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239 Mini Review Mechanical Stress Mechanisms and the Cell An Endothelial Paradigm Peter F. Davies and Satish C. Tripathi There are important physiological and pathological cardiovascular consequences related to endothelial biomechanical properties. The endothelium, however, is not unique in responding to external forces; virtually all cells accommodate or respond to the mechanical environment. (Circulation Research 1993;72:239-245) Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 T he evolution of organisms has been both constrained and facilitated by many internal and external mechanical limitations. Mechanical sensing systems used by primitive cells to ensure survival have been retained, often in modified form, in multicellular animals and in the tissues and organ systems of mammals to form specialized sensors that monitor both the internal state of the organism as well as its interactions with the external environment. Mechanical stresses alter the structural and functional properties of cells (mechanotransduction) at the cellular, molecular, and genetic levels, leading to both rapid responses in the adjacent tissue and slower adaptive changes to a sustained mechanical environment. The latter often results in an alteration of intracellular tension to counterbalance the external stress. Cellular responses to direct mechanical stresses appear to involve an interplay between structural elements and biochemical second messengers. Cell surface proteins and extracellular matrix, linked by transmembrane proteins to the cytoskeleton, activate ion channels and enzymes by mechanical deformation. The signal transduction mechanisms by which mechanical stresses can be converted to electrophysiological and biochemical responses in the sensing cells and the adaptation of such cells to the external forces by altered gene expression is receiving increased attention. A change in the extracellular concentration of bioactive ligands at the cell surface as a result of fluid movement has also been identified as an indirect mechanism of mechanotransduction. Using the paradigm of endothelial cell responses to flow forces, this review attempts to 1) define the nature of direct biomechanical stresses, 2) identify key examples of bioresponses, and 3) discuss signal transduction mechanisms of mechanical stress. From the Department of Pathology (P.F.D.), The University of Chicago, and the Department of Life Sciences (S.C.T.), IIT Research Institute, Chicago. Supported by National Institutes of Health grants HL-15062 and HL-36028 and American Heart Association Grant-in-Aid 91-1557. Address for correspondence: Peter F. Davies, PhD, Department of Pathology, The University of Chicago, MC6079, 5841 S. Maryland Ave., Chicago, IL 60637. Received July 29, 1992; accepted September 30, 1992. Mechanical Stress Stress is force per unit area, which may be manifested as pressure (and compression) acting inward upon a structure, frictional shear at the surface, and tensile reactive forces acting outward, often to equalize an externally imposed stress. Tissues and cells also exhibit definitive stresses characteristic of solid bodies; e.g., torsional stresses resulting from internal shear stresses may be imposed across an entire tissue or organ when subjected to opposing external shearing forces on opposite faces. Hydrostatic forces, both inside and outside the cell, are another example of pressure. Breadth of Mechanical Responses In organisms as diverse as microbes, plants, and animals, the retention of cellular responses to mechanical stress factors reflects the fundamental importance of this process. Stretch-sensitive ion channels occur in Bacillus subtilis, Escherichia coli, rust fungi, yeast, mollusks, insects, amphibians, birds, reptiles, and mammals. In mammals, interconnecting forces on muscles, bones, and connective tissues are additive to the effects of gravity; when gravitational forces are decreased in space, tissues remodel to adapt to the altered load. In the pressurized cardiovascular system, where mechanical demands on the heart and arteries vary enormously, adaptive mechanisms are essential to facilitate the optimal distribution of blood. The endothelium plays a key role in this by regulating vascular tone. Mechanical Stress Responses in Endothelial Cells The arterial endothelium is located at the interface between blood flow and the vessel wall; consequently, it is uniquely exposed to forces that incorporate diverse mechanical characteristics. Blood flow regulates the internal diameter of arteries both acutely, by relaxation/ contraction of smooth muscle cells,' and chronically, by the reorganization of vascular wall cellular and extracellular components.2 In both cases, the presence of the endothelium is required. Thus, the endothelium functions as a mechanically sensitive signal transduction interface between blood and artery