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Int. j. morphol ; 40(3): 760-767, jun. 2022. ilus
Article in English | LILACS-Express | LILACS | ID: biblio-1385669


SUMMARY: Atherosclerosis is a complex disease whose pathogenesis includes endothelial activation, accumulation of lipids in the subendothelium, formation of foam cells, fat bands and formation of atherosclerotic plaque. These complex mechanisms involve different cell populations in the intimate sub-endothelium, and the S-100 protein family plays a role in a number of extracellular and intracellular processes during the development of atherosclerotic lesions. The aim of this study was to determine the phenotypic characteristics of smooth muscle cells and the consequent expression of S100 protein in atherosclerotic altered coronary arteries in advanced stages of atherosclerosis. 19 samples of right atherosclerotic coronary arteries in stages of fibro atheroma (type V lesion) and complicated lesions (type VI lesion) have been analyzed. According to the standard protocol, the following primary antibodies have been used in the immunohistochemical analysis: a-smooth muscle actin (α-SMA), vimentin and S-100 protein. All analyzed samples have been in advanced stages of atherosclerosis, fibro atheroma (stage V lesions) and complicated lesions (type VI lesions). Most of them have had the structure of a complicated lesion with atheroma or fibro atheroma as a basis, subsequently complicated by disruption (subtype VI a), hemorrhage (subtype VI b) or thrombosis (subtype VI c), as well as by the presence of several complications on the same sample. Marked hypocellularity is present in the subendothelium of plaques. Cell population at plaque margins is characterized by immunoreactivity to α-SMA, vimentin, and S100 protein. Some of these cells accumulate lipids and look like foam cells. In the cell population at the margins of the plaques, smooth muscle cells of the synthetic phenotype are present, some of which accumulate lipids and demonstrate S100 immunoreactivity. Summarizing numerous literature data and our results, we could assume that smooth muscle cells, due to their synthetic and proliferative activity in the earlier stages of pathogenesis, as well as the consequent expression of S100 protein, could accumulate lipids in the earlier stages of atherosclerosis which, in advanced stages analyzed in this study, result in immunoreactivity of foam cells of smooth muscle origin to S100 protein.

RESUMEN: La aterosclerosis es una enfermedad compleja cuya patogenia incluye activación endotelial, acumulación de lípidos en el subendotelio, formación de células espumosas, bandas grasas y formación de placa aterosclerótica. Estos complejos mecanismos involucran diferentes poblaciones celulares en el subendotelio íntimo, y la familia de proteínas S-100 juega un papel en varios procesos extracelulares e intracelulares durante el desarrollo de lesiones ateroscleróticas. El objetivo de este estudio fue determinar las características fenotípicas de las células de músculo liso y la consecuente expresión de la proteína S100 en arterias coronarias alteradas ateroscleróticas en estadios avanzados de aterosclerosis. Se analizaron 19 muestras de arterias coronarias ateroscleróticas derechas en estadios de fibroateroma (lesión tipo V) y lesiones complicadas (lesión tipo VI). Según el protocolo estándar, en el análisis inmunohistoquímico se utilizaron los siguientes anticuerpos primarios: α-actina de músculo liso (α-SMA), vimentina y proteína S-100. Todas las muestras analizadas han estado en estadios avanzados de aterosclerosis, fibroateroma (lesiones estadio V) y lesiones complicadas (lesiones tipo VI). La mayoría de ellos han tenido la estructura de una lesión complicada con ateroma o fibroateroma como base, complicada posteriormente por disrupción (subtipo VI a), hemorragia (subtipo VI b) o trombosis (subtipo VI c), así como por la presencia de varias complicaciones en la misma muestra. La hipocelularidad marcada estaba presente en el subendotelio de las placas. La población celular en los márgenes de la placa se caracterizaba por inmunorreactividad a α-SMA, vimentina y proteína S100. Algunas de estas células acumulan lípidos y parecen células espumosas. En la población celular en los márgenes de las placas, estaban presentes las células de músculo liso de fenotipo sintético, algunas de las cuales acumulaban lípidos y mostraban inmunorreactividad S100. Resumiendo numerosos datos de la literatura y nuestros resultados, podríamos suponer que las células del músculo liso, debido a su actividad sintética y proliferativa en las primeras etapas de la patogénesis, así como la consecuente expresión de la proteína S100, podrían acumular lípidos en las primeras etapas de la aterosclerosis que, en estadios avanzados analizados en este estudio, dan como resultado inmunorreactividad de células espumosas de origen muscular liso a la proteína S100.

