Flow shear stress regulates endothelial barrier function and expression of angiogenic factors in a 3D microfluidic tumor vascular model

Endothelial cells lining blood vessels are exposed to various hemodynamic forces associated with blood flow. These include fluid shear, the tangential force derived from the friction of blood flowing across the luminal cell surface, tensile stress due to deformation of the vessel wall by transvascular flow, and normal stress caused by the hydrodynamic pressure differential across the vessel wall. While it is well known that these fluid forces induce changes in endothelial morphology, cytoskeletal remodeling, and altered gene expression, the effect of flow on endothelial organization within the context of the tumor microenvironment is largely unknown. Using a previously established microfluidic tumor vascular model, the objective of this study was to investigate the effect of normal (4 dyn/cm²), low (1 dyn/cm²), and high (10 dyn/cm²) microvascular wall shear stress (WSS) on tumor-endothelial paracrine signaling associated with angiogenesis. It is hypothesized that high WSS will alter the endothelial phenotype such that vascular permeability and tumor-expressed angiogenic factors are reduced. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability relative to mono-cultures. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, gene expression analysis revealed that high WSS (10 dyn/cm²) significantly down-regulates tumor-expressed MMP9, HIF1, VEGFA, ANG1, and ANG2, all of which are important factors implicated in tumor angiogenesis. This result was not observed in tumor mono-cultures or static conditioned media experiments, suggesting a flow-mediated paracrine signaling mechanism exists with surrounding tumor cells that elicits a change in expression of angiogenic factors. Findings from this work have significant implications regarding low blood velocities commonly seen in the tumor vasculature, suggesting high shear stress-regulation of angiogenic activity is lacking in many vessels, thereby driving tumor angiogenesis.     

Fluid-Flow Induced Wall Shear Stress and Epithelial Ovarian Cancer Peritoneal Spreading

Epithelial ovarian cancer (EOC) is usually discovered after extensive metastasis have developed in the peritoneal cavity. The ovarian surface is exposed to peritoneal fluid pressures and shear forces due to the continuous peristaltic motions of the gastro-intestinal system, creating a mechanical micro-environment for the cells. An in vitro experimental model was developed to expose EOC cells to steady fluid flow induced wall shear stresses (WSS). The EOC cells were cultured from OVCAR-3 cell line on denuded amniotic membranes in special wells. Wall shear stresses of 0.5, 1.0 and 1.5 dyne/cm² were applied on the surface of the cells under conditions that mimic the physiological environment, followed by fluorescent stains of actin and β-tubulin fibers. The cytoskeleton response to WSS included cell elongation, stress fibers formation and generation of microtubules. More cytoskeletal components were produced by the cells and arranged in a denser and more organized structure within the cytoplasm. This suggests that WSS may have a significant role in the mechanical regulation of EOC peritoneal spreading.     

Physiological hydrostatic pressure protects endothelial monolayer integrity

Endothelial monolayer integrity is required to maintain endothelial barrier functions and has found to be impaired in several disorders like inflammatory edema, allergic shock, or artherosclerosis. Under physiologic conditions in vivo, endothelial cells are exposed to mechanical forces such as hydrostatic pressure, shear stress, and cyclic stretch. However, insight into the effects of hydrostatic pressure on endothelial cell biology is very limited at present. Therefore, in this study, we tested the hypothesis that physiological hydrostatic pressure protects endothelial monolayer integrity in vitro. We investigated the protective efficacy of hydrostatic pressure in microvascular myocardial endothelial (MyEnd) cells and macrovascular pulmonary artery endothelial cells (PAECs) by the application of selected pharmacological agents known to alter monolayer integrity in the absence or presence of hydrostatic pressure. In both endothelial cell lines, extracellular Ca(2+) depletion by EGTA was followed by a loss of vascular-endothelial cadherin (VE-caherin) immunostaining at cell junctions. However, hydrostatic pressure (15 cmH(2)O) blocked this effect of EGTA. Similarly, cytochalasin D-induced actin depolymerization and intercellular gap formation and cell detachment in response to the Ca(2+)/calmodulin antagonist trifluperazine (TFP) as well as thrombin-induced cell dissociation were also reduced by hydrostatic pressure. Moreover, hydrostatic pressure significantly reduced the loss of VE-cadherin-mediated adhesion in response to EGTA, cytochalasin D, and TFP in MyEnd cells as determined by laser tweezer trapping using VE-cadherin-coated microbeads. In caveolin-1-deficient MyEnd cells, which lack caveolae, hydrostatic pressure did not protect monolayer integrity compromised by EGTA, indicating that caveolae-dependent mechanisms are involved in hydrostatic pressure sensing and signaling.     