wall. Endothelial cells hold a unique position among biomechanically responsive cells because, in addition to pressure and associated stretch and solid mechanical forces, the cells 240 Circulation Research Vol 72, No 2 February 1993 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 are continuously exposed to relatively high fluid shear stresses to which they readily respond. Consequently, they represent an excellent model for studies of the effects of fluid and solid-phase stress on cell function in general.3 When endothelial cells are subjected to shear stress or mechanical stretching, a diverse set of responses are generated; some are extremely fast, whereas others develop over many hours. These are outlined in Table 1 in the approximate order of response time. Rapid changes in ionic conductance, adenylate cyclase activity, inositol trisphosphate generation, and intracellular free calcium in response to mechanical stress are similar to second messenger responses resulting from agonistreceptor coupling, suggesting that they may share common transduction pathways. The rapid responses are followed after several hours by altered gene expression (growth factors, endothelin, and regulators of fibrinolysis) and by major structural reorganization (cytoskeletal redistribution and cell-shape change) if the forces are sustained. How are such stresses manifested in the blood? Blood flow in arteries is generally laminar; however, the vessel geometry near branches, bifurcations, and curved regions promotes flow separation and vortex formation. At such locations, the fluid shear stresses and pressures fluctuate greatly in magnitude and direction over short distances; elsewhere, the forces are unidirectional and pulsatile. A schematic of forces acting on the endothelial cell is shown in Figure 1; the stresses are similar to those imposed on other cells, except that blood flow imparts much higher levels of fluid shear stress than in any other mammalian tissue. In the fluid phase, the force vectors of pressure/stretch and wall shear stress act on the endothelial surface. In endothelium, as in all anchorage-dependent cells, these external mechanical stresses are imposed on a preexisting force equilibrium generated by the cytoskeletal tension.4 Overall, the forces acting on the cell must be balanced by resistance to the action. The forces are transmitted as solid mechanical stresses that are distributed throughout the cell via the cytoskeleton. Intracellular tensile stresses may then be transmitted to adjacent cells and to the underlying extracellular matrix structures via sites of focal adhesion at the ventral cell surface. Thus, the spatial relations of the cell to its neighbors and the surrounding matrix will influence cell tension and mechanotransduction. How then are these interrelated? Stress Transmission and Transduction There is strong evidence that the force transduction mechanisms in endothelial cells, like anchorage-dependent cells in general, are a combination of force transmission via cytoskeletal elements and transduction of the physical forces to biochemical signals at mechanotransducer sites. The most compelling evidence implicates F-actin microfilaments as the principal transmission structure. F-actin also appears to be required for transduction; e.g., in several cell types, stimulation of adenylate cyclase and stretch-activated ion channels in response to deformation is inhibited by disruption of actin microfilaments.5 Endothelial focal adhesion remodeling, cell-shape change, and realignment to flow are all inhibited by drugs that interfere with microfil- ament turnover. In this regard, the endothelium behaves like other anchorage-dependent nontransformed cells. How does the mechanochemical transduction take place such that the mechanisms are consistent with diverse response times? The microfilament network confers tension to the cell, which, together with microtubular rigidity, determines cell shape.4 Actin filaments are anchored in the plasma membrane at several sites (Figure 1), most notably in association with 1) focal adhesions on the ventral surface, 2) intercellular adhesion proteins at the cell periphery, 3) integral membrane proteins at the apical cell surface, and 4) the nuclear membrane. Although the stress transmission pathway is shared, different responses may be determined by the nature of the association between cytoskeleton and mechanotransducers at the different locations in the cell and possibly also by transducers that are independent of the cytoskeleton. Figure 1 illustrates the likely sites of transduction, and Figure 2 summarizes the principal pathways linking stress transmission and transduction. The existence of mechanically activated ion channels6 provides a prototypic mechanism for rapid transduction of stress to an electrophysiological response. Recently, Schultz et a17 have demonstrated that adenylate cyclase purified from