RESUMO
BACKGROUND/AIMS: Neointimal thickening results from inflammation in association with vascular smooth muscle cell (VSMC) proliferation. We studied the role of perivascular adipose tissue (PVAT) on VSMC proliferation and intima-media thickening (IMT) in a rodent model of chronic inflammation. METHODS: The abdominal aorta and surrounding PVAT of tumour necrosis factor (TNF)-α-injected mice were examined 28 days after administration. Plasma and PVAT cytokines were measured with Milliplex™ assays. Inflammatory cells were examined with immunofluorescence. Expression of transforming growth factor (TGF)-ß1, matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 was examined with immunohistochemistry, immunoblotting and zymography. IMT was determined. Cell proliferation and TGF-ß1 mRNA levels were examined after treating VSMC with PVAT homogenates ± MMP-2 inhibitors (batimastat, ARP 100 or TIMP-2) and SB-431542, a selective inhibitor of the TGF-ß-type 1 receptor. RESULTS: Significant increases in CD3, CD68, neutrophils, vascular cell adhesion molecule-1 and MMP-2 in PVAT, and TGF-ß1 and IMT of the aorta of TNF-α-injected mice were observed. PVAT of TNF-α-injected mice significantly up-regulated TGF-ß1 and increased cell proliferation in a dose-dependent manner and was attenuated by SB-431542, batimastat, ARP 100 and TIMP-2. CONCLUSIONS: Our study shows that chronic PVAT inflammation leads to MMP-mediated increase in TGF-ß1 and hence VSMC proliferation.
Assuntos
Tecido Adiposo/fisiopatologia , Aorta Abdominal/patologia , Inflamação/patologia , Fator de Necrose Tumoral alfa/farmacologia , Túnica Íntima/patologia , Túnica Média/patologia , Adipocinas/análise , Tecido Adiposo/química , Tecido Adiposo/efeitos dos fármacos , Animais , Proliferação de Células , Citocinas/análise , Citocinas/sangue , Expressão Gênica , Masculino , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Músculo Liso Vascular/patologia , RNA Mensageiro/análise , Fator de Crescimento Transformador beta1/genéticaRESUMO
BACKGROUND: Although tumor necrosis factor-α (TNF-α) levels are increased in patients with atrial fibrillation (AF), its role in the pathogenesis of AF is unclear. We investigated whether direct delivery of TNF-α could induce atrial fibrosis. METHODS AND RESULTS: TNF-α (4 µg/kg) was injected into the tail vein of 20 male Swiss albino mice (TNF group) and saline into 20 control mice (CON group). The dose was carefully chosen to avoid any significant decrease in left ventricular (LV) function. Animals were killed after 16 weeks and their atria examined for fibrosis. We found increased atrial fibrosis in the TNF group compared with the CON group [372.8±21.5 arbitrary units (a.u.) vs. 56.9±6.5 a.u., respectively, mean±SEM; P<0.0001] and decreased connexin-40 immunofluorescence [7.5±0.4 a.u vs. 40.4±1.9 a.u, respectively; P<0.0001]. Transforming growth factor-ß [TGF-ß: 95.6±1.8 a.u vs. 29.4±5.8 a.u; P<0.001], α-smooth muscle actin (α-SMA: 97.9±13.0 a.u vs. 50.1±18.5 a.u; P<0.05] and matrix metalloproteinase 2 (MMP-2)/GAPDH levels [157.3±26.4 a.u vs. 105.8±13.3 a.u; P<0.05] were also increased in the TNF group. CONCLUSIONS: TNF-α is involved in the pathogenesis of atrial fibrosis and altered connexin-40 expression in mice through the TGF-ß signaling pathway, activation of myofibroblasts and increased secretion of MMPs. Collectively, these changes may contribute to the arrhythmogenic substrate and development of AF.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Função Atrial , Fator de Necrose Tumoral alfa/toxicidade , Actinas/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Função Atrial/efeitos dos fármacos , Conexina 43/metabolismo , Conexinas/metabolismo , Regulação para Baixo , Fibrose , Imunofluorescência , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Injeções Intravenosas , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagem , Proteína alfa-5 de Junções ComunicantesRESUMO
Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.