Humans , Coronary Artery Disease/metabolism , S100 Proteins/metabolism , Myocytes, Smooth Muscle/metabolism , Phenotype
Biol. Res ; 52: 59, 2019. graf
Article in English | LILACS | ID: biblio-1100911


OBJECTIVES: In varicose veins, vascular smooth muscle cells (VSMCs) often shows phenotypic transition and abnormal proliferation and migration. Evidence suggests the FOXC2-Notch pathway may be involved in the pathogenesis of varicose veins. Here, this study aimed to explore the role of long non-coding RNA FOXC2-AS1 (FOXC2 antisense RNA 1) in phenotypic transition, proliferation, and migration of varicose vein-derived VSMCs and to explore whether the FOXC2-Notch pathway was involved in this process. METHODS: The effect of FOXC2-AS1 on the proliferation and migration of human great saphenous vein smooth muscle cells (SV-SMCs) was analyzed using MTT assay and Transwell migration assay, respectively. The levels of contractile marker SM22α and synthetic marker osteopontin were measured by immunohistochemistry and Western blot to assess the phenotypic transition. RESULTS: The human varicose veins showed thickened intima, media and adventitia layers, increased synthetic VSMCs, as well as upregulated FOXC2-AS1 and FOXC2 expression. In vitro assays showed that FOXC2-AS1 overexpression promoted phenotypic transition, proliferation, and migration of SV-SMCs. However, the effect of FOXC2-AS1 overexpression could be abrogated by both FOXC2 silencing and the Notch signaling inhibitor FLI-06. Furthermore, FOXC2-AS1 overexpression activated the Notch pathway by upregulating FOXC2. CONCLUSION: FOXC2-AS1 overexpression promotes phenotypic transition, proliferation, and migration of SV-SMCs, at least partially, by activating the FOXC2-Notch pathway.

Humans , Saphenous Vein/metabolism , Cell Movement/physiology , Myocytes, Smooth Muscle/metabolism , Cell Proliferation/physiology , Forkhead Transcription Factors/metabolism , Phenotype , Saphenous Vein/pathology , Signal Transduction , Up-Regulation , Cells, Cultured , Myocytes, Smooth Muscle/pathology
Rev. Assoc. Med. Bras. (1992) ; 64(4): 361-367, Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-956455


SUMMARY OBJECTIVE: This study aims at investigating the expressions of TOLL-like receptor 4 (TLR-4) and matrix metalloproteinase 9 (MMP-9)/ tissue inhibitor of metalloproteinase 1 (TIMP-1) in pulmonary blood vessels with chronic obstructive pulmonary disease (COPD) and their relationships with pulmonary vascular remodelling (PVR). METHODS: 60 para-tumour tissues were divided into the COPD group and the control group (n=30); the inflammations, pulmonary artery wall area/total artery area (WA%), and wall thickness/vascular outer diameter (WT%) were compared. The expressions of TLR-4, MMP-9/TIMP-1, and PCNA in pulmonary vascular smooth muscle cells were detected, and their relationships with PVR were then analysed. RESULTS: The inflammations (1.6±0.8), WA% (44.0±6.4), and WT% (27.3±3.3) in the COPD group were higher than in the control group (0.3±0.5, 26.1±2.8, 15.6±1.8), and the expressions of TLR-4 (31.4±147) and MMP-9/TIMP-1 (2.2±2.6) were increased compared to the control group (4.7±4.5, 1.9±12). Correlation analysis: TLR-4 and MMP-9/TIMP-1 were positively correlated with the inflammations (r=0.18, P<0.01), WA% (r=0.68, P<0.01), and WT% (r=0.73, P<0.01), as well as positively correlated with the expression of PCNA (r=0.44, P<0.01); the upregulation of TLR-4 was positively correlated with the expressions of MMP-9 and TIMP-1. CONCLUSIONS: The upregulation of TLR-4 in the pulmonary arterial smooth muscle cells of COPD patients could promote the inflammations and the MMP-9 expression, thus causing abnormal degradation of extracellular matrix, so it played an important role in the process of PVR.

