RESUMO
Formins promote actin assembly and are involved in force-dependent cytoskeletal remodeling. However, how force alters the formin functions still needs to be investigated. Here, using atomic force microscopy and biomembrane force probe, we investigated how mechanical force affects formin-mediated actin interactions at the level of single molecular complexes. The biophysical parameters of G-actin/G-actin (GG) or G-actin/F-actin (GF) interactions were measured under force loading in the absence or presence of two C-terminal fragments of the mouse formin mDia1: mDia1Ct that contains formin homology 2 domain (FH2) and diaphanous autoregulatory domain (DAD) and mDia1Ct-ΔDAD that contains only FH2. Under force-free conditions, neither association nor dissociation kinetics of GG and GF interactions were significantly affected by mDia1Ct or mDia1Ct-ΔDAD. Under tensile forces (0-7 pN), the average lifetimes of these bonds were prolonged and molecular complexes were stiffened in the presence of mDia1Ct, indicating mDia1Ct association kinetically stabilizes and mechanically strengthens bonds of the dimer and at the end of the F-actin under force. Interestingly, mDia1Ct-ΔDAD prolonged the lifetime of GF but not GG bond under force, suggesting the DAD domain is critical for mDia1Ct to strengthen GG interaction. These data unravel the mechanochemical coupling in formin-induced actin assembly and provide evidence to understand the initiation of formin-mediated actin elongation and nucleation.
Assuntos
Actinas/metabolismo , Forminas/metabolismo , Animais , Fenômenos Biomecânicos , Membrana Celular/metabolismo , Cinética , Camundongos , Modelos Biológicos , Ligação ProteicaRESUMO
The actin cytoskeleton is subjected to dynamic mechanical forces over time and the history of force loading may serve as mechanical preconditioning. While the actin cytoskeleton is known to be mechanosensitive, the mechanisms underlying force regulation of actin dynamics still need to be elucidated. Here, we investigated actin depolymerization under a range of dynamic tensile forces using atomic force microscopy. Mechanical loading by cyclic tensile forces induced significantly enhanced bond lifetimes and different force-loading histories resulted in different dissociation kinetics in G-actin-G-actin and G-actin-F-actin interactions. Actin subunits at the two ends of filaments formed bonds with distinct kinetics under dynamic force, with cyclic mechanical reinforcement more effective at the pointed end compared to that at the barbed end. Our data demonstrate force-history dependent reinforcement in actin-actin bonds and polarity of the actin depolymerization kinetics under cyclic tensile forces. These properties of actin may be important clues to understanding regulatory mechanisms underlying actin-dependent mechanotransduction and mechanosensitive cytoskeletal dynamics.This article has an associated First Person interview with the first author of the paper.
Assuntos
Actinas/química , Proteínas Aviárias/química , Proteína de Capeamento de Actina CapZ/química , Mecanotransdução Celular , Imagem Individual de Molécula/métodos , Tropomodulina/química , Citoesqueleto de Actina , Actinas/genética , Actinas/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Galinhas , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Microscopia de Força Atômica , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/instrumentação , Estresse Mecânico , Tropomodulina/genética , Tropomodulina/metabolismoRESUMO
The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Using AFM force-clamp experiments, we show that formin controlled by RhoA switches the actin catch-slip bonds to slip-only bonds. SMD simulations reveal that the force does not induce the K113:E195 interaction when formin binds to actin K118 and E117 residues located at the helical segment extending to K113. Actin catch-slip bonds are suppressed by single residue replacements K113E and E195K that interrupt the force-induced K113:E195 interaction; and this suppression is rescued by a K113E/E195K double mutant (E/K) restoring the interaction in the opposite orientation. These results support the biological significance of actin catch bonds, as they corroborate reported observations that RhoA and formin switch force-induced actin cytoskeleton alignment and that either K113E or E195K induces yeast cell growth defects rescued by E/K. Our study demonstrates how the mechano-regulation of actin dynamics is modulated by biochemical signaling molecules, and suggests that actin catch bonds may be important in cell functions.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Lisina/genética , Proteínas dos Microfilamentos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Proteína rhoA de Ligação ao GTP/químicaRESUMO