Assuntos
Autofagia , Músculo Liso Vascular , Animais , Autofagia/fisiologia , Cafeína/metabolismo , Cafeína/farmacologia , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Sequestossoma-1/metabolismo , Via de Sinalização WntRESUMO
Mungbean (Vigna radiata (L.) Wilczek) is a unique species in its ability to fix atmospheric nitrogen, with early maturity, and relatively good drought resistance. We used 454 sequencing technology for transcriptome sequencing. A total of 150 159 and 142 993 reads produced 5 254 and 6 374 large contigs (≥ 500 bp) with an average length of 833 and 853 for Sunhwa and Jangan, respectively. Functional annotation to known sequences yielded 41.34% and 41.74% unigenes for Jangan and Sunhwa. A higher number of simple sequence repeat (SSR) motifs was identified in Jangan (1 630) compared with that of Sunhwa (1 334). A similar SSR distribution pattern was observed in both varieties. A total of 8 249 single nucleotide polymorphisms (SNPs) and indels with 2 098 high-confidence candidates were identified in the two mungbean varieties. The average distance between individual SNPs was approximately 860 bp. Our report demonstrates the utility of transcriptomic data for implementing a functional annotation and development of genetic markers. We also provide large resource sequence data for mungbean improvement programs.
Assuntos
Fabaceae/genética , Repetições Minissatélites , Polimorfismo de Nucleotídeo Único , Perfilação da Expressão Gênica , Análise de Sequência de DNARESUMO
INTRODUCTION: Coronary artery disease (CAD) is the leading cause of death following ischaemic stroke. We aimed to study the prevalence and associations of concomitant CAD among ischaemic stroke patients in Singapore. MATERIALS AND METHODS: We prospectively studied 2686 consecutive Asian ischaemic stroke patients. RESULTS: CAD was prevalent among 24% of the study patients. Older age, hypertension, diabetes, hyperlipidaemia, atrial fibrillation, large stroke and South Asian ethnicity were independently associated with CAD. CONCLUSIONS: The variables found to be associated with CAD are known atherosclerotic risk factors (older age, hypertension, diabetes, hyperlipidaemia) or associations of cardioembolic stroke (atrial fibrillation, large stroke). The over-representation of South Asians with concomitant CAD is consistent with the high burden of CAD in this ethnic group.
Assuntos
Isquemia Encefálica/complicações , Doença da Artéria Coronariana/complicações , Acidente Vascular Cerebral/complicações , Idoso , Isquemia Encefálica/epidemiologia , Doença da Artéria Coronariana/epidemiologia , Feminino , Humanos , Masculino , Prevalência , Estudos Prospectivos , Análise de Regressão , Fatores de Risco , Singapura/epidemiologia , Acidente Vascular Cerebral/epidemiologia , Taxa de Sobrevida , Fatores de TempoRESUMO
Hyperhomocysteinemia (HHcy) is a well-known risk factor for stroke; however, its underlying molecular mechanism remains unclear. Using both mouse and cell culture models, we have provided evidence that impairment of autophagy has a central role in HHcy-induced cellular injury in the mouse brain. We observed accumulation of LC3B-II and p62 that was associated with increased MTOR signaling in human and mouse primary astrocyte cell cultures as well as a diet-induced mouse model of HHcy, HHcy decreased lysosomal membrane protein LAMP2, vacuolar ATPase (ATP6V0A2), and protease cathepsin D, suggesting that lysosomal dysfunction also contributed to the autophagic defect. Moreover, HHcy increased unfolded protein response. Interestingly, Vitamin B supplementation restored autophagic flux, alleviated ER stress, and reversed lysosomal dysfunction due to HHCy. Furthermore, the autophagy inducer, rapamycin was able to relieve ER stress and reverse lysosomal dysfunction caused by HHcy in vitro. Inhibition of autophagy by HHcy exacerbated cellular injury during oxygen and glucose deprivation and reperfusion (OGD/R), and oxidative stress. These effects were prevented by Vitamin B co-treatment, suggesting that it may be helpful in relieving detrimental effects of HHcy in ischemia/reperfusion or oxidative stress. Collectively, these findings show that Vitamin B therapy can reverse defects in cellular autophagy and ER stress due to HHcy; and thus may be a potential treatment to reduce ischemic damage caused by stroke in patients with HHcy.