RESUMO OBJETIVO: Este estudo tem como objetivo investigar as expressões de TOLL-like receptor 4 (TLR-4) e metaloproteinase 9 da matriz (MMP-9)/inibidor de tecido da metaloproteinase 1 (TIMP-1) em vasos sanguíneos pulmonares com doença pulmonar obstrutiva crônica (DPOC) e suas relações com o remodelamento vascular pulmonar (PVR). MÉTODOS: Sessenta tecidos paratumorais foram divididos em grupo COPD e o grupo controle (n = 30). Foram comparadas as inflamações, área da parede da artéria pulmonar/área da artéria total (WA%) e espessura da parede/diâmetro externo vascular (WT%). As expressões de TLR-4, MMP-9/TIMP-1 e PCNA em células de músculo liso vascular pulmonar foram detectadas, e suas relações com PVR foram então analisadas. RESULTADOS: As inflamações (1,6 ± 0,8), WA% (44,0 ± 6,4) e WT% (27,3 ± 3,3) no grupo COPD foram maiores que no grupo controle (0,3 ± 0,5; 26,1 ± 2,8; 15,6 ± 1,8). E as expressões de TLR-4 (31,4 ± 14,7) e MMP-9/TIMP-1 (2,2 ± 2,6) foram aumentadas em relação ao grupo controle (4,7 ± 4,5, 1,9 ± 1,2). Na análise de correlação, TLR-4 e MMP-9/TIMP-1 foram positivamente correlacionadas com as inflamações (r = 0,18; P <0,01), WA% (r = 0,68; P <0,01) e WT% (r = 0,73; P <0,01), bem como correlacionadas positivamente com a expressão de PCNA (r = 0,44; P <0,01). A elevação da TLR-4 foi correlacionada positivamente com as expressões de MMP-9 e TIMP-1. CONCLUSÕES: A regulação positiva do TLR-4 nas células do músculo liso arterial pulmonar de pacientes com DPOC poderia promover as inflamações e a expressão de MMP-9, causando assim uma degradação anormal da matriz extracelular, por isso desempenhou um papel importante no processo de PVR.

Humans , Male , Pulmonary Artery/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Matrix Metalloproteinase 9/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Toll-Like Receptor 4/metabolism , Vascular Remodeling , Reference Values , Immunohistochemistry , Case-Control Studies , Vital Capacity/physiology , Forced Expiratory Volume/physiology , Pulmonary Disease, Chronic Obstructive/physiopathology , Myocytes, Smooth Muscle/metabolism , Hematoxylin , Lung/blood supply , Middle Aged
Acta cir. bras ; 32(5): 350-358, May 2017. tab, graf
Article in English | LILACS | ID: biblio-837705


Abstract Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.

Animals , Male , Flavonoids/pharmacology , Chromones/pharmacology , Fibronectins/metabolism , MAP Kinase Signaling System/drug effects , Collagen Type III/metabolism , Connective Tissue Growth Factor/pharmacology , Pulmonary Artery/cytology , Gene Expression/drug effects , Cells, Cultured , Gene Expression Regulation , Fibronectins/genetics , Rats, Sprague-Dawley , Phosphatidylinositol 3-Kinases/metabolism , Models, Animal , Collagen Type III/genetics , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Connective Tissue Growth Factor/metabolism
Yonsei Medical Journal ; : 373-381, 2016.
Article in English | WPRIM | ID: wpr-147352


PURPOSE: Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. MATERIALS AND METHODS: Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. RESULTS: We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. CONCLUSION: Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.

Animals , Male , Rats , Caspase 3/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Flavonoids/pharmacology , Glucose/metabolism , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 3 , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases , Protein Kinase Inhibitors/pharmacology , Rats, Inbred OLETF , Rats, Long-Evans , Reactive Oxygen Species/metabolism , Rutin/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
Rev. bras. cir. cardiovasc ; 30(6): 644-649, Nov.-Dec. 2015. tab, graf
Article in English | LILACS | ID: lil-774551


ABSTRACT α-smooth muscle actin, encoded by ACTA2 gene, is an isoform of the vascular smooth muscle actins, typically expressed in the vascular smooth muscle cells contributing to vascular motility and contraction. ACTA2 gene mutations cause a diversity of diffuse vasculopathies such as thoracic aortic aneurysms and dissections as well as occlusive vascular diseases, including premature coronary artery disease and ischemic stroke. Dynamics of differentiation-specific α-smooth muscle actin in arterial smooth muscle cells and proliferation of the proteins have been well described. Although a variety of research works have been undertaken in terms of modifications of α-smooth muscle actin and mutations of ACTA2 gene and myosin, the underlying mechanisms towards the pathological processes by way of gene mutations are yet to be clarified. The purpose of the present article is to describe the phenotypes of α-smooth muscle actin and implications of ACTA2 mutations in vasculopathies in order to enhance the understanding of potential mechanisms of aortic and coronary disorders.

