RESUMO
Detection and quantification of differentially expressed genes requires valid and reliable references to control for error variability introduced by preparatory procedures or efficiency of reverse transcription and polymerase chain reaction (PCR) amplification conditions. So-called housekeeping genes are frequently used as endogenous standards, based on the assumption that they are constitutively expressed and independent of experimental conditions. However, if the influence of experimental stimuli is to be analyzed, it cannot a priori be assumed that their expression is unaffected by stimulation. In the present study, the authors studied the expression of different housekeeping genes in the vascular endothelium of intact conduit vessels perfused in a unique biomechanical perfusion model. Ten control gene candidates were investigated by microarray expression analysis. Further, five of these genes were systematically analyzed by real-time reverse transcriptase (RT)-PCR gene quantification and their suitability as reference genes were evaluated. On the basis of these findings, the authors suggest criteria for evaluation of endogenous control genes in vascular perfusion studies.
Assuntos
Endotélio Vascular/fisiologia , Perfilação da Expressão Gênica , Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cordão Umbilical/fisiologia , Pesquisa Biomédica , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Perfusão , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normasRESUMO
OBJECTIVE: By using a computerized vascular perfusion model, we investigated temporal effects of sub-acute pressure elevation on vasomotor behavior and expression of endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) in intact human conduit vessels. METHODS: Paired umbilical veins were perfused during 1.5, 3 and 6 h under high/low intraluminal pressure (40/20 mmHg) and at identical shear stress level of 10 dyn/cm(2). ET-1 and eNOS gene and protein expression was quantified with real-time reverse-transcribed polymerase chain reaction and quantitative immunohistochemistry, respectively. RESULTS: Pressure induced differential temporal regulation patterns of ET-1 and eNOS gene expression. During the high pressure condition, eNOS mRNA was upregulated after 3 h and leveled off after 6 h of perfusion, while ET-1 mRNA was elevated after 6 h perfusion. Immunohistochemistry verified synchronal changes at the protein level. Significant vasodilation was observed after 3 h in the high-pressure system. CONCLUSION: Thus, subacute pressure elevation exerts differential effects on the endothelial eNOS/ET-1 expression, which dynamically regulate the vasomotor tone.
Assuntos
Simulação por Computador , Endotelina-1/genética , Endotélio Vascular/metabolismo , Modelos Cardiovasculares , Óxido Nítrico Sintase/genética , Resistência Vascular , Análise de Variância , Endotelina-1/análise , Endotélio Vascular/química , Expressão Gênica , Humanos , Imuno-Histoquímica , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo III , Perfusão , Pressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
We investigated the effect of shear stress on the expression of tissue-type plasminogen activator (t-PA) in intact human conduit vessels. Human umbilical veins were exposed to high or low shear stress (25 vs < 4 dyn/cm2) at identical intraluminal pressure (20 mmHg) for 1.5, 3, and 6 h in a new computerized biomechanical perfusion system. High shear perfusion induced a marked, time-dependent increase in t-PA immunostaining in both the endothelium and the media. t-PA relative to GAPDH gene expression increased by 54 +/- 14% in high- compared to low-sheared vessels (p = 0.002). By contrast, t-PA release into the perfusion medium was similar in vessels perfused under high or low shear stress conditions. The results show that shear stress independently of pressure is a potent fluid mechanical stimulus for up-regulation of the intracellular storage pool of t-PA in the vascular wall of fresh human conduit vessels. The shear effect is associated with an increased t-PA gene expression.
Assuntos
Líquido Intracelular/química , Ativador de Plasminogênio Tecidual/biossíntese , Veias Umbilicais/fisiologia , DNA Complementar/biossíntese , DNA Complementar/fisiologia , Endotélio Vascular/química , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibrinolíticos/metabolismo , Expressão Gênica/fisiologia , Hemodinâmica , Humanos , Imuno-Histoquímica , Perfusão/instrumentação , Pressão , RNA Mensageiro/biossíntese , RNA Mensageiro/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Fatores de Tempo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Veias Umbilicais/metabolismo , Regulação para Cima/fisiologia , VasodilataçãoRESUMO
We have previously shown distinct effects of shear stress and pressure on transcription of several potent vascular mediators. In the present study, we tested the hypothesis that c-jun and c-fos are regulated differentially by shear and pressure. Intact human umbilical veins were perfused with various combinations of shear and pressure during 1.5, 3 and 6 h. Protein and gene expressions were assessed by immunofluorescence and real-time reverse transcription PCR, respectively. Shear stress and pressure exert differential temporal effects on c-jun and c-fos gene and protein expression, and these immediate-early gene responses appear to be cell-type specific for endothelial and smooth muscle cells.
Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Genes Precoces/genética , Pressão Hidrostática , Estresse Mecânico , Endotélio Vascular/fisiologia , Imunofluorescência , Genes fos/genética , Genes jun/genética , Hemodinâmica/fisiologia , Humanos , Técnicas In Vitro , Músculo Liso/metabolismo , Especificidade de Órgãos , Perfusão , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Veias Umbilicais , Regulação para CimaRESUMO
VEGF is a potent angiogenic factor. We tested the hypothesis that biomechanical forces may regulate VEGF expression. By using a computerized perfusion system, human umbilical veins were exposed to high/low shear stress or intraluminal pressure (25/4 dyn/cm(2) or 40/20 mmHg) for 1.5, 3, or 6 h. Quantification of VEGF gene expression was performed with real-time RT-PCR. VEGF protein was characterized by quantitative immunohistochemistry. All perfusion experiments were performed under identical pH, PO(2), and temperature. Shear stress induced significant biphasic regulation pattern of VEGF (P = 0.0044) with significant downregulation by 45 and 40% after 1.5 and 6 h perfusion, respectively (P = 0.006 and P = 0.0002). The temporal changes of the gene expression were accompanied by synchronal changes at the protein level. High pressure induced transient 25% downregulation of VEGF gene expression after 1.5 h perfusion (P = 0.031). These data provide the first evidence on modulating effects of biomechanical forces on the vascular angiogenic property.
