RESUMO
Mechanobiology at the cellular level is concerned with what phenotypes that cells exhibit to maintain homeostasis in their normal physiological mechanical environment, as well as what phenotypical changes that cells have to make when their environment is altered. Mechanobiology at the molecular level aims to understand the molecular underpinning of how cells sense, respond to, and adapt to mechanical cues in their environment. In this Perspective, we use our work inspired by and in collaboration with Professor Shu Chien as an example with which we connect the mechanobiology between the cellular and molecular levels. We discuss how physical forces acting on intracellular proteins may impact protein-protein interaction, change protein conformation, crosstalk with biochemical signaling molecules, induce mechanotransduction, and alter the cell structure and function.
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
Biomechanical factors profoundly influence the processes of tissue growth, development, maintenance, degeneration, and repair. Regenerative strategies to restore damaged or diseased tissues in vivo and create living tissue replacements in vitro have recently begun to harness advances in understanding of how cells and tissues sense and adapt to their mechanical environment. It is clear that biomechanical considerations will be fundamental to the successful development of clinical therapies based on principles of tissue engineering and regenerative medicine for a broad range of musculoskeletal, cardiovascular, craniofacial, skin, urinary, and neural tissues. Biomechanical stimuli may in fact hold the key to producing regenerated tissues with high strength and endurance. However, many challenges remain, particularly for tissues that function within complex and demanding mechanical environments in vivo. This paper reviews the present role and potential impact of experimental and computational biomechanics in engineering functional tissues using several illustrative examples of past successes and future grand challenges.
Assuntos
Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Fenômenos Biomecânicos , História do Século XX , História do Século XXI , Humanos , Medicina Regenerativa/história , Medicina Regenerativa/tendências , Engenharia Tecidual/história , Engenharia Tecidual/tendênciasRESUMO
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
Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbalpha dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbalpha-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbalpha on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.
Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Plaquetas/metabolismo , Humanos , Microscopia de Força Atômica , Microesferas , Modelos Biológicos , Conformação Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Estresse Mecânico , Doenças de von Willebrand/metabolismoRESUMO
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
Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination. RGDS is a bioactive sequence found in a number of proteins, and P2 is a membrane-activated complex-1 (Mac-1) ligand located in the gamma-chain of the fibrinogen protein. We evaluated, via video microscopy, cell motility by measuring cell displacement from origin and total accumulated distance traveled and then calculated average velocity. Results indicate that although adhesion and shape change were supported by hydrogels containing RGD alone, motility was not. Mac-1-dependent motility was supported on hydrogels containing P2 alone. Motility was enhanced through combined presentation of RGD and P2, engaging Mac-1, alpha(V)beta(3), and beta(1) integrins. Naïve neutrophil motility on combined peptide substrates was dependent on Mac-1, and alpha(4)beta(1) while alpha(6)beta(1) contributed to speed and linear movement. Transmigrated neutrophil motility was dependent on alpha(v)beta(3) and alpha(5)beta(1), and alpha(4)beta(1), alpha(6)beta(1), and Mac-1 contributed to speed and linear motion. Together, the data demonstrate that efficient neutrophil migration, dependent on multi-integrin interaction, is enhanced after transendothelial migration.
Assuntos
Quimiotaxia de Leucócito/imunologia , Células Endoteliais/imunologia , Integrinas/imunologia , Neutrófilos/imunologia , Movimento Celular/imunologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Hidrogéis , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
Endothelial cells synthesize and secrete von Willebrand factor (VWF) multimers, including unusually large forms (ULVWF), which are usually cleaved into smaller multimers found in normal plasma (P-VWF). Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder characterized by systemic attachment of platelets to inadequately cleaved ULVWF multimers. We have compared ULVWF and P-VWF in their capacity to become immobilized onto surfaces in vitro and their ability to mediate platelet adhesion. We have also used functional assays to directly compare ULVWF forms with purified P-VWF in mediating platelet aggregation in solution. At comparable concentrations, ULVWF is more effectively adsorbed onto glass surfaces than P-VWF and supports increased platelet adhesion. ULVWF is also significantly more potent than P-VWF in mediating both shear-induced platelet aggregation and ristocetin-mediated platelet agglutination.