effects of fluid shear stress on gene regulation of vascular cells

REVIEW

Effects of Fluid Shear Stress on Gene Regulation of Vascular Cells

Maria Papadaki† and Suzanne G. Eskin*

Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, Rice University,Houston, Texas 77251, and Cell Biology Department, Texas Biotechnology Corporation, 7000 Fannin, Suite 1920,Houston, Texas 77030

Hemodynamic forces such as fluid shear stress play an active role in many physiologicaland pathophysiological processes of the cardiovascular system. Shear stress resultingfrom blood flow and transmural plasma flux alters the function of vascular cell(primarily endothelial cells), leading to both rapid and slower adaptive tissue responses.Transmission of the shear stress signal throughout the vascular cell involves a complexinterplay between cytoskeletal and biochemical elements and results in changes instructure, metabolism, and gene expression. Herein we review current knowledge onflow-induced mechanotransduction in the vascular endothelial cell and the molecularmechanisms believed responsible for shear-induced endothelial and smooth musclecell gene regulation with an emphasis on signal transduction.

Contents

Introduction 209Signal Mechanotransduction inEndothelial Cells

210

Ion Channels 210G-Protein-Linked Receptors 212Focal Contact Associated Proteinsand Integrins

214

Protein Kinases 215Transcription Factors, ImmediateEarly Genes

217

Flow-Induced Responses of SmoothMuscle Cells

218

Conclusions 219

Introduction

Major hemodynamic forces generated in the vascula-ture include (1) frictional wall shear stress, actingtangentially to the long axis of the vessel due to bloodflow, (2) circumferential stress, which changes vesseldiameter as a consequence of the pulsatility of blood flow,and (3) compressive stress, acting normal to the vesselwall due to hydrostatic pressure (Figure 1) (Dobrin etal., 1989). In vivo studies have demonstrated thatarteries adapt to chronic physiological changes in bloodflow, in that circumference increases under high flow anddecreases under low flow conditions (Davies, 1995; Lang-ille, 1993). Furthermore, altered hemodynamics havebeen implicated in the pathogenesis of atherosclerosis,thrombosis, and restenosis (Glagov, 1994; Glagov et al.,1988; Kohler and Jawien 1992; Nerem and Cornhill,1980). On the basis of the observation that atheroscle-

rotic lesions in humans tend to develop in regions of flowseparation (e.g., in areas near arterial branches, bifurca-tions, large curvatures), fluid shear stress (local gradientsin shear stress together with abnormally low and highabsolute values) has been implicated as the major he-modynamic factor in initiating cardiovascular diseases(Davies, 1995; Eskin and McIntire, 1988).Vascular endothelial cells, found at the lumen of all

blood vessels, serve as a barrier between perfused tissues† Rice University.* Texas Biotechnology Corp. E-mail: [email protected].

Figure 1. Schematic representation of the hemodynamic forcesacting on the three layers of the artery wall. The intimal layeris composed of a monolayer of endothelial cells. The medial layerconsists of multiple layers of smooth muscle cells and thesecreted extracellular matrix, while the adventitial layer ispopulated with smooth muscle cells, fibroblasts, matrix, bloodvessels, and nerves. Endothelial cells are constantly subjectedto fluid shear stress and circumferential stress, while theunderlying layers of smooth muscle cells and fibroblasts areexposed mainly to circumferential stress.