Assuntos
Fatores de Crescimento Endotelial/genética , Regulação da Expressão Gênica , Hemodinâmica , Linfocinas/genética , Veias Umbilicais/metabolismo , Veias Umbilicais/fisiologia , Pressão Sanguínea , Computadores , Regulação para Baixo , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/metabolismo , Humanos , Imuno-Histoquímica , Linfocinas/metabolismo , Músculo Liso/citologia , Músculo Liso/metabolismo , Perfusão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We recently discovered that patients with essential hypertension have a markedly impaired capacity for stimulated release of tissue plasminogen activator (tPA) from vascular endothelium. This defect may reduce the chance of timely spontaneous thrombolysis in case of an atherothrombotic event. We now investigated whether increased intraluminal pressure as such may depress vascular tPA release or downregulate its gene expression. Segments of human umbilical veins were studied in a new computerized vascular perfusion model under steady laminar flow conditions for 3 or 6 hours. Paired segments were perfused at high or physiological intraluminal pressure (40 versus 20 mm Hg) under identical shear stress (10 dyne/cm(2)). Quantitative immunohistochemical evaluation of cellular tPA immunoreactivity was performed on paraffin-embedded 5-microm vascular sections. tPA mRNA in endothelial cells was quantified with reverse transcription real-time TaqMan polymerase chain reaction with GAPDH as endogenous control. Secretion of tPA into perfusion medium was evaluated with SDS-PAGE and Western blotting, followed by densitometric quantification. High-pressure perfusion downregulated tPA gene expression with a 38% decrease in tPA mRNA levels (P=0.01) compared with vessels perfused under normal intraluminal pressure. tPA release into the perfusion medium was markedly suppressed by high pressure (P<0.01 ANOVA). The intracellular storage pool of tPA was reduced after 6 but not 3 hours. Thus, elevated intraluminal pressure downregulates tPA gene and protein expression and inhibits its release from the endothelium independently of shear stress. The defective capacity for stimulated tPA release that we demonstrated in patients with essential hypertension might thus be an effect of the elevated intraluminal pressure per se.
Assuntos
Ativador de Plasminogênio Tecidual/metabolismo , Veias Umbilicais/metabolismo , Pressão Sanguínea , Regulação para Baixo , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Estresse Mecânico , Ativador de Plasminogênio Tecidual/genética , Veias Umbilicais/patologia , Veias Umbilicais/fisiopatologiaRESUMO
This study investigated the effects of shear stress on gene expression of prostacyclin synthesis-related enzymes cyclooxygenases (COX-1 and COX-2), prostacyclin synthase (PGS), and thromboxane synthase (TXS) and their metabolites prostaglandin (PGI(2)) and thromboxane A(2) (TXA(2)) in endothelium of intact conduit vessels. Paired human umbilical veins were perfused at high/low shear stress (25/<4 dyn/cm(2)) at identical intraluminal pressure (20 mmHg) for 1.5, 3, or 6 hours in a computerized vascular model. High shear perfusion induced a significant, monophasic upregulation of PGS and TXS gene expressions after 6 hours. COX-1 and COX-2 mRNA showed a biphasic response with peaks at 1.5 and 6 hours, with a nadir level at 3 hours. Shear-induced gene expression was associated with a significantly greater accumulation of 6-keto prostaglandin F(1alpha) and TXA(2) in the perfusion medium. Thus, shear stress independently of perfusion pressure alters the expression of prostacyclin synthesis-related enzymes and the biosynthesis of their vasoactive metabolites.
Assuntos
Eicosanoides/genética , Eicosanoides/metabolismo , Endotélio Vascular/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Sequência de Bases , Fenômenos Biomecânicos , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Primers do DNA/genética , Epoprostenol/metabolismo , Feminino , Expressão Gênica , Humanos , Técnicas In Vitro , Isoenzimas/genética , Proteínas de Membrana , Modelos Cardiovasculares , Perfusão , Gravidez , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Mecânico , Tromboxano A2/metabolismo , Tromboxano-A Sintase/genética , Veias Umbilicais/metabolismoRESUMO
We have developed a new computerized biomechanical ex vivo perfusion system for intact conduit vessels in which a wide range of combinations of intraluminal pressure, fluid flow and shear stress could be set and maintained at target levels in mammalian conduit vessels under controlled metabolic conditions. Mean wall shear stress is calculated using the formula: Accuracy of the wall shear stress calculation was validated by ultrasonographic imaging of the vessel radius. In a series of simulation experiments, the hemodynamic homeostasis functions of the system were challenged by generating a wide range of vascular resistance in artificial vessels and by pharmacologically induced changes in vascular tone in intact human vessels. Despite rapid changes in vessel resistance, shear stress and pressure, or flow and pressure were maintained well at target levels. Shear- and pressure-stimulated production of the vasodilator prostaglandin E2 (PGE2) was used to validate the biological relevance of the model. PGE2 release was significantly more stimulated by high (25 dyn/cm2) compared to low (<4 dyn/cm2) shear (ANOVA, p = 0.012). High compared to low intraluminal pressure depressed the production of PGE2 (ANOVA, p = 0.019). In summary, the computerized perfusion model appears to offer new possibilities of investigating the complex interplay between fluid mechanics and the vascular wall.