Humans , Actins/genetics , Aortic Diseases/metabolism , Coronary Disease/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Actins/metabolism , Aortic Diseases/genetics , Coronary Disease/genetics , Gene Expression , Muscle Contraction/physiology , Mutation/genetics , Phenotype
Ciênc. Saúde Colet. (Impr.) ; 20(3): 841-850, marc. 2015. tab, graf
Article in English | LILACS | ID: lil-742239


This study sought to confirm the structure and to investigate the psychometric properties of an experimental Portuguese version of the York Cardiac Beliefs Questionnaire (YCBQ) in a general population sample. It also set out to identify the prevalent misconceptions in the community and to assess the differences according to socio-demographic characteristics. It involved a cross-sectional survey in which both test and validation samples were collected (n = 476), including participants aged between 18 and 40, recruited via e-mail and social networks. The Confirmatory Factor Analysis on both samples suggested a shorter, three factor version of the YCBQ. Also, misconceptions differed significantly according to sociodemographic variables. The validation of the YCBQ for samples in the community constitutes an important starting point to promote research on misconceptions held in the community by specific groups, as well as to provide key points for health promotion.

Este estudo teve como objetivo confirmar a estrutura e investigar as propriedades psicométricas de uma versão experimental portuguesa do York Cardiac Beliefs Questionnaire numa amostra da população geral; identificar as crenças erróneas mais fortes na comunidade; e avaliar as diferenças de acordo com características sociodemográficas. Trata-se de um estudo transversal com uma amostra de teste e outra de validação, incluindo um total de 476 participantes, com idade entre 18 e 40 anos, recrutados via e-mail e nas redes sociais. A Análise Fatorial Confirmatória em ambas as amostras indicou uma versão reduzida do YCBQ de três factores. As crenças erróneas diferiram significativamente de acordo com as variáveis sociodemográficas. A validação do YCBQ para amostras da comunidade constitui um importante ponto de partida para promover a investigação sobre crenças erróneas em grupos específicos da comunidade, assim como fornecer indicadores relevantes para a promoção da saúde.

Humans , Calcium/metabolism , Inflammation/metabolism , /metabolism , Membrane Proteins/metabolism , Muscle, Smooth/metabolism , Neoplasm Proteins/metabolism , Respiratory System/embryology , Tumor Necrosis Factor-alpha/metabolism , Binding Sites , Calcium Channels/metabolism , Cell Membrane/metabolism , Cells, Cultured , Myocytes, Smooth Muscle/metabolism , Sarcoplasmic Reticulum/metabolism
Article in English | WPRIM | ID: wpr-57306


Scoparone, which is a major constituent of Artemisia capillaries, has been identified as an anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant and anti-inflammatory drug, and it is used for the traditional treatment of neonatal jaundice. Therefore, we hypothesized that scoparone could suppress the proliferation of VSMCs by interfering with STAT3 signaling. We found that the proliferation of these cells was significantly attenuated by scoparone in a dose-dependent manner. Scoparone markedly reduced the serum-stimulated accumulation of cells in the S phase and concomitantly increased the proportion of cells in the G0/G1 phase, which was consistent with the reduced expression of cyclin D1, phosphorylated Rb and survivin in the VSMCs. Cell adhesion markers, such as MCP-1 and ICAM-1, were significantly reduced by scoparone. Interestingly, this compound attenuated the increase in cyclin D promoter activity by inhibiting the activities of both the WT and active forms of STAT3. Similarly, the expression of a cell proliferation marker induced by PDGF was decreased by scoparone with no change in the phosphorylation of JAK2 or Src. On the basis of the immunofluorescence staining results, STAT3 proteins phosphorylated by PDGF were predominantly localized to the nucleus and were markedly reduced in the scoparone-treated cells. In summary, scoparone blocks the accumulation of STAT3 transported from the cytosol to the nucleus, leading to the suppression of VSMC proliferation through G1 phase arrest and the inhibition of Rb phosphorylation. This activity occurs independent of the form of STAT3 and upstream of kinases, such as Jak and Src, which are correlated with abnormal vascular remodeling due to the presence of an excess of growth factors following vascular injury. These data provide convincing evidence that scoparone may be a new preventative agent for the treatment of cardiovascular diseases.

Animals , Humans , Rats , Active Transport, Cell Nucleus , Biomarkers , Cell Cycle Proteins/genetics , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Coumarins/pharmacology , Gene Expression Regulation/drug effects , Hep G2 Cells , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Proto-Oncogene Proteins c-sis/metabolism , STAT3 Transcription Factor/genetics , Signal Transduction/drug effects , Transcription, Genetic
Acta cir. bras ; 29(supl.1): 39-44, 2014. tab, graf
Article in English | LILACS | ID: lil-720397


