Effect of combined cyclic stretch and fluid shear stress on endothelial cell morphological responses

Endothelial cells in vivo are normally subjected to multiple mechanical stimuli such as stretch and fluid shear stress (FSS) but because each stimulus induces magnitude-dependent morphologic responses, the relative importance of each stimulus in producing the normal in vivo state is not clear Using cultured human aortic endothelial cells, this study first determined equipotent levels of cyclic stretch, steady FSS, and oscillatory FSS with respect to the time course of cell orientation. We then tested whether these levels of stimuli were equipotent in combination with each other by imposing simultaneous cyclic stretch and steady FSS or cyclic stretch and oscillatory FSS so as to reinforce or counteract the cells' orientation responses. Equipotent levels of the three stimuli were 2% cyclic stretch at 2%/s, 80 dynes/cm2 steady FSS and 20 +/- 10 dynes/cm2 oscillatory FSS at 20 dyne/cm2-s. When applied in reinforcing fashion, cyclic stretch and oscillatory, but not steady, FSS were additive. Both pairs of stimuli canceled when applied in counteracting fashion. These results indicate that this level of cyclic stretch and oscillatory FSS sum algebraically so that they are indeed equipotent. In addition, oscillatory FSS is a stronger stimulus than steady FSS for inducing cell orientation. Moreover, arterial endothelial cells in vivo are likely receiving a stronger stretch than FSS stimulus.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.     

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow-induced stimulation of nuclear factor kappaB (NF-kappaB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Regulation of endothelial connexin40 expression by shear stress

Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.     

Effect on endothelial cell gene expression of shear stress, oxygen concentration, and low-density lipoprotein as studied by a novel flow cell culture system

A new cell culture system has been developed that reflects the vascular microenvironment. By means of this system the cultured cells are exposed not only to shear stress by the circulating culture medium, but also to an oxygen concentration gradient and certain critical blood components such as low-density lipoprotein (LDL) and monocytes. DNA microarray analysis was performed for human umbilical vein endothelial cells cultured in this system in the absence and presence of laminar flow at a low shear stress, 0.2 dyn/cm(2). In addition to shear stress, either an oxygen concentration gradient, or LDL (1 mg/ml), or both were applied. Many Nrf-2-regulating genes, such as heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, solute carrier family 7 No. 11, and glutamate-cysteine ligase modifier subunit, were induced by laminar flow at very low shear stress regardless of the additional conditions. Certain genes were specifically affected by exposure to the oxygen gradient and/or LDL under shear stress, but the degree was very low. These results suggest that shear stress is the most critical factor affecting gene expression in endothelial cells and that Nrf-2-regulating proteins may contribute to protecting endothelial cells against other vascular stress. This system should provide highly relevant and useful information about both vascular physiology and pathology, in the latter on such urgent matters as the specific steps involved in atherogenesis.     

Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype

Vascular endothelium, the cellular monolayer lining the entire cardiovascular system, is exposed to a variety of biochemical and biomechanical stimuli. Fluid shear stresses generated by blood flow in the vasculature can profoundly influence the phenotype of the endothelium by regulating the activity of certain flow-sensitive proteins (for example, enzymes), as well as by modulating gene expression. The finding that specific fluid mechanical forces can alter endothelial structure and function has provided a framework for a mechanistic understanding of flow-dependent processes, ranging from vascular remodeling in response to hemodynamic changes, to the initiation and localization of chronic vascular diseases such as atherosclerosis.     