Assuntos
Autofagia/efeitos dos fármacos , Suplementos Nutricionais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hiper-Homocisteinemia/patologia , Vitamina B 12/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dieta , Ácido Fólico/farmacologia , Glucose/deficiência , Humanos , Hiper-Homocisteinemia/tratamento farmacológico , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Oxigênio , Traumatismo por Reperfusão/patologia , Proteína Sequestossoma-1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Vitamina B 12/uso terapêuticoRESUMO
An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate assay. TNF-α-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-α-induced upregulation of interleukin (IL)-1ß and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/ß and p65, degradation of IkappaBα, binding of NF-kappaB to its binding motif, and upregulation of IL-1ß and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1ß and VCAM-1, in human cardiomyocytes.
Assuntos
Miócitos Cardíacos/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Acetofenonas/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Quinase I-kappa B/metabolismo , Interleucina-1beta/genética , Miócitos Cardíacos/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
The diagnosis and management of patients with acute coronary syndrome (ACS) have evolved dramatically over the past decade. Biomarkers play an important role in the diagnosis of ACS, especially in unstable angina and non-ST-segment elevation myocardial infarction. Among these, cardiac troponin and creatine kinase appear to be the most sensitive and specific markers of myocardial injury. Recent studies have revealed several novel biomarkers. Elevated levels of C-reactive protein and interleukin-6 are strong independent markers of increased mortality among patients with ACS. However, the ideal biomarkers that offer early detection, risk stratification, selection of therapy, monitoring disease progression, and treatment efficacy remain to be elucidated. This review assesses limitations and contemporary needs for biomarkers in the context of diagnosis of ACS. It also discusses the newly developing technologies for novel biomarkers or novel biomarker protein signatures discovery, and importance of point-of-care testing for future management.
Assuntos
Síndrome Coronariana Aguda/sangue , Troponina I/sangue , Troponina T/sangue , Síndrome Coronariana Aguda/patologia , Biomarcadores/sangue , Creatina Quinase/sangue , Eletrocardiografia , Humanos , Mioglobina/sangue , Necrose/sangue , Estresse Oxidativo , Ativação PlaquetáriaRESUMO
This paper presents a novel microfluidic system for rapid label-free detection of endothelial progenitor cells (EPCs) from small volumes of white blood cells samples, to obtain a bedside cardiovascular diagnostic solution. The system was built on a single 1 cm(2) microelectrode array silicon chip, integrated with negative dielectrophoresis for cell trapping, surface immunochemistry for selective cell capture, and fluidics for cell washing and impedance detection. The level of circulating EPC level in blood is a biomarker of clinical interest, linked to the assessment of risk factors in cardiovascular diseases which are a major global concern. Rare EPCs are usually detected through in vitro culture or flow cytometry, which are too time-consuming to bring timely reports in acute diseases. Although microfluidics approaches have enabled reduced processing time and enhanced portability, their sensitivity and processing volumes are still inadequate for rare cell detection at a bedside setting. Using small highly sensitive microelectrodes, our novel integrated system achieved the detection of 720 EPCs in a small 12 microl sample of 72,000 peripheral blood mononuclear cells (PBMC), i.e. equivalent to a concentration of EPCs of 0.1% of 100 microl blood. This demonstrated that clinically significant level of EPCs (<0.5% of PBMC) could be detected for the first time on a detection system at bedside set-up, showing great potential in applications for point-of-care diagnosis.