209Biotechnol. Prog. 1997, 13, 209−221

S8756-7938(97)00029-5 CCC: $14.00 © 1997 American Chemical Society and American Institute of Chemical Engineers

and flowing blood. They are believed to act as sensorsof the local changes in blood flow (Figure 1) (Davies, 1995;Patrick et al., 1995). In vivo studies indicate thatendothelial cells are sensitive to the magnitude of theapplied shear stress and respond by changing theirmorphology and function (Davies, 1995; Langille andAdamson, 1981). However, it is not possible to accuratelymeasure hemodynamic forces and monitor all physicaland biochemical variables in animals. For this reasonin vitro studies are performed, in which cultured cellsare subjected to well-controlled shear stresses for variousintervals, in circuits which can be assayed on-line bydigital processing of video microscope images for studiesof morphological changes. Simultaneously, alterationsin gene expression and protein production can be deter-mined from the shear stressed cells perfused withcontrolled volumes of circulating medium (Papadaki andMcIntire, 1997; Tran-Son-Tay, 1993). In Table 1, weinclude some significant findings reported over the past15 years in the flow-induced morphological and functionalresponses of endothelial cells. In addition to endothelialcells, the responses of vascular smooth muscle cells tofluid shear stress are briefly discussed.

Signal Mechanotransduction in EndothelialCells

The mechanisms by which endothelial cells identifyshear stress forces and convert them to electrophysiologi-cal and biochemical responses remain unclear (Davies,1995; Nollert et al., 1992; Patrick et al., 1995). Moleculesat the luminal cell surface are candidate flow sensorssince they are in direct contact with the flowing blood.These molecules can be activated directly by physicaldisplacement (conformational change) or indirectly bymass transfer gradients (which change ligand-receptorinteractions) (Davies and Tripathi, 1993; Nollert et al.,1991). Such membrane structures or mechanoreceptorsinclude ion channels, G-protein linked receptors, tyrosinekinase receptors, and integrins (Davies, 1995). Thesemechanoreceptors can generate a biochemical cascade ofresponses (centralized mode of mechanotransduction) atthe cytoplasmic face of the cell membrane, by secondmessengers, activation of protein kinases followed byactivation of cytosolic transcription factors, and/or regu-lation of gene transcription in the nucleus (Figure 2)(Patrick et al., 1995). Another way shear stress may betransduced is through interactions of activated mecha-noreceptors with cytoskeletal elements (Figure 2). Usinga decentralized mode of mechanotransduction, transmis-sion could occur through any connection to the cytosk-eleton (e.g., focal adhesion sites, cell-cell junctions,nuclear membrane), and it may lead to a greater diversityof cell responses. There is evidence that mechanotrans-duction in anchorage-dependent cells is a combinationof force transmission through biochemical and cytoskel-etal elements (Figure 2) (Davies, 1995; Davies andTripathi, 1993).Some of the responses generated by intracellular

signaling molecules (“second messengers”) are rapid, onthe order of seconds or minutes (such as changes in ionpermeability, inositol trisphosphate generation, intrac-ellular free Ca2+, and adenylate cyclase activity), whereasothers develop over many hours (altered gene expression,cytoskeletal redistribution, and cell shape change) (Daviesand Tripathi, 1993; Resnick and Gimbrone, 1995). Re-cently it has been proposed that Ca2+-dependent mech-anisms may play an important role in the rapid re-sponses, while Ca2+-independent mechanisms are criticalin the slower sustained responses (Berk et al., 1995).Figure 3 illustrates possible signaling pathways through

which shear stress could transmit signals into the celland induce changes in metabolism and regulate geneexpression. As shown, it is unlikely for a single signaltransduction pathway to be activated, without involvingthe other pathways. Each of the following sectionsfocuses on different hypothetical shear stress activatedmechanoreceptors and the signal transduction pathwaysassociated with them.Ion Channels. The lipid bilayer of cell membranes

has high permeability for hydrophobic and small polarmolecules while it is highly impermeable to ions/chargedmolecules, a function which is crucial for the cell tomaintain different concentrations of solutes from thosein the extracellular fluid. For example, the extracellularconcentrations of K+ and Ca2+ are 5 and 2 mM, respec-tively, while the intracellular concentrations of K+ andCa2+ are 140 and 1 × 10-4 mM, respectively. Specializedmembrane proteins (channels and transporters) areresponsible for the transfer of a specific ion across thecell membrane. Channel proteins do not bind the ion butform hydrophobic pores across the cell membrane, whichopen to allow transport, while transporters bind to theion and change their conformation to effect transfer ofthe bound ion. The mode of transport for all channelsand some carrier proteins is facilitated diffusion, where

Maria Papadaki received her Ph.D. in the Department ofChemical Engineering (1997), Rice University, Houston, TX.She received a Diploma in Chemical Engineering (1992) fromthe Aristotle University of Thessaloniki, Greece. Her re-search interests are in the areas of cellular and tissueengineering.

