A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells

Vascular endothelial cells (ECs) are constantly subjected to blood flow-induced shear stress and the influences of neighboring smooth muscle cells (SMCs). In the present study, a coculture flow system was developed to study the effect of shear stress on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC side subjected to the flow condition. When ECs were exposed to a shear stress of 12 dynes/cm2 for 24 h, the cocultured SMCs tended to orient perpendicularly to the flow direction. This perpendicular orientation of the cocultured SMCs to flow direction was not observed when ECs were exposed to a shear stress of 2 dynes/cm2. Under the static condition, long and parallel actin bundles were observed in the central regions of the cocultured SMCs, whereas the actin filaments localized mainly at the periphery of the cocultured ECs. After 24 h of flow application, the cocultured ECs displayed very long, well-organized, parallel actin stress fibers aligned with the flow direction in the central regions of the cells. Immunostaining of platelet endothelial cell adhesion molecule-1 confirmed the elongation and alignment of the cocultured ECs with the flow direction. Coculture with SMCs under static condition induced EC gene expressions of growth-related oncogene-alpha and monocyte chemotactic protein-1, and shear stress was found to abolish these SMC-induced gene expressions. Our results suggest that shear stress may serve as a down-regulator for the pathophysiologically relevant gene expression in ECs cocultured with SMCs.     

Dynamic modeling for shear stress induced ATP release from vascular endothelial cells

A dynamic model is proposed for shear stress induced adenosine triphosphate (ATP) release from endothelial cells (ECs). The dynamic behavior of the ATP/ADP concentration at the endothelial surface by viscous shear flow is investigated through simulation studies based on the dynamic ATP release model. The numerical results demonstrate that the ATP/ADP concentration against time at endothelium-fluid interface predicted by the dynamic ATP release model is more consistent with the experimental observations than that predicted by previous static ATP release model.     

Proteomic analysis of vascular endothelial cells in response to laminar shear stress

Isotope-coded affinity tags (cICAT) coupled with mass spectrometric analysis is one of the leading technologies for quantitative proteomic profiling and protein quantification. We performed proteomic analysis of bovine aortic endothelial cells (BAEC) in response to laminar shear stress using cICAT labeling coupled with LC-MS/MS. Protein expressions in BAEC under 15 dynes/cm2 of shear stress for 10 min, 3 h, and 6 h were compared with matched stationary controls. Analysis of each sample produced 1800-2400 proteins at >or=75% confidence level. We found 142, 213, and 186 candidate proteins that were up- or down-regulated by at least two-fold after 10 min, 3 h, and 6 h of shear stress, respectively. Some of these proteins have known cellular functions and they encompass many signaling pathways. The signaling pathways that respond to shear stress include those of integrins, G-protein-coupled receptors, glutamate receptors, PI3K/AKT, apoptosis, Notch and cAMP-mediated signaling pathways. The validity of the mass spectrometric analysis was also confirmed by Western blot and confocal immunofluorescence microscopy. The present quantitative proteomic analysis suggests novel potential regulatory mechanisms in vascular endothelial cells in response to shear stress. These results provide preliminary footprints for further studies on the signaling mechanisms induced by shear stress.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

Molecular basis of the effects of shear stress on vascular endothelial cells

Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells (ECs) are subjected to the shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular functions, e.g., proliferation, apoptosis, migration, permeability, and remodeling, as well as gene expression. The ECs use multiple sensing mechanisms to detect changes in mechanical forces, leading to the activation of signaling networks. The cytoskeleton provides a structural framework for the EC to transmit mechanical forces between its luminal, abluminal and junctional surfaces and its interior, including the cytoplasm, the nucleus, and focal adhesion sites. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows. In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.     

Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells.

The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.     

Effect of the endothelial surface layer on transmission of fluid shear stress to endothelial cells

Responses of vascular endothelial cells to mechanical shear stresses resulting from blood flow are involved in regulation of blood flow, in structural adaptation of vessels, and in vascular disease. Interior surfaces of blood vessels are lined with a layer of bound or adsorbed macromolecules, known as the endothelial surface layer (ESL). In vivo investigations have shown that this layer has a width of order 1 microm, that it substantially impedes plasma flow, and that it excludes flowing red blood cells. Here, the effect of the ESL on transmission of shear stress to endothelial cells is examined using a theoretical model. The layer is assumed to consist of a matrix of molecular chains extending from the surface, held in tension by a slight increase in colloid osmotic pressure relative to that in free-flowing plasma. It is shown that, under physiological conditions, shear stress is transmitted to the endothelial surface almost entirely by the matrix, and fluid shear stresses on endothelial cell membranes are very small. Rapid fluctuations in shear stress are strongly attenuated by the layer. The ESL may therefore play an important role in sensing of shear stress by endothelial cells.     

A model for shear stress-induced deformation of a flow sensor on the surface of vascular endothelial cells

