase and osteopontin (OPN) proteins in the cells. RESULTS: Both apocynin and eplerenone significantly decreased SBP, and markedly improved diastolic dysfunction in Ang II-induced hypertensive mice, accompanied with ameliorated oxidative stress and cardiac fibrosis. In the Ang II-treated cardiac fibroblasts, the expression levels of NOX4 and OPN proteins were markedly upregulated. Both Apocynin and eplerenone significantly suppressed the increased expression levels of NOX4 and OPN proteins in the Ang II-treated cells. In all the experiments, apocynin and eplerenone produced comparable effects. Co-administration of the two agents did not produce synergic effects. CONCLUSION: Apocynin produces cardioproteictive effects comparable to those of eplerenone. The beneficial effects of apocynin on myocardial oxidative stress and cardiac fibrosis might be mediated partly through a pathway involving NADPH oxidase and OPN.
Assuntos
Acetofenonas/uso terapêutico , Angiotensina II/farmacologia , Cardiomegalia/prevenção & controle , Cardiotônicos/uso terapêutico , Diástole/efeitos dos fármacos , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Acetofenonas/administração & dosagem , Acetofenonas/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiotônicos/administração & dosagem , Cardiotônicos/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Diástole/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Frequência Cardíaca/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismoRESUMO
OBJECTIVE: To examine the role of perivascular adipose tissue (PVAT)-derived factors in the regulation of adventitial fibroblast (AF) function in vitro and in vivo. METHODS AND RESULTS: PVAT is an active component of blood vessels. Bioactive substances released from PVAT play regulatory roles in vascular function. However, their effects on vascular AFs remain unclear. PVAT-conditioned medium stimulated AF migration using a transwell technique, and differentiation was evaluated by α-smooth muscle-actin induction. We identified the secretome of PVAT by liquid chromatography-tandem mass spectrometry. One of the major secretory proteins in PVAT is complement 3 (C3). The C3 antagonist and neutralizing antibody attenuated PVAT-conditioned medium-induced AF migration and differentiation. Similar to PVAT-conditioned medium, C3 recombinant protein stimulated AF migration and differentiation. We demonstrated that the effects of PVAT-derived C3 were mediated by the c-Jun N-terminal kinase pathway. Moreover, we found morphological changes in perivascular adipocytes and increased expression of C3 in PVAT that was tightly associated with adventitial thickening and myofibroblast clustering around PVAT in deoxycorticosterone acetate-salt hypertensive rats. CONCLUSIONS: PVAT-derived C3 stimulated AF migration and differentiation via the c-Jun N-terminal kinase pathway. PVAT-derived C3 may contribute to adventitial remodeling in a deoxycorticosterone acetate-salt hypertensive model.
Assuntos
Tecido Adiposo/metabolismo , Complemento C3/metabolismo , Tecido Conjuntivo/metabolismo , Desoxicorticosterona/análogos & derivados , Fibroblastos/metabolismo , Hipertensão/metabolismo , Comunicação Parácrina , Cloreto de Sódio na Dieta , Tecido Adiposo/patologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Diferenciação Celular , Movimento Celular , Forma Celular , Células Cultivadas , Cromatografia Líquida , Tecido Conjuntivo/patologia , Meios de Cultura/metabolismo , Modelos Animais de Doenças , Fibroblastos/patologia , Hipertensão/etiologia , Hipertensão/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
AIM: To identify proteins that could potentially be involved in adventitial remodeling in vascular adventitial fibroblasts (AFs) from spontaneously hypertensive rats (SHR). METHODS: AFs were isolated from thoracic aortas of 4-, 8-, 16-, and 24-week-old male SHR and Wistar-Kyoto (WKY) rats and cultured to passage 4. Proteomic differential expression profiles between SHR-AFs and WKY-AFs were investigated using 2-D electrophoresis (2-DE), whereas gel image analysis was processed using Image Master 2D Platinum. Protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Expression levels of annexin A1 in AFs and aortas from SHR and WKY rats were detected with Western blotting and immunofluorescence techniques. RESULTS: In 4-, 8-, 16-, and 24-week-old SHR-AFs, 49, 59, 54, and 69 protein spots were found to have significant differences from the age-matched WKY-AFs. Fourteen spots with the same changes in patterns were analyzed in 4-, 8-, 16-, and 24-week-old SHR-AFs with mass spectrometry. Except for cytoskeleton proteins such as tubulin beta 5, it was found that annexin A1, translation elongation factor Tu, endoplasmic reticulum protein 29 and calcium-binding protein 1 were expressed in vascular AFs and their levels changed significantly in SHR-AFs compared with those in WKY-AFs. A decrease in annexin A1 in SHR-AFs was confirmed with Western blotting and immunofluorescence staining at the cell and tissue levels. CONCLUSION: The application of proteomic techniques revealed a number of novel proteins involved in adventitial remodeling of AFs from SHR, which provide new mechanisms responsible for the occurrence and development of hypertension and potential targets for influencing vascular remodeling in hypertension.