Suzanne G. Eskin has been Director of Cell Biology atTexas Biotechnology Corp. in Houston, TX, since 1992. Shereceived a Ph.D. in Zoology from University of Texas atAustin. She has been Associate Professor of Medicine atUniversity of Texas Medical School at Houston and AssociateProfessor of Surgery at Baylor College of Medicine. Herresearch interests are response of vascular cells to mechan-ical forces and cellular responses to growth factors.

210 Biotechnol. Prog., 1997, Vol. 13, No. 3

the net driving force for the solute is its electrochemicalgradient across the membrane. Carrier proteins can alsoactively transport a solute against its electrochemicalgradient, through coupling with energy sources (ATP andGTP).Mechanotransducing ion channels of varying degrees

of selectivity are present in endothelial cells (Figure 4).Electrophysiological studies have shown that exposureof endothelial cells to physiological levels of fluid shearstress for 10 s activated a K+ channel (“shear-stress-activated K+ channel”), which was not rapidly desensi-tized, and the magnitude and duration of which wasshear stress dependent (Alevriadou et al., 1993; Olesenet al., 1988). Blockage of K+ channels significantlyinhibited the shear-stress-induced increase in transform-ing growth factor â1 (TGF-â1) gene transcription, provid-ing evidence that K+ channels play a role in flow-stimulated gene expression (Ohno et al., 1995). This K+

channel appears to be specific to endothelial cells sinceit was not found in vascular smooth muscle cells or atrialmyocytes. Recent studies provided evidence that activa-tion of a K+ channel is associated with G protein coupling(Cooke et al., 1991; Ohno et al., 1993). There is someevidence that activation of K+ channels in endothelialcells results in hyperpolarization, which can lead toincreased Ca2+ influx through a Ca2+-permeable channelwhich is voltage independent (Olesen and Bundgaard,1993; Olesen et al., 1988). Ca2+ influx can activate manydownstream signaling pathways, one of which stimulatesnitric oxide (NO) production and subsequently vasodila-tion via activation of guanylate cyclase (GC) and produc-tion of cyclic guanosine monophosphate (cGMP) (Ohnoet al., 1993). However, there are contradictory reportsin the literature on whether NO production depends onK+ channel activation (Gooch and Frangos, 1996; Ohnoet al., 1995). Although hyperpolarization results in Ca2+

Figure 2. Model of initiation of signal transduction in endothelial cells, showing two major types of mechanoreceptors, those whichtransmit signals through interactions with the cytoskeleton and those which elicit cascades of biochemical signaling after activation.

Table 1. Shear-Stress-Mediated Endothelium Responses

effect refs

K+ channel activation Alevriadou et al., 1993; Olesen and Bundgaard, 1993; Olesen et al., 1988G-protein activation Gudi et al., 1996; Kuchan et al., 1994transient elevation in IP3 and sustained PGI2 release Bhagyalakshmi et al., 1992; Frangos et al., 1985, 1988; Nollert et al., 1990increases in NO release and cGMP production Kuchan and Frangos, 1994; Kuchan et al., 1994decreases in intracellular pH Patrick and McIntire, 1995; Ziegelstein et al., 1992stimulation of the ras-ERK1/2 pathway, ras-JNK,

phosphorylation of small heat shock proteinsIshida et al., 1996; Jo et al., 1997; Li, S., et al., 1996; Li, Y.-S., et al., 1996;