As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catch-slip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Substituição de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Galinhas , Humanos , Microscopia de Força Atômica , Mutação de Sentido Incorreto , Coelhos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm(2) or 1 dyne/cm(2)) or reversing shear stress (time average 1 dyne/cm(2)) for 24 h. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure. Unlike other shear stress regimes, reversing shear stress alone enhanced monocyte adhesion, which may be associated with increased H(2)O(2) and superoxide together with relatively low levels of nitric oxide (NO) production. L-N(G)-Nitroarginine methyl ester (L-NAME) treatment increased monocyte adhesion under 15 dynes/cm(2) and under reversing shear stress. After reversing shear stress, monocyte adhesion dramatically increased with heparinase III treatment followed by a zinc scavenger. Static culture experiments supported the reduction of monocyte adhesion by zinc following endothelial cell cytokine activation. These results suggest that endothelial cell zinc levels are important for the inhibition of monocyte adhesion to endothelial cells, and may be one of the key factors in the early stages of atherogenesis.
Assuntos
Células Endoteliais/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Óxido Nítrico/farmacologia , Zinco/farmacologia , Aterosclerose/etiologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Engenharia Biomédica , Adesão Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Modelos Biológicos , Polissacarídeo-Liases/farmacologia , Resistência ao Cisalhamento , Estresse Mecânico , Superóxidos/metabolismo , Zinco/deficiência , Zinco/metabolismoRESUMO
We examined the effects of fluid shear stress on metallothionein (MT) gene and protein expression and intracellular free zinc in mouse aorta and in human umbilical vein endothelial cells (HUVECs). Immunostaining of the endothelial surface of mouse aorta revealed increased expression of MT protein in the lesser curvature of the aorta relative to the descending thoracic aorta. HUVECs were exposed to high steady shear stress (15 dyn/cm(2)), low steady shear stress (1 dyn/cm(2)), or reversing shear stress (mean of 1 dyn/cm(2), 1 Hz) for 24 h. Gene expression of three MT-1 isoforms, MT-2A, and zinc transporter-1 was upregulated by low steady shear stress and reversing shear stress. HUVECs exposed to 15 dyn/cm(2) had increased levels of free zinc compared with cells under other shear stress regimes and static conditions. The increase in free zinc was partially blocked with an inhibitor of nitric oxide synthesis, suggesting a role for shear stress-induced endothelial nitric oxide synthase activity. Cells subjected to reversing shear stress in zinc-supplemented media (50 µM ZnSO(4)) had increased intracellular free zinc, reduced surface intercellular adhesion molecule-1 expression, and reduced monocyte adhesion compared with cells exposed to reversing shear stress in normal media. The sensitivity of intracellular free zinc to differences in shear stress suggests that intracellular zinc levels are important in the regulation of the endothelium and in the progression of vascular disease.
Assuntos
Células Endoteliais/metabolismo , Metalotioneína/biossíntese , Resistência ao Cisalhamento , Estresse Mecânico , Zinco/metabolismo , Animais , Aorta/metabolismo , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Zinco/análiseRESUMO
This study addresses whether pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction in human umbilical vein endothelial cells (HUVEC) or human aortic endothelial cells (HAEC). mRNA and protein expression of c-Myc under physiological (6-10%) and pathological cyclic strain conditions (20%) were studied. Both c-Myc mRNA and protein expression increased 2-3-fold in HUVEC cyclically strained at 20%. c-Myc protein increased 4-fold in HAEC. In HUVEC, expression of mRNA peaked at 1.5-2 h. Subsequently, the effect of modulating c-Myc on potential downstream gene targets was determined. A small molecular weight compound that binds to and stabilizes the silencer element in the c-Myc promoter attenuates cyclic strain-induced c-Myc transcription by about 50%. This compound also modulates c-Myc downstream gene targets that may be instrumental in induction of vascular disease. Cyclic strain-induced gene expression of vascular endothelial growth factor, proliferating cell nuclear antigen and heat shock protein 60 are attenuated by this compound. These results offer a possible mechanism and promising clinical treatment for vascular diseases initiated by increased cyclic strain.
