血流剪切力在动脉粥样硬化形成中的作用

血管内皮位于血管壁和血液的界面,直接与血流接触而持续受血流剪切力的作用。血管内皮细胞能感受血流机械力的变化,通过激活相应的信号通路调节血管内皮和平滑肌的结构和功能。研究发现,血液流动力的形式与动脉粥样硬化的发生发展有密切的关系。本综述将就血流剪切力与动脉粥样硬化的相互关系及作用机制的最新研究进展作简要介绍。     

Role of Cbl in shear-activation of PI 3-kinase and JNK in endothelial cells

Fluid shear stress can activate PI-3 kinase and JNK in vascular endothelial cells. This study was designed to establish the role of Cbl as an upstream molecule in the shear stress activation of PI-3 kinase and JNK. Confluent monolayers of bovine aortic endothelial cells (BAECs) were subjected to a shear stress of 12 dyn/cm(2) over intervals ranging from 0.5 to 30 min. Shear stress increased Cbl phosphorylation to 2.9-fold of control and Cbl association with the regulatory PI-3 kinase subunit p85 to 5.4-fold. The PI-3 kinase activity measured in Cbl-immunoprecipitated complexes increased to 11.7-fold in response to shear, suggesting that the shear stress activation of PI-3 kinase involves its association with Cbl. Furthermore, the shear stress induction of JNK was attenuated by a negative mutant of Cbl. Finally, shear stress caused an activation of PI 3-kinase only in BAECs seeded onto fibronectin, vitronectin, or laminin, but not poly-l-lysine. Our results suggest that Cbl plays a critical role in the shear stress induction of PI 3-kinase and JNK activities, and that this shear-induced activation requires the interaction of endothelial integrins with extracellular matrix proteins.     

Selective modulation of endothelial cell [Ca2+]i response to flow by the onset rate of shear stress

The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration ([Ca2+]i) increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime.     

Shear stress augments the endothelial cell differentiation marker expression in late EPCs by upregulating integrins

Vascular endothelial cell injury has been implicated in the onset of atherosclerosis. A number of previous studies have demonstrated that endothelial progenitor cells (EPCs), in particular late EPCs, play important roles in endothelial maintenance and repair. Recent evidence has revealed shear stress as a key regulator for EPC differentiation. However, the detailed events that contribute to the shear stress-induced EPC differentiation, in particular the mechanisms of mechanotransduction, remain to be identified. The present study was undertaken to further confirm the effects of shear stress on the late EPC differentiation, and to investigate the role of integrins in this procedure. Shear stress was observed to increase the expression of endothelial cell differentiation markers, such as vWF and CD31, in late EPCs isolated from rat bone marrow. Shear stress moreover enhanced the mRNA expression of integrin subunits β(1) and β(3) in a time-dependent manner, and also upregulated specific integrins in late EPCs plated on substrates containing various extracellular matrix (ECM) proteins. In addition, the shear stress-induced vWF and CD31 expression were found to be related to the levels of integrin β(1) and β(3), and were inhibited in late EPCs treated with RGD peptide (Gly-Arg-Gly-Asp-Asn-Pro, GRGDNP) that blocks the binding of integrins to the extracellular matrix. Additionally, this increase was also attenuated by both anti-β(1) integrin and anti-β(3) integrin antibodies. The integrin subunits β(1) and β(3) thus play important roles in regulating the shear stress-induced endothelial cell differentiation marker expression in late EPCs. This may provide novel insights into the mechanisms of mechanotransduction in shear stress-mediated late EPC differentiation.     

Endothelial mechanosensors: the gatekeepers of vascular homeostasis and adaptation under mechanical stress

Endothelial cells (ECs) not only serve as a barrier between blood and extravascular space to modulate the exchange of fluid, macromolecules and cells, but also play a critical role in regulation of vascular homeostasis and adaptation under mechanical stimulus via intrinsic mechanotransduction. Recently, with the dissection of microdomains responsible for cellular responsiveness to mechanical stimulus, a lot of mechanosensing molecules (mechanosensors) and pathways have been identified in ECs. In addition, there is growing evidence that endothelial mechanosensors not only serve as key vascular gatekeepers, but also contribute to the pathogenesis of various vascular disorders. This review focuses on recent findings in endothelial mechanosensors in subcellular microdomains and their roles in regulation of physiological and pathological functions under mechanical stress.     

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.     