Takahashi and Berk, 1996; Tseng et al., 1995activation of transcription factors NFκ-B, c-jun, c-fos Khachigian et al., 1995; Lan et al., 1994increases in c-myc, c-fos, c-jun mRNA Hsieh et al., 1993; Ranjan and Diamond, 1993increases in PDGF-A, B mRNA Hsieh et al., 1991, 1992biphasic response of MCP-1 mRNA Shyy et al., 1994increases in tPA mRNA and protein Diamond et al., 1989, 1990increases in TGF-â1 mRNA and protein Ohno et al., 1995decrease in VCAM-1 and transient increases in

ICAM-1 mRNA and protein levelsNagel et al., 1994; Ohtsuka et al., 1993; Sampath et al., 1995

decrease in ET-1 mRNA and protein at arteriallevels of shear stress

Kuchan and Frangos, 1993; Malek et al., 1993; Malek and Izumo, 1995;Nollert et al., 1991; Sharefkin et al., 1991

SSRE responsible for shear-induced regulation of PDGF-A gene Resnick et al., 1993; Resnick and Gimbrone, 1995TRE responsible for MCP-1 gene flow regulation Shyy et al., 1995bfocal adhesion contacts, F-actin and cytoskeleton remodeling,

cell alignment in flow directionBarbee et al., 1994; Davies, 1995; Davies et al., 1994; Dewey et al., 1981;

Eskin et al., 1984; Ives et al., 1986; Levesque and Nerem, 1985decrease in cell proliferation Dewey et al., 1981; Levesque et al., 1989

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influx in endothelial cells, the reverse is true in smoothmuscle cells in which hyperpolarization inactivates anddepolarization activates voltage-gated calcium channels(Davies, 1995). Another potential mechanosensitivechannel in endothelial cells is a recently identifiedadenylate cyclase (AC) K+ channel, in which the hyper-polarized state of the enzyme regulates adenosine 3′,5′-monophosphate (cAMP) production (Schultz et al., 1992).It is unclear whether ion channel activation is secondaryto activation of upstream mechanoreceptors or whetherit results from conformational alteration of the proteinsthat comprise the ion channel. Stretch-activated ionchannels were not activated by shear stress in endothelialcells (Davies, 1995).In addition to K+ and Ca2+ channels, transporters such

as Na+/H+, HCO3-/Cl-, and Cl-/Na+HCO3

- exchangers,which tightly control intracellular pH levels (to 7.2),

might play a role in flow-induced changes in endothelialmetabolism. There is increasing evidence that changesin intracellular pH levels may serve as second mes-sengers for the cell (Patrick and McIntire, 1995). Fluidshear-stress-induced cytosolic acidification in endothelialcells is probably due to HCO3

-/Cl- exchanger activation(Patrick and McIntire, 1995; Ziegelstein et al., 1992).G-Protein-Linked Receptors. G-protein-linked re-

ceptors, which belong to a receptor family with seventransmembrane domains, alter the concentration ofsecond messengers, which in turn influence the behaviorof other target proteins in the cell. The interactionsbetween the receptor and second messengers are medi-ated through enzymes or ion channels, which are acti-vated by GTP-binding regulatory proteins (or G-proteins).G proteins are heterotrimers composed of an R chainloosely bound to a âγ dimer. The âγ dimer anchors the

Figure 3. Schematic representation of the multiple “shear-stress-responsive” signaling pathways, their crosstalk, and integration.Activation of one or more hypothetical mechanoreceptors elicits a sequence of biochemical and cytoskeletal events, which can leadto downstream changes in the cell metabolism and gene expression. Mechanoreceptors and the signaling pathways associated withthem are presented with matching colors and are described in separate sections in the text.