PURPOSE: To investigate the effect of dietary lipid quantity and/or quality on penis morphology in adult rats. METHODS: Thirty-eight male Wistar rats were divided into 4 groups: normal lipid diet (NL), high-fat diet rich in saturated fatty acids (HF-S), high-fat diet rich in polyunsaturated fatty acids (HF-P), and high-fat diet rich in saturated and polyunsaturated fatty acids (HF-SP). Blood samples were collected and the penises were removed for histomorphometrical and immunohistochemical analysis. RESULTS: All high-fat diets promoted an increase in the body mass (p<0.0001). The HF-S and HF-SP groups presented hyperglycemia (p=0.0060), hyperinsulinemia (p=0.0030), and hypercholesterolemia (p=0.0020). Concerning the penis, the high-fat diets led to an increase in the collagen fibers (p<0.0001) and smooth muscle cell density area (p=0.0027), and a decline in the sinusoidal space density area (p<0.0001) and corpus cavernosum cell proliferation (p=0.0003). CONCLUSION: Diets rich in saturated and/or polyunsaturated fatty acids promoted overweight and induced penile changes in rodent models, which may lead to the development of erectile dysfunction. .

Animals , Male , Diet, High-Fat/adverse effects , Dietary Fats/adverse effects , Erectile Dysfunction/etiology , Fatty Acids, Monounsaturated/adverse effects , Penis/pathology , Actins/analysis , Collagen/analysis , Dietary Fats/metabolism , Fatty Acids, Monounsaturated/metabolism , Models, Animal , Myocytes, Smooth Muscle/metabolism , Overweight/metabolism , Proliferating Cell Nuclear Antigen/analysis , Random Allocation , Rats, Wistar , Risk Factors
Indian J Biochem Biophys ; 2013 Oct; 50(5): 419-427
Article in English | IMSEAR | ID: sea-150251


There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate, PKC activity using an assay kit and assay of Na+/K+ATPase activity spectrophotometrically. We purified m-calpain and PKCα by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCα by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCα (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCα in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCα increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2+]i-mCalpain-PKCα signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.

Amino Acid Sequence , Animals , Calpain/metabolism , Cattle , Caveolae/drug effects , Caveolae/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Molecular Sequence Data , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Ouabain/pharmacology , Protein Kinase C-alpha/chemistry , Protein Kinase C-alpha/metabolism , Proteolysis/drug effects , Pulmonary Artery/cytology , Sodium-Potassium-Exchanging ATPase/metabolism
Biocell ; 35(3): 71-79, Dec. 2011. tab
Article in English | LILACS | ID: lil-653213


Apigenin, a nonmutagenic flavonoid, has been shown to possess free radical scavenging activities, anticarcinogenic properties, antioxidant and anti-inflammatory effects. Recently, apigenin was reported to cause gastric relaxation in murine. To assess possible effects of apigenin on migration of bladder smooth muscle (SM) cell, we isolated SM cells from peri-cancer tissue of human bladder and established a cell model that was capable to overexpress transiently MEKK1 (MEK kinase 1). Results showed that overexpression of active human MEKK1 by adenoviruses infection induced migration of human bladder smooth muscle (hBSM) cells and phosphorylation of MAPKs, ERK, JNK and p38, which are the downstream molecules of MEKK1. Then, hBSM cell overexpressing MEKK1 were exposed to apigenin (50 microM). Our data indicated that apigenin inhibited significantly activation/phosphorylation of MAPKs and migration of hBSM cells induced by MEKK1 overexpression. Besides, apigenin inhibited actin polymerization, which underlines muscle contraction and cell migration. The results suggest that apigenin inhibits activation of MAPKs and thereby the cell migration. The mechanism might be that apigenin blocks signal transmission from MEKK1 to MAPKs.

Humans , Animals , Rats , Apigenin/pharmacology , Flavonoids , MAP Kinase Kinase Kinase 1/metabolism , Myocytes, Smooth Muscle , Myocytes, Smooth Muscle/metabolism , Cell Movement , Urinary Bladder , Urinary Bladder/metabolism , Cells, Cultured , Phosphorylation , Immunoblotting , MAP Kinase Kinase Kinase 1/genetics , Mitogen-Activated Protein Kinases/metabolism
Arq. bras. cardiol ; 97(1): 40-45, jul. 2011. ilus, tab
Article in Portuguese | LILACS | ID: lil-597662