Rotating wall vessel as a newin vitro shear stress generation system: Application to rat coronary endothelial cell cultures

In this paper, we describe a simple new design for the application of controlled, top-hat profiled wall shear stress forces in a way that is independent of hydrostatic pressure and oxygen tension, based on a rotating wall vessel system. This system has been applied to the culture of rat coronary endothelial cells obtained with a Langendorff-derived procedure isolation. Endothelial cells are immunopurified on the basis of RECA expression, and conservation of endothelial phenotype has been assessed on the basis of morphology, RECA and von Willebrand factor expressions and diI-Ac-LDL uptake. Shear stress induced by the rotating wall vessel was measured using a mathematical formula specifically designed for this type of model, and its impact on coronary endothelial cells was evaluated. Shear stress produced cell orientation parallel to the flux direction, elevated NO production and decreased monocyte adhesion. Cells were kept viable and functional for at least 4 days under shear. This simple design allows the handling and management of numerous vials in parallel and appears to be suitable for large-scale studies of both the acute and chronic impact of modulation of the physico-chemical environment on endothelial cell physiology and function.     

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H₂O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H₂O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H₂O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H₂O for 7 days contained at ansferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. (3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley-Liss, Inc.     

Differentiation of stem/progenitor cells into vascular cells in response to fluid mechanical forces

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.     

Exogenous and endogenous force regulation of endothelial cell behavior

Endothelial cells live in a dynamic environment where they are constantly exposed to external hemodynamic forces and generate cytoskeletal-based endogenous forces. These exogenous and endogenous forces are critical regulators of endothelial cell health and blood vessel maintenance at all generations of the vascular system, from large arteries to capillary beds. The first part of this review highlights the role of the primary exogenous hemodynamic forces of shear, cyclic strain, and pressure forces in mediating endothelial cell response. We then discuss the emergent role of the mechanical properties of the extracellular matrix and of cellular endogenous force generation on endothelial cell function, implicating substrate stiffness and cellular traction stresses as important mediators of endothelial cell health. The intersection of exogenous and endogenous forces on endothelial cell function is discussed, suggesting some of the many remaining questions in the field of endothelial mechanobiology.     

Oscillatory shear stress and hydrostatic pressure modulate cell-matrix attachment proteins in cultured endothelial cells

Summary Endothelial cells (ECs) may behave as hemodynamic sensors, translating mechanical information from the blood flow into biochemical signals, which may then be transmitted to underlying smooth muscle cells. The extracellular matrix (ECM), which provides adherence and integrity for the endothelium, may serve an important signaling function in vascular diseases such as atherogenesis, which has been shown to be promoted by low and oscillating shear stresses. In this study, confluent bovine aortic ECs (BAECs) were exposed to an oscillatory shear stress or to a hydrostatic pressure of 40 mmHg for time periods of 12 to 48 h. Parallel control cultures were maintained in static condition. Although ECs exposed to hydrostatic pressure or to oscillatory flow had a polygonal morphology similar to that of control cultures, these cells possessed more numerous central stress fibers and exhibited a partial loss of peripheral bands of actin, in comparison to static cells. In EC cultures exposed to oscillatory flow or hydrostatic pressure, extracellular fibronectin (Fn) fibrils were more numerous than in static cultures. Concomitantly, a dramatic clustering ofα ₅β₁ Fn receptors and of the focal contact-associated proteins vinculin and talin occurred. Laminin (Ln) and collagen type IV formed a network of thin fibrils in static cultures, which condensed into thicker fibers when BAECs were exposed to oscillatory shear stress or hydrostatic pressure. The ECM-associated levels of Fn and Ln were found to be from 1.5-to 5-fold greater in cultures exposed to oscillatory shear stress or pressure for 12 and 48 h, than in static cultures. The changes in the organization and composition of ECM and focal contacts reported here suggest that ECs exposed to oscillatory shear stress or hydrostatic pressure may have different functional characteristics from cells in static culture, even though ECs in either environment exhibit a similar morphology.     