Renal tubular fluid shear stress promotes endothelial cell activation

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. In this study, we hypothesized that changes in urinary FSS represent a tubular aggression that contributes to the development of inflammation, a key event in progression of nephropathies. Human renal tubular cells (HK-2) were exposed to FSS for 30 min at 0.01 Pa. Treatment of human endothelial cells (HMEC-1) with the resulting conditioned medium (FSS-CM) increased C-C chemokine ligand 2 (CCL2) and tumor necrosis factor (TNF)-α protein secretion, increased endothelial vascular adhesion molecule-1 (VCAM-1) mRNA expression and stimulated adhesion of human (THP-1) monocytes to the endothelial monolayer. These effects were TNF-α dependent as they were abolished by neutralization of TNF-α. Interestingly, the origin of TNF-α was not epithelial, but resulted from autocrine endothelial production. However, in contrast to short term FSS, long term FSS (5 h) induced the release of the key inflammatory proteins CCL2 and TNF-α directly from tubular cells. In conclusion, these results suggest for the first time that urinary FSS can contribute to the inflammatory state involved in initiation/perpetuation of renal diseases.     

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.     

Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism

Mammalian cells responds to physical forces by altering their growth rate, morphology, metabolism, and genetic expression. We have studied the mechanism by which these cells detect the presence of mechanical stress and convert this force into intracellular signals. As our model systems, we have studied cultured human endothelial cells, which line the blood vessels and forms the interface between the blood and the vessel wall. These cell responds within minutes to the initiation of flow by increasing their arachidonic acid metabolism and increasing the level of the intracellular second messengers inositol trisphosphate and calcium ion concentration. With continued exposure to arterial levels of wall shear stress for up to 24 h, endothelial cells increase the expression of tissue plasminogen activator (tPA) and tPA messenger RNA (mRNA) and decrease the expression of endothelin peptide and endothelin mRNA. Since the initiation of flow also causes enhanced convective mass transfer to the endothelial cell monolayer, we have investigated the role of enhanced convection of adenosine trisphosphate (ATP) to the cell surface in eliciting a cellular response by monitoring cytosolic calcium concentrations on the single cell level and by computing the concentration profile of ATP in a parallel-plate flow geometry. Our result demonstrate that endothelial cells respond in very specific ways to the initiation of flow and that mass transfer and fluid shear stress can both play a role in the modulation of intracellular signal transduction and metabolism.     

Effects of pulsatile shear stress on nitric oxide production and endothelial cell nitric oxide synthase expression by ovine fetoplacental artery endothelial cells

Placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression increase during pregnancy. Shear stress, the frictional force exerted on endothelial cells by blood flow, stimulates vessel dilation, endothelial NO production, and eNOS expression. In order to study the effects of pulsatile flow/shear stress, we adapted Cellco CELLMAX artificial capillary modules to study ovine fetoplacental artery endothelial (OFPAE) cells for NO production and eNOS expression. OFPAE cells were grown in the artificial capillary modules at 3 dynes/cm2. Confluent cells were then exposed to 10, 15, or 25 dynes/cm2 for up to 24 h. NO production by OFPAE cells exposed to pulsatile shear stress was inhibited to nondetectable levels by the NOS inhibitor l-NMMA and reversed by excess NOS substrate l-arginine. NO production and expression of eNOS mRNA and protein by OFPAE cells were elevated by shear stress in a graded fashion (P < 0.05). The rise in NO production with 25 dynes/cm2 shear stress (8-fold) was greater (P < 0.05) than that observed for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). The acute shear stress-induced rise in NO production by OFPAE cells was via eNOS activation, whereas the prolonged NO rise occurred by elevations in both eNOS expression and enzyme activation. Thus, elevations of placental blood flow and physiologic shear stress may be partly responsible for the increases in placental arterial endothelial eNOS expression and NO production during pregnancy.     

Effects of wall shear stress and its gradient on tumor cell adhesion in curved microvessels