Paramecium flagella can act as a K' channel in which the hyperpolarized state of the enzyme regulates cAMP production. Thus, ion flux and generation of an important biochemical second messenger are regulated by an enzyme that exhibits some of the functional characteristics of an ion channel. Since in many cells adenylate cyclase activation occurs in response to mechanical stimulation,5 a direct effect of stress on integral membrane enzymes and ion channels may generate second messenger activity by similar mechanisms. Stretch-activated,6 stretch-inactivated,8 and shear stress-activated9 ion channels are candidates for direct activation. Are cytoskeletal elements necessary for ion channel responses? Although the presence of cytoskeleton is not energetically necessary to activate the ion channels (stretch activated channels can solely use the free energy stored in the transmembrane electrochemical gradient and are sensitive to the tension imposed by membrane lipids6), disruption of actin microfilaments inhibits their responses. It is unclear whether the cytoskeleton-channel association is by direct linkage or is an indirect effect of changing the "background" cytoskeletal tension exerted on the membrane in general. Other membrane proteins, both integral as well as those linked to the glycocalyx extension of the cell surface, could act as flow sensors that, on deformation, activate membrane enzymes and channels. Integral membrane proteins of the integrin superfamily are the most extensively studied sites of microfilament anchorage to the plasma membrane. The integrins are linked to F-actin on the cytoplasmic side via a-actinin, talin, and vinculin.10 On the cell surface, integrins bind to the extracellular matrix, thereby facilitating a series of protein-protein interactions extending from outside the cell to the filamentous cytoskeleton. Endothelial cell shape and DNA synthesis are regulated by integrin-extracellular matrix associations that determine the tensional integrity (tensegrity) of the cell. 4 When the endothelial cell is Davies and Tripathi Endothelial Stress Mechanisms 241 Shear Stress (Endothielial cell) Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 1. SAC / SIC 2. Shear stress JK DAG 1P3 K+ Na+K + cytoskeleton Ca++ 3. Focal adhesions FIGURE 1. Diagrams showing mechanical stress sites in cells. SAC, stretch-activated ion channel; SIC, stretch-inactivated ion channel; IMP, integral membrane protein; PL, phospholipid; DAG, diacylglycerol; IP3, inositol 1,4,5trisphosphate; prots., proteins; EC-CAM, endothelial cell to cell adhesion molecule; PECAM, platelet endothelial cell adhesion molecule. Adenyate Cyclwse (K1) 4. Cell-cell 5. Nucleus F-Acdin Nuclear L pore Link prots. Integrins = 7 matrix exposed to directional fluid shear stress, the force distribution throughout the cytoskeleton must ultimately be balanced by resistance at the focal adhesion sites if the cell is to remain stationary. Endothelial focal adhesions are directionally remodeled by shear stress," consistent with the tensegrity hypothesis of mechanochemical transduction proposed by Ingber4 in 1991. But do integrins function as mechanotransducers as well as mechanotransmitters? Recent evidence suggests that the integrins may elicit biochemical signals by tyrosine kinase phosphorylation as well as by interaction with closely associated intracellular proteins. Endothelial integrin proteins are composed primarily of the subunits a,43. The P subunit binds a-actinin, which in turn binds to F-actin. Tyrosine-phosphorylated proteins (including integrins, talin, vinculin, a-actinin, and paxillin) and the cytoplasmic tyrosine kinase pp60src have been localized to focal adhesions.12 Since tyrosine phosphorylation is a prominent chemical transducer for receptor signaling, induc- tion of phosphorylation by mechanical stress at these sites in endothelial cells would demonstrate convergence of mechanotransduction and hormone receptor transduction pathways in the cell. Stimulation of phosphatidylinositol metabolism, a prominent response to shear stress and stretch,13 is likely to be linked to plasma membrane protein deformation. Association of the actin-binding protein profilin with specific phosphoinositides has recently been shown to inhibit phospholipase C,14 thus providing a direct link between phospholipid metabolism and the cytoskeleton. The consequences of phospholipase activation and the release of inositol trisphosphate and diacylglycerol are immediate (Ca'+ influx and mobilization), rapid (Ca'+ as second messenger regulates rapid gene expression in excitable cells15), and delayed (protein kinase C activation leading to altered gene expression'6). Whatever the transduction system, the fundamental unambiguous signal is the stress force acting on the 242 Circulation Research Vol 72, No 2 February 1993 TABLE 1. Mechanical Stress Responses in Endothelial Cells Force LSS (0.2-16.5 dyne/cm2) LSS (10-120 dyne/cm2) Suction (pressure, stretch) (10-20 mm Hg) Mechanical poking and dimpling LSS (8 dyne/cm2) Flow through microcarrier bed LSS (30 and 60 dyne/cm2) Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 LSS (0.2-4.0 dyne/cm2) Effect K' channel activation, hyperpolarization (whole-cell recording) Hyperpolarization (Vm-sensitive dyes) Activation of nonselective cation channels (membrane patch) Intracellular Ca2+ rise (fluo-3) 50-fold increase in release of nitric oxide Release of ATP, acetylcholine, and substance P Transient elevation of 1P3, biphasic (BAECs) Intracellular Ca2+ rise, Ca 2+ oscillations Cell type and response time BAECs (msec) BPAECs (steady state at 60 seconds) PAECs (msec) Earliest response to flow, related to vasorelaxation Endothelial stretch-activated channels HUVECs (seconds) BAECs (seconds) Stretch-activated Ca2+ channels, depolarization Flow-mediated vasorelaxation HUVECs (seconds) Neurotransmitter release BAECs and HUVECs (15-30 seconds), major peak at 5 minutes (BAECs) BAECs (15-40 Phosphoinositides and Ca2+ as second messengers for shear stress transduction seconds) Cyclic strain (24%), deformation (1 Hz) Transient elevation of IP3 HSVECs LSS (pulsatile) (mean, 10 Pulsed PGI2 release HUVECs (<1 minute) LSS (0.9 and 14.0 dyne/cm2) Sustained PGI2 release at lower rate than pulsatile flow HUVECs (2 minutes) Cyclic stretching, osmotic swelling LSS (10 dyne/cm2) LSS (10 dyne/cm2) Activation of adenylate cyclase Induction of c-myc expression Directional remodeling of focal adhesion sites, realignment with flow (>8 hours) Tension control of cell shape, pH, and growth via ECM-integrin binding 10-fold enhancement of PDGF-A mRNA, 2-3 fold increase of PDGF-B expression Pinocytosis stimulated, adaptation by 6 hours BAECs and HUVECs dyne/cm2) Modulation of inherent cell tension LSS (0-51 dyne/cm2) LSS (>5 dyne/cm2) LSS (10 dyne/cm2) LSS (5 dyne/cm2) LSS (15 and 25 dyne/cm2) LSS (15 and 25 dyne/cm2) Turbulent flow (average shear stress, 1.5-15.0 dyne/cm2) LSS (>5 dyne/cm2) LSS (>5 dyne/cm2 and in vivo) Induction of c-fos and c-jun Endothelin mRNA and protein secretion stimulated t-PA mRNA expression and secretion stimulated PAI-1 mRNA expression and secretion elevated Cell proliferation in quiescent monolayer Cell alignment in direction of flow, function of time and magnitude of shear stress Cytoskeletal and fibronectin rearrangement Significance Earliest response to flow, related to vasorelaxation Phosphoinositides and Ca 2+ as second messengers for shear stress transduction Phosphoinositides as second messengers for strain deformation PGI2 regulation of vascular tone, antithrombotic properties PGI2 regulation of vascular tone, antithrombotic properties cAMP as second messenger (minutes) BAECs (minutes) BAECs (minutes) Early growth response gene Cell attachment sites as transmitters and/or transducers of stress Capillary EC (<1 hour) Integrins regulate cell growth via cell tension HUVECs (PDGF-A peak, 1.5-2 hours) Enhanced mitogen secretion, regulation of SMC growth BAECs (<2 hours) HUVECs (5 hours) Plasma membrane vesicle formation rate transiently elevated Growth response genes Regulation of vasoconstriction Enhancement of fibrinolytic HUVECs (?) Antagonizes t-PA effects BAECs (>3 hours) Loss of contact inhibition of growth by disturbed flow Minimizes drag on cell BAECs (2 hours) PAECs (peak at 2-4 hours) activity All types (>6 hours) All types (>6 hours) Associated with cell realignment Davies and Tripathi Endothelial Stress Mechanisms 243 TABLE 1. (continued) Force Cyclic biaxial deformation (0.78-12%, 1-Hz frequency, 20-24% strain, 0.9-1.0 Hz) LSS (24 dyne/cm2+20 mm Hg hydrostatic pressure) Effect Cell realignment perpendicular to strain, protein synthesis increase, F-actin redistribution perpendicular to strain Downregulation of fibronectin synthesis Cell type and response time BPAECs (>7 hours) HUVECs and HSVECs (15 minutes) Significance Stretching of artery by blood pulsation, separation of strain and shear stress effects Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Altered cell adhesion, platelet-endothelium interactions Disturbed laminar flow (flow Regional cell cycle BAECs (12 hours) Steep shear gradient separation, vortex, stimulation in confluent stimulation of cell turnover, reattachment) (0-10 dyne/cm2) monolayer focal hemodynamic effects LSS (10-85 dyne/cm2) Mechanical stiffness of cell BAECs (24 hours) Decreased deformability of surface proportional to extent subplasma membrane cortical of realignment to flow complex LSS (30 and 60 dyne/cm2) LDL metabolism stimulated BAECs (24 hours) Endothelial cholesterol balance Cyclic biaxial stretch (3 Inhibition of collagen Inverse relation related to BAECs (5 days) endothelial repair cycles/min, 24% deformation) synthesis and stimulation of cell growth mechanisms LSS, laminar shear stress; BAECs, bovine aortic endothelial cells; Vm, membrane potential; BPAECs, bovine pulmonary