Fluid mechanical shear stress elicits humoral, metabolic, and structural responses in vascular endothelial cells (ECs); however, the mechanisms involved in shear stress sensing and transduction remain incompletely understood. Beyond being responsive to shear stress, ECs distinguish among and respond differently to different types of shear stress. Recent observations suggest that endothelial shear stress sensing may occur through direct interaction of the flow with cell-surface structures that act as primary flow sensors. This paper presents a mathematical model for the shear stress-induced deformation of a flow sensor on the EC surface. The sensor is modeled as a cytoskeleton-coupled viscoelastic structure exhibiting standard linear solid behavior. Since ECs respond differently to different types of flow, the deformation and resulting velocity of the sensor in response to steady, non-reversing pulsatile, and oscillatory flow have been studied. Furthermore, the sensitivity of the results to changes in various model parameters including the magnitude of applied shear stress, the constants that characterize the viscoelastic behavior, and the pulsatile flow frequency (f) has been investigated. The results have demonstrated that in response to a suddenly applied shear stress, the sensor exhibits a level of instantaneous deformation followed by gradual creeping to the long-term response. The peak deformation increases linearly with the magnitude of the applied shear stress and decreases for viscoelastic constants that correspond to stiffer sensors. While the sensor deformation depends on f for low f values, the deformation becomes f -independent above a critical threshold frequency. Finally, the peak sensor deformation is considerably larger for steady and non-reversing pulsatile flow than for oscillatory flow. If the extent of sensor deformation correlates with the intensity of flow-mediated endothelial signaling, then our results suggest possible mechanisms by which ECs distinguish among steady, non-reversing pulsatile, and oscillatory shear stress.     

血管内皮细胞内质网应激

内质网是调控细胞内膜型/分泌型蛋白质合成、钙稳态和细胞凋亡的重要细胞器,多种因素影响内质网稳态、触发内质网应激。适当的内质网应激通过激活未折叠蛋白反应促进内质网紊乱的恢复,但过度内质网应激触发内质网相关凋亡途径,参与多种疾病的发生。血管内皮细胞具有高度发达的内质网,对内质网应激非常敏感,本文综述血管内皮细胞内质网应激反应及其在血管损伤相关疾病中的作用。     

A mechanical model for morphological response of endothelial cells under combined wall shear stress and cyclic stretch loadings

The shape and morphology of endothelial cells (ECs) lining the blood vessels are a good indicator for atheroprone and atheroprotected sites. ECs of blood vessels experience both wall shear stress (WSS) and cyclic stretch (CS). These mechanical stimuli influence the shape and morphology of ECs. A few models have been proposed for predicting the morphology of ECs under WSS or CS. In the present study, a mathematical cell population model is developed to simulate the morphology of ECs under combined WSS and CS conditions. The model considers the cytoskeletal filaments, cell–cell interactions, and cell–extracellular matrix interactions. In addition, the reorientation and polymerization of microfilaments are implemented in the model. The simulations are performed for different conditions: without mechanical stimuli, under pure WSS, under pure CS, and under combined WSS and CS. The results are represented as shape and morphology of ECs, shape index, and angle of orientation. The model is validated qualitatively and quantitatively with several experimental studies, and good agreement with experimental studies is achieved. To the best of our knowledge, it is the first model for predicting the morphology of ECs under combined WSS and CS condition. The model can be used to indicate the atheroprone regions of a patient’s artery.     

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.     

Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells

Vascular endothelial cells (ECs) distinguish among and respond differently to different types of fluid mechanical shear stress. Elucidating the mechanisms governing this differential responsiveness is the key to understanding why early atherosclerotic lesions localize preferentially in arterial regions exposed to low and/or oscillatory flow. An early and very rapid endothelial response to flow is the activation of flow-sensitive K⁺ and Cl^– channels that respectively hyperpolarize and depolarize the cell membrane and regulate several important endothelial responses to flow. We have used whole cell current- and voltage-clamp techniques to demonstrate that flow-sensitive hyperpolarizing and depolarizing currents respond differently to different types of shear stress in cultured bovine aortic ECs. A steady shear stress level of 10 dyn/cm² activated both currents leading to rapid membrane hyperpolarization that was subsequently reversed to depolarization. In contrast, a steady shear stress of 1 dyn/cm² only activated the hyperpolarizing current. A purely oscillatory shear stress of 0 ± 10 dyn/cm² with an oscillation frequency of either 1 or 0.2 Hz activated the hyperpolarizing current but only minimally the depolarizing current, whereas a 5-Hz oscillation activated neither current. These results demonstrate for the first time that flow-activated ion currents exhibit different sensitivities to shear stress magnitude and oscillation frequency. We propose that flow-sensitive ion channels constitute components of an integrated mechanosensing system that, through the aggregate effect of ion channel activation on cell membrane potential, enables ECs to distinguish among different types of flow. ion channels; atherosclerosis; mechanotransduction     

Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro

Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1(+)) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and G(2)-M phases of the cell cycle than Flk-1(+) ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle alpha-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1(+) ES cells into the endothelial cell lineage. The shear stressed Flk-1(+) ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-1(+) ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-1(+) ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-1(+) ES cells.     

Secretome of human endothelial cells under shear stress

Endothelial cells are exposed to different types of shear stress which triggers the secretion of subsets of proteins. In this study, we analyzed the secretome of endothelial cells under static, laminar, and oscillatory flow. To differentiate between endogenously expressed and added proteins, isolated human umbilical vein endothelial cells were labeled with l-Lysine-(13)C(6),(15)N(2) and l-Arginine-(13)C(6),(15)N(4). Shear stress was applied for 24 h using a cone-and-plate viscometer. Proteins from the supernatants were isolated, trypsinized, and finally analyzed using LC-MS/MS (LTQ). Under static control condition 395 proteins could be identified, of which 78 proteins were assigned to the secretome according to Swiss-Prot database. Under laminar shear stress conditions, 327 proteins (83 secreted) and under oscillatory shear stress 507 proteins (79 secreted) were measured. We were able to identify 6 proteins specific for control conditions, 8 proteins specific for laminar shear stress, and 5 proteins specific for oscillatory shear stress. In addition, we identified flow-specific secretion patterns like the increased secretion of cell adhesion proteins and of proteins involved in protein binding. In conclusion, the identification of shear stress specific secreted proteins (101 under different flow conditions) emphasizes the role of endothelial cells in modulating the plasma composition according to the physiological requirements.