Assuntos
Fibroblastos/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas/metabolismo , Envelhecimento/metabolismo , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/patologia , Hipertensão/patologia , Técnicas In Vitro , Masculino , Músculo Liso Vascular/patologia , Proteômica , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Especificidade da EspécieRESUMO
AIMS: N-acetylcysteine (NAC) has a protective effect against vascular dysfunction by decreasing the level of reactive oxygen species (ROS) in experimental and human hypertension. This study was designed to examine whether NAC would relax vascular rings in vitro via nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway, extracellular Ca2+ and/or K+ channels. MAIN METHODS: Rat aortic arteries were mounted in an organ bath, contracted with 0.1, 0.5 or 1 micromol/L phenylephrine to plateau, and the vasodilatory effect of NAC was examined in the absence or presence of ROS scavengers, inhibitors of NO-cGMP pathway or K+ channels. Vascular smooth muscle cells (VSMCs) were loaded with a calcium sensitive fluorescent dye fluo-3 AM, and [Ca2+](i) was determined with laser-scanning confocal microscopy. KEY FINDINGS: NAC (0.1-4 mmol/L) dose-dependently relaxed rat aorta pre-contracted with phenylephrine. Endothelium removal, endothelial nitric oxide synthase inhibitor N(omega)-Nitro-l-arginine (L-NNA) (100 micromol/L) or soluble guanylyl cyclase (sGC) inhibitor (ODQ) (10 micromol/L) did not affect NAC-induced vasodilation. In contrast, NAC-induced vasodilation was blunted after extracellular calcium was removed and calcium imaging showed that 4 mmol/L NAC quickly decreased [Ca2+](i) in fluo-3 AM loaded VSMCs. NAC-induced vasodilation was significantly reduced in the presence of voltage-gated K+ channels (Kv) inhibitor 4-aminopyridine (4-AP). SIGNIFICANCE: The vasodilatory effect of NAC may be explained at least partly by activation of voltage-gated K+ channels.