FUNDAMENTO: Mecanismos subjacentes a anormalidades vasculares na obesidade ainda não estão completamente esclarecidos. OBJETIVO: Foi avaliada a via do óxido nítrico/L-arginina na resposta vascular de ratos obesos por dieta rica em gordura, enfocando as células endoteliais e do músculo liso. MÉTODOS: Ratos com 30 dias de vida foram divididos em 2 grupos: controle (C) e obeso (OB, ratos sob dieta rica em gordura por 30 semanas). Após 30 semanas, foram registrados o peso corporal, índice de adiposidade, pressão arterial e perfis metabólicos e endócrinos dos animais. Foram obtidas curvas para noradrelanina na ausência e presença de inibidor de óxido nítrico sintase (L-NAME, 3x10-4M) em aorta torácica intacta e com desnudamento em ratos C e OB. RESULTADOS: As medidas de peso corporal, índice de adiposidade, leptina e insulina aumentaram nos ratos OB, enquanto a pressão arterial permaneceu inalterada. A obesidade também produziu tolerância à glicose e resistência à insulina. A reatividade à noradrenalina da aorta intacta foi similar em ratos C e OB. A presença de L-NAME produziu um aumento similar nas respostas máximas, mas um desvio maior à esquerda das respostas nas aortas intactas dos ratos C em relação aos ratos OB [EC50 (x10-7M): C = 1,84 (0,83-4,07), O = 2,49 (1,41-4,38); presença de L-NAME C = 0,02 (0,01-0,04)*, O = 0,21 (0,11-0,40)*†,*p < 0,05 vs controle respectivo,†p < 0,05 vs controle mais L-NAME, n = 6-7]. Nenhum dos protocolos alterou a reatividade à noradrenalina de aortas com desnudamento. CONCLUSÃO: A obesidade induzida por dieta rica em gordura promove alterações metabólicas e vasculares. A alteração vascular envolveu uma melhora da via endotelial L-arginina/NO provavelmente relacionada à hiperinsulinemia e hiperleptinemia induzidas por dieta. A maior resistência aos efeitos do L-NAME na aorta de ratos obesos diz respeito a menor vulnerabilidade de indivíduos obesos na presença de patologias associadas que causam danos à atividade do sistema NO.

BACKGROUND: Mechanisms underlying vascular abnormalities in obesity remain to be completely clarified. OBJECTIVE: L-arginine/nitric oxide pathway was evaluated on vascular response of high-fat diet-obese rats, focusing on endothelial and smooth muscle cells. METHODS: 30-day-old rats were divided in two groups: control (C) and obese (OB, high-fat diet for 30 weeks). After 30 weeks, body weight, adiposity index, blood pressure, and metabolic and endocrine profiles of the animals were recorded. Curves to noradrenaline were obtained in absence and presence of nitric oxide synthase inhibitor (L-NAME, 3x10-4M) on intact and denuded thoracic aorta from C and OB rats. RESULTS: Body weight, adiposity index, leptin and insulin levels were increased in OB, while blood pressure was unchanged. Obesity also produced glucose tolerance and insulin resistance. Reactivity to noradrenaline of intact aorta was similar in C and OB rats. L-NAME presence produced a similar increase in maximal responses, but a higher leftward shift of noradrenaline responses in intact aorta from C than in OB rats [EC50 (x10-7M): C = 1.84 (0.83-4.07), O = 2.49 (1.41-4.38); L-NAME presence C = 0.02 (0.01-0.04)*, O = 0.21 (0.11-0.40)*†,*p < 0.05 vs respective control, †p < 0.05 vs control plus L-NAME, n = 6-7]. None of the protocols altered the reactivity to noradrenaline of denuded aortas. CONCLUSION: High-fat diet-induced obesity promotes metabolic and vascular alterations. The vascular alteration involved an endothelial L-arginine/NO pathway improvement was probably correlated to diet-induced hyperinsulinemia and hyperleptinemia. The greater resistance to L-NAME effects in aorta of obese rats raises concerns about the lower cardiovascular vulnerability of obese individuals in the presence of associated pathologies that impair NO-system activity.