artery endothelial cells; PAECs, porcine aortic endothelial cells; HUVECs, human umbilical vein endothelial cells; 1P3, inositol trisphosphate; HSVECs, human saphenous vein endothelial cells; PGI2, prostaglandin I2 (prostacyclin); t-PA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor-i; ECM, extracellular matrix; EC, endothelial cell; PDGF-A and PDGF-B, platelet-derived growth factor A and B chains; SMC, vascular smooth muscle cell; LDL, low density lipoprotein. (A list of references relating to the above table may be obtained by writing to the authors.) primary transducer. How does the physical deformation of a membrane protein or cytoskeletal component lead to a biochemical response? Conformational changes within the proteins appear to be the likeliest mechanism. The intrinsic tension within the cell generated by cytoskeletal anchoring and the molecular interactive forces of components in the plasma membrane provide HUVECs (12 and 48 hours) a framework against which extrinsic forces act. An existing thermodynamic equilibrium would be disturbed by deformation and a new more favorable one would be established that alters the conformation of the proteins. Thus, conformational changes could occur 1) directly within the mechanotransducer at the plasma membrane (ion channel, enzyme, and integrin), 2) within proteins Transduction 0 ce FIGURE 2. Diagram showing mechanisms of mechanical stress transmission and transduction in cells. ECM, extracellular matrix; DAG, diacylglycerol; PKC, protein kinase C; IP3, inositol 1,4,5-trisphosphate. Cor.t s:3 k~ 'I L + IOaysequilibnarI fee Cyokeeo -j> |Cytoskeq-letoi| Intermed. filaments Microfilaments Microtubules Protein Iconformationail changes | 244 Circulation Research Vol 72, No 2 February 1993 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 associated with integral membrane proteins but not in the membrane, (a-actinin, talin, vinculin, and paxillin), or 3) within the cytoskeletal proteins themselves, leading to changes in the subunit dissociation rates of actin and tubulin. These are not mutually exclusive mechanisms, and as noted above, all active cell responses to stress require assembled microfilaments. The transducer-actin interaction must therefore be physically very close or be mediated by intermediate metabolites. In some cases, GTP-binding proteins, classically associated with the cytoplasmic domain of receptors, can influence mechanotransduction. While deformation-induced activation of adenylate cyclase in S49 mouse lymphoma cells occurs independent of GTP-binding proteins,5 tubulin and Gi protein complex can inhibit adenylate cyclase.'7 Evidence for direct binding of cytoskeleton to ion channels is lacking and needs to be addressed. At present, the integrins are the best example of cytoskeletal-transducer coupling, particularly in focal adhesion sites. When a stress stimulus is sustained, gene regulatory changes often follow the immediate responses; several examples for endothelial cells are included in Table 1. The mechanisms probably involve protein kinase C activation. For example, the immediate early genes c-fos and c-myc and the late responsive genes a-actin and ,B-myosin heavy chain are induced in cardiac myocytes by mechanical loading.16 Both c-fos and actin responses were associated with activated protein kinase C and were suppressed by downregulation of the enzyme. These responses did not appear to involve Ca`+. In contrast, rapid "touch-sensitive" gene expression occurs in the plant Arabidopsis by mechanisms involving Ca ` / calmodulin.18 Ten to 30 minutes after stimulation, mRNA levels for four touch-induced genes coding for calmodulin-related proteins increased 100-fold. Diverse biochemical pathways therefore appear to influence gene expression, and these are derived from the immediate responses (inositol trisphosphate and diacylglycerol). In what ways does the cell determine if a mechanical response will lead to gene expression? Perhaps the mechanical properties of the cell itself or the tissue in which it is imbedded may act as a filter that is differentially responsive to selective frequencies. Indirect Mechanisms of Stress Responses Although this review is focused on direct mechanical perturbation of cells, mechanical forces acting on the fluid around the cell can also stimulate intracellular responses; e.g., changes of flow influence the endothelial boundary layer concentrations of adenine nucleotides by altering the mass transport of exogenous or endogenous agonists and consequently influence their interactions with cell surface receptors.19 This indirect mechanism may contribute to some cellular responses that are considered to result from direct deformation. Analyses of the relative contributions of the two types of mechanisms to endothelial flow responses are currently proceeding in several laboratories. Concluding Remarks There has been a large increase of interest in