Ionizing radiation increases adhesiveness of human aortic endothelial cells via a chemokine-dependent mechanism

Exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Since radiation also induces inflammation, a possible mechanism is a change in the adhesiveness of vascular endothelial cells, triggering pro-atherogenic accumulation of leukocytes. To investigate this mechanism at the cellular level, the effect of X rays on adhesiveness of cultured human aortic endothelial cells (HAECs) was determined. HAECs were grown as monolayers and exposed to 0 to 30 Gy X rays, followed by measurement of adhesiveness under physiological shear stress using a flow chamber adhesion assay. Twenty-four hours after irradiation, HAEC adhesiveness was increased, with a peak effect at 15 Gy. Radiation had no significant effect on surface expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Antibody blockade of the leukocyte integrin receptors for ICAM-1 and VCAM-1, however, abolished the radiation-induced adhesiveness. Since these leukocyte integrins can be activated by chemokines presented on the endothelial cell surface, the effect of pertussis toxin (PTX), an inhibitor of chemokine-mediated integrin activation, was tested. PTX specifically inhibited radiation-induced adhesiveness, with no significant effect on nonirradiated cells. Therefore, radiation induces increased adhesiveness of aortic endothelial cells through chemokine-dependent signaling from endothelial cells to leukocytes, even in the absence of increased expression of the adhesion molecules involved.     

Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G₂/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses. shear stress; nitric oxide; cell cycle     

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) affects RhoA activation in endothelial cells

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) expressed in endothelial cells (ECs) plays an important role in angiogenesis due to its involvement in migration, proliferation and network formation. Here we examined the biological function of PILSAP with respect to EC morphogenesis and the related intracellular signaling for this process. When mouse endothelial MSS31 cells were cultured, a dominant negative PILSAP mutant converted cell shape to disk-like morphology, blocked stress fiber formation, and augmented membrane ruffling in random directions. These phenotypic changes led us to test whether PILSAP affected activities of Rho family small G-proteins. Abrogation of PILSAP enzymatic activity or its expression attenuated RhoA but not Rac1 activation during cell adhesion. This attenuation of RhoA activation was also evident when G-protein coupled receptors such as proteinase-activated receptor or lysophosphatidic acid receptor were activated in ECs. These results indicate that PILSAP affects RhoA activation and that influences the proper function of ECs.     

Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFκB: Differential role of the shear stress

Cancer metastasis is a multistep process involving cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of tumor cells (TCs). In this study, cell adhesion molecule (CAM) expression was investigated using an in vitro assay, in which TCs were seeded onto an endothelial cell (ECs) monolayer and cocultured during 5 h. Flow cytometry, confocal microscopy as well as western blot analysis indicated that endothelial ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular Adhesion Molecule-1) and E-selectin were up-regulated after TC-EC coculture, whereas no change was observed for CAMs expression in tumor cells. This increased CAMs expression required tight contact between TCs and ECs. Incubation of ECs with the pyrrolidine-dithiocarbamate NFκB inhibitor prior to coculture, fully prevented coculture-induced expression of endothelial CAMs. Using specific blocking antibodies we showed an implication of ICAM-1 and VCAM-1 for TCs extravasation and VCAM-1 for adhesion. Moreover, fluid flow experiments revealed that high shear stress totally abolished coculture-induced as well as TNFα-induced CAMs over-expression. This study suggests that TCs could act as a potent inflammatory stimulus on ECs by inducing CAMs expression via NFκB activation, and that this action can be modulated by shear stress.     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner

We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity.     

Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment

Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin alpha(v)beta3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear-induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of flow. The results therefore define the role of integrins and Rho in a pathway leading to endothelial cell adaptation to flow.     

In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(V)beta(3) integrin for tumor imaging

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(V)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(V)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both 99mTc peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of alpha(V)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.     

JNK2 promotes endothelial cell alignment under flow

Endothelial cells in straight, unbranched segments of arteries elongate and align in the direction of flow, a feature which is highly correlated with reduced atherosclerosis in these regions. The mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) is activated by flow and is linked to inflammatory gene expression and apoptosis. We previously showed that JNK activation by flow is mediated by integrins and is observed in cells plated on fibronectin but not on collagen or basement membrane proteins. We now show thatJNK2 activation in response to laminar shear stress is biphasic, with an early peak and a later peak. Activated JNK localizes to focal adhesions at the ends of actin stress fibers, correlates with integrin activation and requires integrin binding to the extracellular matrix. Reducing JNK2 activation by siRNA inhibits alignment in response to shear stress. Cells on collagen, where JNK activity is low, align slowly. These data show that an inflammatory pathway facilitates adaptation to laminar flow, thereby revealing an unexpected connection between adaptation and inflammatory pathways.     

Fluid shear stress‐induced JNK activity leads to actin remodeling for cell alignment

Fluid shear stress (FSS) exerted on endothelial cell (EC) surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N‐terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in ECs exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm² FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm² flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 min after flow onset with an eightfold activity increase compared to cells in static culture. FSS‐induced phospho‐JNK co‐localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS‐induced actin structure and distribution as a response to FSS. Our results indicate that FSS‐induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in ECs. J. Cell. Physiol. 226: 110–121, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.