Design of a novel rotating wall bioreactor for the in vitro simulation of the mechanical environment of the endothelial function

Endothelial cells (ECs), besides being a permeability barrier between the blood and vessel wall, perform many important functions, e.g., cell migration, remodeling, proliferation, and the production, secretion and metabolism of biochemical substances, as well as the regulation of contractility of vascular smooth muscle cells (SMCs). Their function is modulated by chemical ligands as well as mechanical factors. The mechanical stresses acting on the vessel wall include the normal and circumferential stresses that result from the action of blood pressure, the shear stress that acts parallel to the luminal surface of the vessel due to blood flow and the magnitude and orientation of the gravitation field. The aim of this work was to design and construct a novel bioreactor for the stimulation of endothelial cells in vitro with a combination of mechanical factors that simulates their in vivo environment.     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

Hydrostatic pressure influences morphology and expression of VE-cadherin of vascular endothelial cells

Bovine aortic endothelial cells (BAECs) were exposed to hydrostatic pressures of 50, 100, and 150 mmHg and changes in morphology and expression of vascular endothelial (VE)-cadherin were studied. After exposure to hydrostatic pressure, BAECs exhibited elongated and tortuous shape without predominant orientation, together with the development of centrally located, thick stress fibers. Pressured BAECs also exhibited a multilayered structure unlike those under control conditions and showed a significant increase in proliferation compared with control cells. Western blot analysis demonstrated that protein level of VE-cadherin were significantly lower under pressure conditions than under control conditions. Inhibition of VE-cadherin expression, using an antibody to VE-cadherin, induced the formation of numerous randomly distributed intercellular gaps, elongated and tortuous shapes, and multilayering. These responses were similar to those of pressured BAECs. The exposure of BAECs to hydrostatic pressure may therefore downregulate the expression of VE-cadherin, resulting in loss of contact inhibition followed by increased proliferation and formation of a multilayered structure.     

Effects of vibration on differentiation of cultured PC12 cells

Different types of physiological‐mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non‐physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano‐vibration system was designed to produce nanometer‐scale vibration. The frequency and amplitude of the nano‐vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation. Biotechnol. Bioeng. 2011; 108:592–599. © 2010 Wiley Periodicals, Inc.     

Effect of the stress phase angle on the strain energy density of the endothelial plasma membrane

Endothelial cells are simultaneously exposed to the mechanical forces of fluid wall shear stress (WSS) imposed by blood flow and solid circumferential stress (CS) induced by the blood vessel's elastic response to the pressure pulse. Experiments have demonstrated that these combined forces induce unique endothelial biomolecular responses that are not characteristic of either driving force alone and that the temporal phase angle between WSS and CS, referred to as the stress phase angle, modulates endothelial responses. In this article, we provide the first theoretical model to examine the combined forces of WSS and CS on a model of the endothelial cell plasma membrane. We focus on the strain energy density of the membrane that modulates the opening of ion channels that can mediate signal transduction. The model shows a significant influence of the stress phase angle on the strain energy density at the upstream and downstream ends of the cell where mechanotransduction is most likely to occur.     

Primary cilia as biomechanical sensors in regulating endothelial function

Depending on the pattern of blood flow to which they are exposed and their proliferative status, vascular endothelial cells can present a primary cilium into the flow compartment of a blood vessel. The cilium modifies the response of endothelial cells to biomechanical forces. Shear stress, which is the drag force exerted by blood flow, is best studied in this respect. Here we review the structural composition of the endothelial cilia and the current status of knowledge about the relation between the presence of primary cilia on endothelial cells and the shear stress to which they are exposed.     

Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.     

The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the innermost surfaces of blood vessels and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein that displaces endogenous nesprins from the nuclear envelope and disrupts nuclear–cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell–cell adhesion and barrier function, as well as altered cell–matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH–expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis.     

Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article. we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells

A central aspect of cellular mechanochemical signaling is a change of cytoskeletal tension upon the imposition of exogenous forces. Here we report measurements of the spatiotemporal distribution of mechanical strain in the intermediate filament cytoskeleton of endothelial cells computed from the relative displacement of endogenous green fluorescent protein (GFP)-vimentin before and after onset of shear stress. Quantitative image analysis permitted computation of the principal values and orientations of Lagrangian strain from 3-D high-resolution fluorescence intensity distributions that described intermediate filament positions. Spatially localized peaks in intermediate filament strain were repositioned after onset of shear stress. The orientation of principal strain indicated that mechanical stretching was induced across cell boundaries. This novel approach for intracellular strain mapping using an endogenous reporter demonstrates force transfer from the lumenal surface throughout the cell.