mechanotransduction over the last few years as investigators in well-developed research areas, particularly cytoskeletal, endothelial, cardiac, and focal adhesion biology and signal transduction, have begun to study mechanical stress mechanisms in cells, often in collaboration with physical scientists. The importance of hemodynamic forces has focused much attention on the endothelium, but only recently has attention narrowed from the observational to the mechanistic level. The role of cytoskeleton and focal adhesion proteins is likely to be particularly important in these shear responsive cells. Are phosphorylation events altered by flow? Can mechanotransduction be influenced by integrin subunit manipulation (e.g., truncation or antibody block)? Can the precise topography of the apical cell surface and its deformation by flow be measured in real time to determine shear stresses localized to specific domains? Could there be tensionrelated conformational changes in the nucleus, transmitted via cytoskeletal anchorage to the nuclear membrane? Within tissues, can the tension distribution be more precisely evaluated throughout a cell in relation to the matrix that surrounds it? In the near future, cloning of the genes that code for stretch- and shearsensitive ion channels will provide valuable probes, and parallels between hormone/growth factor transduction and mechanotransduction should continue to be a rich source for comparative studies. References 1. Pohl U, Holtz J, Busse R, Bassenge E: Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 1986;8:37-47 2. Langille BL, O'Donnell F: Reductions in arterial diameter produced by chronic decreases in blood flow are endotheliumdependent. Science 1986;231:405-407 3. Davies PF: How do vascular endothelial cells respond to flow? News Physiol Sci 1989;4:22-25 4. Ingber D: Integrins as mechanochemical transducers. Curr Opin Cell Biol 1991;3:841-848 5. Watson PA: Function follows form: Generation of intracellular signals by cell deformation. FASEB J 1991;5:2013-2019 6. Guharay F, Sachs F: Stretch-activated single ion channel currents in tissue cultured embryonic chick skeletal muscle cells. J Physiol (Lond) 1984;352:685-701 7. Schultz JE, Klumpp S, Benz R, Schurhoff-Goeters WJ, Schmid A: Regulation of adenylyl cyclase from paramecium by an intrinsic potassium conductance. Science 1992;255:600-603 8. Morris CE, Sigurdson WS: Stretch-inactivated ion channels coexist with stretch-activated ion channels. Science 1989;243:807-809 9. Olesen SP, Clapham DE, Davies PF: Hemodynamic shear stress activates a K' current in vascular endothelial cells. Nature 1988; 331:168-170 10. Burridge K, Fath K, Kelly T, Nuckells G, Turner C: Focal adhesions: Transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 1988;4:487-525 11. Robotewskyj A, Dull RO, Griem ML, Davies PF: Dynamics of focal adhesion site remodelling in living endothelial cells in response to shear stress forces using confocal image analysis. (abstract) FASEB J 1991;5:A527 12. Horvath AR, Elmore MA, Kellie S: Differential tyrosine-specific phosphorylation of integrin in Rous sarcoma virus transformed cells with differing transformed phenotypes. Oncogene 1990;5: 1349-1357 13. Nollert MU, Eskin SG, Mclntire LV: Shear stress increases inositol triphosphate levels in human endothelial cells. Biochem Biophys Res Commun 1990;170:281-287 14. Goldschmidt-Clermont PJ, Machesky LM, Baldassare JJ, Pollard TD: The actin-binding protein profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C. Science 1990;247:1575-1578 15. Sheng M, McFadden G, Greenberg ME: Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB. Neuron 1990;4:571-582 16. Komuro I, Katoh Y, Kaida T, Shibazaki Y, Kurabayashi M, Hoh E, Takaku F, Yazaki Y: Mechanical loading stimulates cell hypertro- phy and specific gene expression in cultured rat cardiac myocytes: Davies and Tripathi Endothelial Stress Mechanisms Possible role of protein kinase C activation. J Biol Chem 1991;266: 1265-1268 17. Rasenick MM, Wang N, Yan K: Specific associations between tubulin and G proteins: Participation of cytoskeletal elements in cellular signal transduction. Adv Second Messenger Phosphoprotein Res 1990;24:381-386 245 18. Braam J, Davis RW: Rain, wind and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis. Cell 1990;60:357-364 19. Dull RO, Davies PF: Flow modulation of agonist (ATP)-response (Ca++) coupling in vascular endothelial cells. Am J Physiol 1991; 261:H149-H151 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Mechanical stress mechanisms and the cell. An endothelial paradigm. P F Davies and S C Tripathi Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Circ Res. 1993;72:239-245 doi: 10.1161/01.RES.72.2.239 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1993 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/72/2/239 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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