Assuntos
Células Endoteliais/metabolismo , Mecanotransdução Celular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sítios de Ligação , Células Cultivadas , Chaperonina 60/metabolismo , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Estresse Mecânico , Fatores de Tempo , Ativação Transcricional , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
To simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress to mimic the hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average: 1 dyn/cm(2), maximum: +11 dyn/cm(2), minimum: -11 dyn/cm(2), 1 Hz), arterial steady shear stress (15 dyn/cm(2)), and low steady shear stress (1 dyn/cm(2)) on gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed that most differentially expressed genes were similarly regulated by all three shear stress regimens compared with static culture. Comparisons of the three shear stress regimens to each other identified 138 genes regulated by low average shear stress and 22 genes regulated by fluid reversal. Low average shear stress induced increased cell proliferation compared with high shear stress. Only reversing shear stress exposure induced monocyte adhesion. The adhesion of monocytes was partially inhibited by the incubation of endothelial cells with ICAM-1 blocking antibody. Increased heparan sulfate proteoglycan expression was observed on the surface of cells exposed to reversing shear stress. Heparinase III treatment significantly reduced monocyte adhesion. Our results suggest that low steady shear stress is the major impetus for differential gene expression and cell proliferation, whereas reversing flow regulates monocyte adhesion.
Assuntos
Aterosclerose/patologia , Aterosclerose/fisiopatologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiopatologia , Fluxo Sanguíneo Regional/fisiologia , Estresse Mecânico , Aterosclerose/metabolismo , Fenômenos Biomecânicos , Adesão Celular/fisiologia , Proliferação de Células , Células Cultivadas , Endotélio Vascular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Tempo , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo , Veias Umbilicais/fisiopatologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
We investigated the hypothesis that transmigration drives monocyte transcriptional changes. Using Agilent whole human genome microarrays, we identified over 692 differentially expressed genes (2x, P<0.05) in freshly isolated human monocytes following 1.5 h of transmigration across IL-1beta-stimulated ECs compared with untreated monocytes. Genes up-regulated by monocyte transmigration belong to a number of over-represented functional groups including immune response and inhibition of apoptosis. qRT-PCR confirmed increased expression of MCP-1 and -3 and of NAIP following monocyte transmigration. Additionally, quantification of Annexin V binding revealed a reduction in apoptosis following monocyte transmigration. Comparison of gene expression in transmigrated monocytes with additional controls (monocytes that failed to transmigrate and monocytes incubated beneath stimulated ECs) revealed 89 differentially expressed genes, which were controlled by the process of diapedesis. Functional annotation of these genes showed down-regulation of antimicrobial genes (e.g., alpha-defensin down 50x, cathelicidin down 9x, and CTSG down 3x). qRT-PCR confirmed down-regulation of these genes. Immunoblots confirmed that monocyte diapedesis down-regulates alpha-defensin protein expression. However, transmigrated monocytes were functional and retained intact cytokine and chemokine release upon TLR ligand exposure. Overall, these data indicate that the process of monocyte transmigration across stimulated ECs promotes further monocyte recruitment and inhibits monocyte apoptosis. Unexpectedly, following transmigration, monocytes displayed reduced antimicrobial protein expression.