G protein to the cytoplasmic face of the plasma mem-brane, while the R chain exchanges bound GDP for GTPupon receptor activation, which then leads to dissociationof the R, â, and γ subunits. A number of differentG-protein families, each of them associated with differentfamilies of R, â, and γ subunits, have been identified invarious cells. G proteins associated with transmembranereceptors that are involved in downstream signal trans-duction in endothelial cells include (a) Gq proteins, whichactivate phospholipase C (PLC), (b) Gs proteins or stimu-latory G proteins, which activate Ca2+ channels and AC,and (c) Gi proteins or inhibitory G proteins, which inhibit

AC and are thought to be involved in the stimulation ofK+ channels in endothelial cells (Simon et al., 1991).One of the earliest responses after initiation of flow is

the activation of phospholipase C (PLC). PLC stimulatesmany cellular responses by breaking down phosphati-dylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Figure 5).IP3 diffuses rapidly to the cytosol from the cell membraneand releases Ca2+ to the cytosol from an intracellularcalcium-sequestering compartment. However, most in-vestigators have been unable to find any significantincreases in the bulk cytosolic Ca2+ in endothelial cells

Figure 4. Ion channels and ion exchangers as possible mechanotransducers. Activation of K+ channels induces hyperpolarization,which can lead to activation of Ca2+-permeable channels. The resulting Ca2+ influx can activate a Ca2+-calmodulin-dependent nitricoxide synthase, which catalyzes the formation of NO from L-arginine in the presence of several cofactors. The adenylate cyclase K+

channel may be involved in the production of cAMP. The ion exchangers or transporters may also be shear-stress-sensitive receptors.

Figure 5. G-protein-linked receptors as possible mechanotransducers and downstream signaling associated with their activation.Activation of G-protein-linked receptors activates PLC, which breaks down PIP2 to produce IP3 and DAG. IP3 stimulates Ca2+ releaseinto the cytosol from calcium-sequestering compartments; while DAG can activate PKC, can be cleaved to produce AA, or can formPA. Little is known about the activation of AC/GC-associated G-protein-coupled receptors.


that were exposed to shear stress in the absence ofcalcium mobilizing agonists (such as adenosine triphos-phate (ATP) or serum) (Dull and Davies, 1991; Mo et al.,1991; Nollert and McIntire, 1992), and only a fewinvestigators report a flow-dependent stimulation ofintracellular Ca2+ (Geiger et al., 1992). It may be thatfluid shear stress causes changes in local Ca2+ concentra-tions in the vicinity of the cell membrane without anyincrease in bulk cytosolic Ca2+ concentration (Nollert etal., 1991). Diacylglycerol activation is transient, andwithin seconds, DAG can either (1) be further cleaved torelease free arachidonic acid (AA), which is subsequentlyconverted to prostacyclin (PGI2) and related lipid signal-ing molecules, (2) be phosphorylated to form phosphati-date (PA), or (3) activate protein kinase C (PKC) (serine/threonine-specific protein kinase) (Berthiaume andFrangos, 1993; Panaro and McIntire, 1993). Activationof PKC is in most cases Ca2+ dependent (although eachof the eight PKC isozymes have different Ca2+ affinities).PKC can phosphorylate many proteins with differentfunctions in the cell and can increase the transcriptionof certain genes. For example, there is evidence thatPKC activates the plasma membrane Na+/H+ exchangerthat controls intracellular pH.In endothelial cells exposure to shear stress stimulates

PGI2 release (Frangos et al., 1985, 1988). Furthermore,it has been shown that shear stress induces transientincreases in PLC and IP3 in endothelial cells, whichimplies that PKC is activated by flow (Berthiaume andFrangos, 1993; Frangos et al., 1985). Tseng et al. haverecently shown that the PKC isozyme activated by shearstress, and necessary for the activation of a downstreammitogen-activated protein kinase (MAPK), was PKC-εand/or PKC-ú and did not require increases in intracel-lular Ca2+ (Ca2+ independent) for its activation (Tsenget al., 1995). PKC activation plays an important role incell response to flow, since many flow-induced transcrip-tion factors and proteins have been found to depend onPKC (Berthiaume and Frangos, 1993; Davies, 1995;Tseng et al., 1995).The earliest shear-stress-induced signal transduction