FUNDAMENTO: Los mecanismos subyacentes a las anormalidades vasculares en la obesidad todavía no están completamente aclarados. OBJETIVO: Se evaluó la vía del óxido nítrico/L-arginina en la respuesta vascular de ratones obesos por dieta rica en grasa, concentrándonos en las células endoteliales y en el músculo liso. MÉTODOS: Ratones con 30 días de vida que fueron divididos en 2 grupos: control (C) y obeso (OB, ratones bajo dieta rica en grasa durante 30 semanas). Después de 30 semanas, fueron registrados el peso corporal, el índice de adiposidad, la presión arterial y los perfiles metabólicos y endocrinos de los animales. Fueron obtenidas las curvas para noradrelanina en ausencia y en presencia del inhibidor de óxido nítrico sintasa (L-NAME, 3x10-4M), en la aorta torácica intacta y con denudación de los ratones C y OB. RESULTADOS: Las medidas de peso corporal, índice de adiposidad, leptina e insulina aumentaron en los ratones OB, mientras que la presión arterial permaneció inalterada. La obesidad también produjo una tolerancia a la glucosa y una resistencia a la insulina. La reactividad a la noradrenalina de la aorta intacta fue similar en los ratones C y OB. La presencia de L-NAME generó un aumento similar en las respuestas máximas, pero una desviación mayor a la izquierda de las respuestas en las aortas intactas de los ratones C con relación a los ratones OB [EC50 (x10-7M): C = 1,84 (0,83-4,07), O = 2,49 (1,41-4,38); presencia de L-NAME C = 0,02 (0,01-0,04)*, O = 0,21 (0,11-0,40)*†,*p < 0,05 vs control respectivo, †p < 0,05 vs control más L-NAME, n = 6-7]. Ninguno de los protocolos alteró la reactividad a la noradrenalina de las aortas con denudación. CONCLUSIÓN: La obesidad inducida por dieta rica en grasa genera alteraciones metabólicas y vasculares. La alteración vascular conllevó a una mejoría de la vía endotelial L-arginina/NO tal vez relacionada con la hiperinsulinemia e hiperleptinemia inducidas por dieta. La mayor resistencia a los efectos del L-NAME en la aorta de ratones obesos, se refiere a una menor vulnerabilidad de individuos obesos en presencia de patologías asociadas que causan daños a la actividad del sistema NO.

Animals , Male , Rats , Aorta, Thoracic/metabolism , Diet, High-Fat/adverse effects , Endothelium, Vascular/metabolism , NG-Nitroarginine Methyl Ester/metabolism , Nitric Oxide/metabolism , Obesity/metabolism , Adiposity , Aorta, Thoracic/physiopathology , Blood Pressure , Body Weight , Blood Glucose/analysis , Endothelium, Vascular/physiopathology , Epinephrine/metabolism , Myocytes, Smooth Muscle/metabolism , Obesity/physiopathology , Rats, Wistar , Time Factors
Article in English | WPRIM | ID: wpr-174320


Advanced glycation endproducts (AGEs)-induced vascular smooth muscle cell (VSMCs) proliferation and formation of reactive oxygen species (ROS) are emerging as one of the important mechanisms of diabetic vasculopathy but little is known about the antioxidative action of HMG CoA reductase inhibitor (statin) on AGEs. We hypothesized that statin might reduce AGEs-induced intracellular ROS of VSMCs and analyzed the possible mechanism of action of statin in AGEs-induced cellular signaling. Aortic smooth muscle cell of Sprague-Dawley rat (RASMC) culture was done using the different levels of AGEs stimulation in the presence or absence of statin. The proliferation of RASMC, ROS formation and cellular signaling was evaluated and neointimal formation after balloon injury in diabetic rats was analyzed. Increasing concentration of AGEs stimulation was associated with increased RASMC proliferation and increased ROS formation and they were decreased with statin in a dose-dependent manner. Increased NF-kappaB p65, phosphorylated ERK, phosphorylated p38 MAPK, cyclooxygenase-2, and c-jun by AGEs stimulation were noted and their expression was inhibited by statin. Neointimal formation after balloon injury was much thicker in diabetic rats than the sham-treated group but less neointimal growth was observed in those treated with statin after balloon injury. Increased ROS formation, subsequent activation of MAPK system and increased VSMC proliferation may be possible mechanisms of diabetic vasculopathy induced by AGEs and statin may play a key role in the treatment of AGEs-induced diabetic atherosclerosis.

Animals , Male , Rats , Aorta/metabolism , Cell Proliferation/drug effects , Cyclooxygenase 2/metabolism , Diabetes Mellitus, Experimental/drug therapy , Diabetic Angiopathies/drug therapy , /metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Myocytes, Smooth Muscle/metabolism , Oxidative Stress/drug effects , Proto-Oncogene Proteins c-jun/metabolism , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Simvastatin/pharmacology , Transcription Factor RelA/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
Article in English | WPRIM | ID: wpr-37030


Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)- BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.

Humans , Bronchi/metabolism , Cell Proliferation , Cells, Cultured , Chemokine CCL2/metabolism , Chemokine CCL3/metabolism , Cytokines/metabolism , Gene Expression Regulation , Glycoproteins/physiology , Intercellular Signaling Peptides and Proteins/physiology , Interleukin-4/metabolism , Models, Biological , Myocytes, Smooth Muscle/metabolism , Vascular Endothelial Growth Factor A/metabolism
Braz. j. med. biol. res ; 39(6): 739-748, June 2006. graf
Article in English | LILACS | ID: lil-428284


Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70 percent by several inhibitors of the RyR, including ryanodine (10 µM), dantrolene (10 µM), and ruthenium red (10 µM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 µM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80 percent. Pre-incubation of cells with 100 µM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 µM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80 percent. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.