Assuntos
Apoptose/imunologia , Movimento Celular/imunologia , Endotélio Vascular/imunologia , Regulação da Expressão Gênica/imunologia , Monócitos/imunologia , alfa-Defensinas/imunologia , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Monócitos/citologia , Monócitos/metabolismo , Fatores de Tempo , alfa-Defensinas/biossínteseRESUMO
AIMS: CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions. METHODS AND RESULTS: We investigated CYP1A1 and CYP1B1 expressions under different durations, levels, and patterns of shear stress. CYP1A1 and CYP1B1 mRNA, protein, and enzymatic activity were maximally up-regulated at > or =24 h of arterial levels of shear stress (15-25 dynes/cm2). Expression of both genes was significantly attenuated by reversing shear stress when compared with 15 dynes/cm2 steady shear stress. Small interfering RNA knockdown of CYP1A1 resulted in significantly reduced CYP1B1 and thrombospondin-1 expression, genes regulated by the aryl hydrocarbon receptor (AhR). Immunostaining of human coronary arteries showed constitutive CYP1A1 and CYP1B1 protein expressions in endothelial cells. Immunostaining of mouse aorta showed nuclear localization of AhR and increased expression of CYP1A1 in the descending thoracic aorta, whereas reduced nuclear localization of AhR and attenuated CYP1A1 expression were observed in the lesser curvature of the aortic arch. CONCLUSION: CYP1A1 and CYP1B1 gene and protein expressions vary with time, magnitude, and pattern of shear stress. Increased CYP1A1 gene expression modulates AhR-regulated genes. Based on our in vitro reversing flow data and in vivo immunostained mouse aorta, we suggest that increased expression of both genes reflects an anti-atherogenic endothelial cell phenotype.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/enzimologia , Animais , Aorta/enzimologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Aterosclerose/enzimologia , Aterosclerose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Vasos Coronários/enzimologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fluxo Pulsátil , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Fluxo Sanguíneo Regional , Estresse Mecânico , Trombospondina 1/metabolismo , Fatores de TempoRESUMO
During the inflammatory response, endothelial cell (EC) functions and mechanics change dramatically. To understand these responses, the authors analyzed changes in EC gene expression in an in vitro model of inflammation using cDNA microarrays. After interleukin-1 beta (IL1beta) stimulation, over 2500 genes were differentially expressed, of which approximately 2000 had not been previously identified by microarray studies of IL1beta stimulation in human umbilical vein endothelial cells (HUVECs). Functional grouping of these genes according to gene ontologies revealed genes associated with apoptosis, cell cycle, nuclear factor (NF)-kappa B cascade, chemotaxis, and immune response. Interestingly, claudin-1, known to exist in endothelial cell-cell junctions was up-regulated, but claudin-5 and occludin, which also exist in EC junctions, were down-regulated. Pre-b-cell colony enhancing factor (PBEF), a cytokine which may play a role in regulating endothelial permeability, was also up-regulated following IL1beta stimulation. Neutrophil transmigration across IL1beta-stimulated ECs did not induce changes in EC gene expression as strongly as IL1beta stimulation alone. Nineteen genes after 1 h and 22 genes after 3 h of neutrophil application were differentially expressed. These results indicate that, in terms of transcriptional effects on ECs, neutrophil transmigration is a relatively small perturbation in comparison to the background of large scale changes induced in ECs by cytokine stimulation. Supplementary materials are available for this article. Go to the publisher's online edition of Endothelium for the following free supplementary resources: supplementary figures and tables.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Expressão Gênica , Interleucina-1beta/farmacologia , Neutrófilos/fisiologia , Adulto , Técnicas de Cultura de Células , Movimento Celular , Células Cultivadas , Claudina-1 , Claudina-5 , Citocinas/genética , Citocinas/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/citologia , Humanos , Inflamação/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