Assuntos
Acetilcisteína/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Vasodilatação/efeitos dos fármacos , Animais , Cálcio/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Guanilato Ciclase/antagonistas & inibidores , Contração Muscular/efeitos dos fármacos , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Oxidiazóis/farmacologia , Fenilefrina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Proteína Quinase C/metabolismo , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Vasoconstritores/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
1. Angiotensin (Ang) II-mediated oxidative stress may be important in enhanced adventitial fibroblast collagen formation. The aim of the present study was to test whether PPAR-gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ2) and pioglitazone could alter AngII-induced collagen type I formation in vascular adventitial fibroblasts via reactive oxygen species (ROS). 2. Vascular adventitial fibroblasts were isolated from rat thoracic aortas of male Sprague-Dawley rats and treated with different concentrations of AngII for different periods of time. The expression of collagen type I induced by AngII was examined by western blot. Expression of PPAR-gamma mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR). Intracellular ROS generation was measured by flow cytometry. Activation of transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1 was assessed by an electrophoretic mobility shift assay. 3. Angiotensin II increased expression of collagen type I in a time- and dose-dependent manner in adventitial fibroblasts. In addition, AngII stimulated intracellular generation of ROS in adventitial fibroblasts. Pretreatment of cells with 15d-PGJ2 and pioglitazone attenuated collagen type I expression and generation of ROS induced by AngII, respectively. Moreover, we observed that N-acetylcysteine inhibited collagen type I expression induced by AngII as did the PPAR-gamma agonists. Angiotensin II treatment activated the redox-sensitive transcription factors NF-kappaB and AP-1, whereas pretreatment with 15d-PGJ2 and pioglitazone reduced the AngII-induced DNA-binding activity of NF-kappaB but not AP-1. 4 Our data demonstrate that the PPAR-gamma agonists 15d-PGJ2 and pioglitazone attenuate AngII-mediated collagen type I expression in adventitial fibroblasts, which may be mediated by the modulation of ROS release and the redox-sensitive transcription factor NF-kappaB.
Assuntos
Angiotensina II/metabolismo , Colágeno Tipo I/metabolismo , Tecido Conjuntivo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/agonistas , Prostaglandina D2/análogos & derivados , Tiazolidinedionas/farmacologia , Acetilcisteína/farmacologia , Animais , Células Cultivadas , Tecido Conjuntivo/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Sequestradores de Radicais Livres/farmacologia , Masculino , NF-kappa B/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Pioglitazona , Prostaglandina D2/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismoRESUMO
Our previous study demonstrated that TGF-beta1 could induce the differentiation of vascular adventitial fibroblasts (AFs) to myofibroblasts (MFs). The aim of this study was to identify the genes which might be responsible for the cell phenotypic change using genechips. Cultured rat AFs were treated with TGF-beta1 (10 ng/ml) for 0 min, 5 min, 15 min, 2 h, 12 h and 24 h, respectively. Then the cells were gathered to prepare total RNA. We examined TGF-beta1-induced gene expression profiling using Affymetrix oligonucleotide microarrays and analyzed data by GCOS1.2 software. Moreover, expressional similarity was measured by hierarchical clustering. Some of genechip results were confirmed by real-time quantitative RT-PCR. Microarray analysis identified 2121 genes with a 2-fold change or above after TGF-beta1 stimulation. 1318 genes showed a greater than 2-fold increase and 761 genes were reduced 2 folds or more at mRNA levels, whereas a small portion of the total regulated genes (42 genes) displayed dynamically up- and down-regulated pattern. Genes were further segregated for early (peak at 5 min, 15 min and/or 2 h), late (peak at 12 h and/or 24 h), and sustained (2-fold change or above at five time points) temporal response groups according to the time of their peak expression level. Among 1318 up-regulated genes, 333 genes (25.3%) responded rapidly to TGF-beta1 and 159 genes (12.1%) responded in a sustained manner. Most genes (826, 62.6%) were regulated at 12 h or later. For the 761 down-regulated genes, numbers of early and late responsive genes were 335 (44%) and 267 (36.1%), respectively. There were also 159 genes, 19.9% of total down-regulated genes, decreased at five time points treated by TGF-beta1. The results suggested that the gene expressions of secreted phosphoprotein 1 (APP1) and Rho-associated coiled-coil forming kinase 2 (ROCK2) had the same trends as alpha-smooth muscle-actin, a marker of MF differentiation. In addition, the gene expression of potassium voltage-gated channel, Shal-related family and member 2 (KCND2) was up-regulated. Furthermore, it was found that endothelin 1 (EDN1), some complement components, NADPH oxidase 4 (NOX4) and NAD(P)H dehydrogenase, quinone 1 (NQO1) might be involved in MF differentiation. Using microarrary technique, we confirmed some genes that have been identified by other techniques were implicated in MF differentiation and observed new genes involved in this process. Our results suggest that gene expression profiling study is helpful in identifying genes and pathways potentially involved in cell differentiation.