Female , Humans , Calcium/metabolism , Cyclic ADP-Ribose/metabolism , Myocytes, Smooth Muscle/metabolism , Myometrium/cytology , Blotting, Western , Calcium/pharmacology , Cyclic ADP-Ribose/antagonists & inhibitors , Cyclic ADP-Ribose/pharmacology , Immunohistochemistry , Immunoprecipitation , Myocytes, Smooth Muscle/drug effects , Myometrium/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Signal Transduction , Transient Receptor Potential Channels/metabolism
Acta cir. bras ; 21(supl.1): 54-57, 2006.
Article in English, Portuguese | LILACS | ID: lil-438807


The cirrhosis represents the final stage of several chronic hepatic diseases and it is characterized by the presence of fibrosis and morphologic conversion from the normal hepatic architecture into structurally abnormal nodules. In the evolution of the disease there is loss of the normal vascular relationship and portal hypertension. There are also regenerative hepatocelular alterations that become more prominent with the progression of the disease. The liver transplantation continues to be the only therapeutic option in cases of disease in terminal phase. The hepatic stellate cells (HSC) are perisinusoidal cells that store vitamin A and produce growth factors, citocins, prostaglandins and other bioactive substances. They can suffer an activation process that convert them to cells with a phenotype similar to myofibroblasts. When activated, they present increased capacity of proliferation, mobility, contractility and synthesis of collagen and other components of extracelular matrix. They possess cytoplasmic processes adhered to sinusoids and can affect the sinusoidal blood flow. HSC are important in pathogenesis of fibrosis and portal hypertension.

A cirrose representa o estágio final de diversas doenças hepáticas crônicas e é caracterizada pela presença de fibrose e conversão da arquitetura hepática normal em nódulos estruturalmente anormais. Na evolução da doença ocorre perda da relação vascular normal e hipertensão portal. Há também alterações regenerativas hepatocelulares que se tornam mais proeminentes com a progressão da doença. O transplante hepático permanece como a única opção terapêutica nos casos de doença em fase terminal. As células estreladas hepáticas (CEH) são células perisinusoidais que armazenam vitamina A e produzem fatores de crescimento, citocinas, prostaglandinas e outras substâncias bioativas. Podem sofrer um processo de ativação para um fenótipo semelhante a miofibroblastos. Quando ativadas apresentam maior capacidade de proliferação, motilidade, contractilidade, síntese de colágeno e componentes da matriz extracelular. Possuem processos citoplasmáticos aderidos aos sinusóides e podem afetar o fluxo sangüíneo sinusoidal. As CEH são importantes na patogênese da fibrose e hipertensão portal.

Humans , Adult , Hepatocytes/metabolism , Kupffer Cells/metabolism , Liver Cirrhosis/physiopathology , Liver/metabolism , Cell Proliferation , Disease Progression , Extracellular Matrix/metabolism , Hepatocyte Growth Factor/metabolism , Hepatocytes/cytology , Hypertension, Portal/complications , Kupffer Cells/cytology , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Liver Failure/complications , Liver/cytology , Myocytes, Smooth Muscle/metabolism , Paracrine Communication/physiology , Platelet-Derived Growth Factor/metabolism
Article in English | WPRIM | ID: wpr-23950


A 19-yr-old woman with a previous history of a mass of the right ciliary body presented with a decreased visual acuity of right eye. Clinicoradiologic examinations suggested a recurrent mass of the ciliary body. Enucleation of the right eye was performed under the impression of malignant tumor. On microscopic examination, the tumor was a mesectodermal leiomyoma of the ciliary body. On immunohistochemistry, the tumor cells were reactive to smooth muscle actin and vimentin, but not reactive to cytokeratin, S-100 protein, neurofilament, desmin, epithelial membrane antigen, HMB-45, glial fibrillary acidic protein, and synaptophysin. Electron microscopy revealed numerous thin longitudinally placed myofilaments and focal densities in the cytoplasms. In the review of the literature, only 27 cases of mesectodermal leiomyoma of the ciliary body were reported, however, there was no report of recurrent cases. Mesectodermal leiomyoma should be differentiated from other orbital spindle-cell tumors such as amelanotic melanomas and glial tumors. Immunohistochemical and electron microscopic studies may be useful for the correct diagnosis by showing smooth muscle differentiation in the tumor cells.

Adult , Female , Humans , Actins/biosynthesis , Cell Differentiation , Ciliary Body/pathology , Cytoplasm/metabolism , Immunohistochemistry , Leiomyoma/diagnosis , Microscopy, Electron , Myocytes, Smooth Muscle/metabolism , Recurrence , Uveal Neoplasms/diagnosis , Vimentin/biosynthesis