Pulsations in arterial blood flow expose the endothelium to diverse mechanical forces that may differentially regulate endothelial cell (EC) phenotype. We postulated that pulsatile non-reversing shear stress (typical of the common carotid artery), would produce a more "athero-protective" gene expression pattern compared with steady shear stress of the same mean value. Transcriptional analysis of human umbilical vein endothelial cells (HUVEC) subjected to 24 h of pulsatile shear stress (average = 13 dyne/cm(2), range = 7-25 dyne/cm(2); 1 Hz) or steady shear stress (13 dyne/cm(2)) identified approximately 200 differentially expressed genes. Hierarchical cluster analysis indicated that HUVEC respond similarly to both types of shear stress (Pearson correlation coefficient = 0.785). However, categorization of the differentially expressed genes with Ingenuity Pathways Analysis and with Expression Analysis Systematic Explorer revealed possible differences in nitric oxide (NO) production and signaling. Consistent with gene expression analysis, pulsatile shear stress significantly attenuated NO production relative to steady shear stress (0.77 +/- 0.08, p < 0.01) in HUVEC without significantly altering the levels of intracellular reactive oxygen species (0.95 +/- 0.14, p = 0.65). These results demonstrate that the common carotid flow waveform elicits subtle changes in HUVEC responses to arterial levels of shear stress, which lead to differences in NO production.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Cardiovasculares , Óxido Nítrico/metabolismo , Fluxo Pulsátil/fisiologia , Células Cultivadas , Humanos , Resistência ao CisalhamentoRESUMO
The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H(2)O(2) production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H(2)O(2) levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC.
Assuntos
Aorta/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesão Celular , Células Endoteliais/metabolismo , Mecanotransdução Celular , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Veias Umbilicais/metabolismo , Aorta/citologia , Catalase/metabolismo , Forma Celular , Células Cultivadas , Glutationa Peroxidase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Fenótipo , Fluxo Pulsátil , Estresse Mecânico , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Veias Umbilicais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Pulsations in blood flow alter gene and protein expressions in endothelial cells (EC). A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier-Stokes equation solution. Following 24 h of steady flow (SF: 13 dyne/cm2), pulsatile arterial flow (AF: average = 13 dyne/cm2, range = 7-25 dyne/cm2) or static conditions, heme oxygenase-1 (HO-1) and prostaglandin H synthase-2 (PGHS-2) mRNA and protein expressions from human umbilical vein endothelial cells were measured. Relative to steady flow, pulsatile arterial flow significantly attenuated mRNA upregulation of HO-1 (SF: 7.26 +/- 2.70-fold over static, AF: 4.84 +/- 0.37-fold over static; p < 0.01) and PGHS-2 (SF: 6.11+/-1.79-fold over static, AF: 3.54+/-0.79-fold over static; p < 0.001). Pulsatile arterial flow (4.57+/-0.81-fold over static, p < 0.01) also significantly reduced the steady-flow-induced HO-1 protein upregulation (7.99 +/- 1.29-fold over static). These findings reveal that EC can discriminate between different flow patterns of the same average magnitude and respond at the molecular level.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Células Endoteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Heme Oxigenase-1/biossíntese , Modelos Cardiovasculares , Artéria Carótida Primitiva/fisiologia , Células Cultivadas , Simulação por Computador , Células Endoteliais/citologia , Estresse MecânicoRESUMO
Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle alpha-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased alpha-actin.