Assuntos
Contagem de Células/instrumentação , Separação Celular/instrumentação , Células Endoteliais/citologia , Imunoensaio/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Células-Tronco/citologia , Técnicas Biossensoriais/instrumentação , Eletroforese/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
Cymbidium spp. are popular flowering plants. Assessment of the genetic diversity in cultivated Cymbidium facilitates conservation of germplasm and subsequent cultivar improvement. Thus, it is important to develop more efficient polymorphic DNA markers. Although more motifs (403) were identified and more primers (206) were designed in the genomic library compared to the cDNA library, a larger number of successful primers were obtained from the cDNA library (59.9 percent) than from genomic DNA library (51.1 percent). However, higher PIC and gene diversity were identified in genomic SSRs. The average allele number per locus was also higher in genomic SSRs (7.3) than EST-SSRs (5.2), among the 24 evaluated Cymbidium accessions. AT/TA was comparatively high in EST-SSRs, while this motif was not as common in genomic SSRs. The CTT/AAG/TCT/AGA/TTC/GAA and TGC/GCA/GCT/AGC/CTG/CAG motifs were the most abundant tri-nucleotide sequences in EST-SSRs, while GTT/AAC/TGT/ACA/TTG/CAA was the most frequent in genomic SSRs. The number of repeats ranged from 3 to 12 in EST-SSRs. Currently, 52 novel polymorphic SSR markers have been evaluated, which will be useful for germplasm assessments, core set construction, evaluation of genetic diversity, and marker assisted selection (MAS) based Cymbidium breeding.
Assuntos
DNA Complementar , Repetições de Microssatélites , Orchidaceae/genética , Biblioteca Gênica , Marcadores Genéticos , Variação Genética , Polimorfismo GenéticoRESUMO
In total, 18 simple sequence repeat (SSR) markers were used to analyze the genetic diversity and population structure of 59 accessions of cultivated strawberry (Fragaria x ananassa Duch.) from Korea, Germany, United States, United Kingdom, and Japan. In total, 101 alleles were detected with an average of 5.6 per locus and 21 specific alleles were identified. Notably, one genotype (Blonoli from Germany) possessed a maximum of 10 different unique alleles specific to each genotype. The gene diversity varied from 0.027 (EMPaEKO1B) to 0.791 (CFACT110), with an average value of 0.509. PIC values ranged from 0.026 to 0.762 (average 0.454). A model-based structure analysis revealed the presence of two populations. The accessions that were clearly assigned to a single population in which > 70 percent of their inferred ancestry was derived from one of the model-based populations. However, two accessions (3.4 percent) in the sample were categorized as having admixed ancestry. Here, we report detailed information on commercially grown strawberry accessions from five different origins using SSR markers. These results couldbe used for broadening the genetic base of commercially grown varieties.
Assuntos
Fragaria/genética , Variação Genética , Repetições de Microssatélites , Marcadores Genéticos , Genética Populacional , Polimorfismo GenéticoRESUMO
Pathogenic PINK1 mutations have been described in PARK6-linked Parkinson's disease (PD) patients of Asian origin. However, data on the frequency of PINK1 mutations in sporadic early-onset Parkinson's disease (EOPD) Asian patients are lacking. The objectives of this study were to report the frequency of PINK1 mutations of sporadic EOPD in an Asian cohort comprising of ethnic Chinese, Malays, and Indians, and to highlight a PINK1-positive patient who presented with restless legs symptoms. Eighty consecutive sporadic EOPD patients from the movement disorder clinics of two major tertiary institutions in the country were included. We performed sequence analysis of all the coding and exon-intron junctions of the PINK1 using specific primer sets. In addition, we genotyped polymorphisms detected from the analysis in a group of sporadic PD patients and controls. Three different mutations (two homozygous nonsense and one heterozygous missense) in the putative kinase domain were found in three patients, giving a 3.7% frequency of PINK1 mutations in our EOPD cohort. All the mutations were absent in 200 healthy controls. One patient with a novel homozygous nonsense PINK1 mutation presented unusually with restless legs symptoms. Separately, analysis of the frequency of four PINK1 polymorphisms in a group of sporadic PD and controls did not reveal any significant differences. We highlight a 3.7% frequency of PINK1 mutations in an Asian cohort (ethnic Chinese, Malay, and Indian) of EOPD. The phenotypic spectrum associated with PINK1-positive patients may be wider than previously reported. Polymorphisms of PINK1 do not appear to modulate risk of PD in our population.