Tumor cell adhesion to vessel walls in the microcirculation is one critical step in cancer metastasis. In this paper, the hypothesis that tumor cells prefer to adhere at the microvessels with localized shear stresses and their gradients, such as in the curved microvessels, was examined both experimentally and computationally. Our in vivo experiments were performed on the microvessels (post-capillary venules, 30–50 μm diameter) of rat mesentery. A straight or curved microvessel was cannulated and perfused with tumor cells by a glass micropipette at a velocity of ~1mm/s. At less than 10 min after perfusion, there was a significant difference in cell adhesion to the straight and curved vessel walls. In 60 min, the averaged adhesion rate in the curved vessels (n = 14) was ~1.5-fold of that in the straight vessels (n = 19). In 51 curved segments, 45% of cell adhesion was initiated at the inner side, 25% at outer side, and 30% at both sides of the curved vessels. To investigate the mechanical mechanism by which tumor cells prefer adhering at curved sites, we performed a computational study, in which the fluid dynamics was carried out by the lattice Boltzmann method , and the tumor cell dynamics was governed by the Newton’s law of translation and rotation. A modified adhesive dynamics model that included the influence of wall shear stress/gradient on the association/dissociation rates of tumor cell adhesion was proposed, in which the positive wall shear stress/gradient jump would enhance tumor cell adhesion while the negative wall shear stress/gradient jump would weaken tumor cell adhesion. It was found that the wall shear stress/gradient, over a threshold, had significant contribution to tumor cell adhesion by activating or inactivating cell adhesion molecules. Our results elucidated why the tumor cell adhesion prefers to occur at the positive curvature of curved microvessels with very low Reynolds number (in the order of 10⁻²) laminar flow.     

A disk-type apparatus for applying fluid shear stress on cultured endothelial cell

To study the effect of fluid shear stress on cultured endothelial cells, we have developed an apparatus for the stress creation, which consists of a stainless steel disk driven by an electric DC motor and a stage to place a culture dish and to adjust the distance between the disk and the dish. When the disk is rotated, a concentric fluid movement occurs in the culture medium in the dish and exerts the shear stress on the endothelial cells cultured on the bottom of the dish. A theoretical analyses concerning the induced concentric flow velocity predicted that when the angular velocity of the disk rotation (omega) is slow enough to maintain a Reynolds' number of the order of 10, the exerted wall shear stress tau w on the endothelial cell monolayer is given for a constant as tau w = mu r omega/d where mu is the viscosity of the medium, d the distance from the plate to the monolayer and r the radial distance from the center of the dish. When omega is varied in a sinusoidal mode tau w also becomes sinusoidal, thus allowing to apply a pulsatile stress. In vitro experiments carried out to examine the validity of the theoretical results, using a suspension of polystyrene as a tracer with the ordinary culture medium and 99% ethanol, revealed excellent agreement of the measured velocity profiles with the predicted ones. The results demonstrated that the present apparatus can create both the steady and pulsatile wall shear stress on the culture cell layer as expected, unless Reynolds' number greatly exceeds the level of 10.     

Effects of laminar shear stress on IL-8 mRNA expression in endothelial cells

In order to demonstrate that IL-8 mRNA expression in endothelial cells is not only regulated by chemical factors, but also by mechanical factors, in this article, after pretreating cultured human umbilical vein endothelial cells (HUVECs) with shear stress for different time, we employed both RT-PCR to assay IL-8 mRNA expression and immunocytochemical staining to detect NF-kappaB activation in HUVECs. We found that: (i) IL-8 mRNA expressed little in HUVECs untreated or pretreated with low laminar shear stress for 0.5 hour; IL-8 mRNA expression was increased when HUVECs were pretreated with low laminar shear stress for 1 hour, and increased further when pretreated for 2 hours; (ii) the immunoreactivity of NF-kappaB p65 in the nuclei of HUVECs untreated or pretreated with low laminar shear stress for 0.5 hour was negative, while it became weak positive in the nuclei of HUVECs pretreated with shear stress for 1 hour and positive in the nuclei of HUVECs pretreated for 2 hours. The results imply that low laminar shear stress was capable of inducing IL-8 gene expression and activating NF-kappaB, which were both time-dependent. The induction of IL-8 gene expression by laminar shear stress is probably due to the activation of NF-kappaB. We suggest that IL-8 mRNA expression in endothelial cells induced by low shear stress may play a key role in the pathogenesis and development of both inflammation and arterioatherosclerosis.