Assuntos
Movimento Celular , Proliferação de Células , Proteínas de Membrana Transportadoras/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Fosfoproteínas/metabolismo , Biossíntese de Proteínas , Animais , Células Cultivadas , Proteínas de Membrana Transportadoras/deficiência , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Cadeias Pesadas de Miosina/biossíntese , NADPH Oxidases/deficiência , Fosfoproteínas/deficiência , Espécies Reativas de Oxigênio/metabolismo , Tropomiosina/biossíntese , Doenças Vasculares/metabolismo , Doenças Vasculares/terapiaRESUMO
Microarrays were utilized to determine gene expression of vascular endothelial cells (ECs) subjected to mechanical stretch for insight into the role of strain in vascular pathophysiology. Over 4,000 genes were screened for expression changes resulting from cyclic strain (10%, 1 Hz) of human umbilical vein ECs for 6 and 24 h. Comparison of t-statistics and adjusted P values identified genes having significantly different expression between strained and static cells but not between strained and motion control. Relative to static, 6 h of cyclic stretch upregulated two genes and downregulated two genes, whereas 24 h of cyclic stretch upregulated eight genes but downregulated no genes. However, incorporating the motion control revealed that fluid agitation over the cells, rather than strain, is the primary regulator of differential expression. Furthermore, no gene exceeded a threefold change when comparing cyclic strain to either static or motion control. Quantitative real-time polymerase chain reaction confirmed the dominance of fluid agitation in gene regulation with the exception of heat shock protein 10 at 24 h and plasminogen activator inhibitor 1 at 6 h. Taken together, the small number of differentially expressed genes and their low fold expression levels indicate that cyclic strain is a weak inducer of gene regulation in ECs. However, many of the differentially expressed genes possess antioxidant properties, suggesting that oxidative mechanisms direct EC adaptation to cyclic stretch.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Antioxidantes/metabolismo , Células Cultivadas , DNA Complementar/metabolismo , Regulação para Baixo , Expressão Gênica , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Humanos , Hipertensão/metabolismo , Modelos Estatísticos , Movimento , Ativadores de Plasminogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Fatores de Tempo , Veias Umbilicais/metabolismo , Regulação para CimaRESUMO
Third-passage human umbilical vein endothelial cells (HUVECs) or fifth-passage human aortic endothelial cells (HAECs) were subjected to 25 dynes/cm(2) for 24 h in a parallel-plate flow system. Matched control cells were maintained in static conditions. Total RNA was isolated and pooled from six to eight slides per experiment. Changes in gene expression were analyzed by Northern blots and reverse transcriptase-polymerase chain reaction. Fold changes were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) values. In HUVECs, arterial levels of shear stress increased mRNA expression of Cytochrome P450 1A1 (CYP1A1) 10.8 +/- 2.1-fold, and CYP1B1 23.1 +/- 3.7-fold; whereas connective tissue growth factor (CTGF) expression was unchanged and endothelin-1 (ET-1) mRNA expression was decreased 0.7 +/- 0.05-fold. The authors determined whether these changes were induced by beta-naphthoflavone, a polyaromatic hydrocarbon, and whether they occurred in HAECs. beta-Naphthoflavone up-regulated CYP1A1 18.3 +/- 4.2-fold, and CYP1B1 4.1 +/- 0.3-fold in HUVECs. Shear stress up-regulated CYP1A1 6.3 +/- 0.4-fold and CYP1B1 51.1 +/- 2.1-fold in HAECs. In addition, the authors examined CYP1A1 and CYP1B1 proteins translated from these genes. Experiments identical to those described above were performed and the cells harvested for protein identification by Western blot of CYP1A1 and CYP1B1. Protein levels of CYP1A1 in HUVECs were up-regulated under shear stress, whereas protein levels of CYP1B1 were not.
Assuntos
Citocromo P-450 CYP1A1/genética , Sistema Enzimático do Citocromo P-450/genética , Células Endoteliais/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Regulação para Cima/fisiologia , Hidrocarboneto de Aril Hidroxilases , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Meios de Cultivo Condicionados/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/efeitos dos fármacos , Endotelina-1/genética , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Estresse Mecânico , Regulação para Cima/efeitos dos fármacos , beta-Naftoflavona/farmacologiaRESUMO
The cDNA microarray is an extremely beneficial tool for study of differential gene expression in the cardiovascular system. This technique is used in many different applications including drug discovery, environmental science, and the effects of mechanical forces on vascular cell phenotype. The paper reviews work by others, and describes our study on effects of shear stress on vascular endothelial cells. These microarray studies verified earlier findings using Northern and polymerase chain reaction (PCR) analyses in this area; and also found previously unidentified differentially expressed genes, leading to new hypotheses regarding how cells and tissues respond to biochemical and mechanical stimuli.