event reported in human umbilical vein endothelial cells(HUVEC) was the activation of membrane associatedG-proteins, which occurred during the first second of flowexposure (Gudi et al., 1996). These investigators pro-posed that fluid-flow activated G proteins either directlyor indirectly through G-protein-coupled receptors. IP3-induced Ca2+ release from intracellular stores occurredsubsequent to G-protein activation. Since no mecha-nosensitive receptor has been yet identified, it may bethat the plasma membrane itself responds to shear stressby increasing fluidity of the lipid bilayer (Berthiaume andFrangos, 1994; Gudi et al., 1996). Perturbations due toincreased membrane fluidity can directly activate Gproteins on the cytosolic side of the cell. The finding thatthe initial burst production of NO in shear stressedendothelial cells depends upon the activation of Gproteins correlates well with the above hypothesis (Kuchanand Frangos, 1994).ATP is converted into cAMP through AC activation

from a G-protein-linked receptor. cAMP can activate avariety of protein kinases to regulate cellular function(Nollert et al., 1991), or it may be involved in the controlof transendothelial permeability and pinocytosis (Ber-thiaume and Frangos, 1993). Reich et al. reported thatcAMP levels in human endothelial cells increase uponexposure to shear stress (Reich et al., 1990), while Maleket al. did not observe any shear-stress-induced regulationof cAMP (Malek et al., 1993). Although it is not knownwhether guanylate cyclase (GC) is directly activated byshear, increased production of NO by shear stress in

endothelial cells has been associated with increased(cGMP) (Kuchan and Frangos, 1994).Focal Contact Associated Proteins and Integrins.

Many studies have demonstrated that unidirectionalsteady shear stress causes cytoskeletal reorganizationand alignment of endothelial cells in the direction of flow(Davies, 1995; Dewey et al., 1981; Eskin et al., 1984; Iveset al., 1986). The redistribution of F-actin stress fibersor microfilaments (MF), microtubules (MT), and inter-mediate filaments (IF) in response to shear stress, inaddition to the findings that disruption of the microfila-ments by drugs inhibited flow-induced intracellularresponses (Davies and Tripathi, 1993; Malek and Izumo,1996; Morita et al., 1993), provide strong evidence thatthe cytoskeleton is a major mechanotransducer. Asdiscussed previously, contact sites (e.g., focal adhesioncontacts, cell-cell contacts, nuclear membrane) betweenthe cytoskeleton and mechanoreceptors are structurallyconnected, which implies that signal transduction throughthe cytoskeleton is decentralized. A tensegrity model oftension which considers the distribution of shear stressat multiple sites throughout the cell that are mechani-cally coupled to the cytoskeleton has been developed(Ingber et al., 1994). In fact, experimental studiesdemonstrated that the stiffness of the cytoskeletonincreased linearly with increasing shear stress, as pre-dicted by the model (Wang et al., 1993).Focal adhesions are the most intensively studied as

potential mechanotransducers to the cytoskeleton. Theyprovide a continuum between the cytoskeleton and ex-tracellular membrane proteins, and they are formed atthe sites of linkage of the cytoplasmic sides of integrinswith cytoskeletal proteins. Focal adhesions are respon-sible for cell adhesion and they play a role in cellsignaling, morphology, proliferation, migration, and dif-ferentiation (Davies, 1995). Shear stress causes rapidremodeling in focal adhesion structure in endothelialcells, while no net change in cell adhesion is observed(Davies et al., 1994). Integrins are R/â heterodimertransmembrane glycoproteins, which act as adhesionreceptors. Both subunits span the plasma membrane,with the R subunit determining the binding specificityof the receptor to extracellular matrix components (In-gber et al., 1994). In endothelial cells, the most commonintegrins are the receptors for the matrix proteins fi-bronectin, laminin, and vitronectin (Davies, 1995). In-tegrin activation occurs through interactions of theintegrin receptor with extracellular matrix ligands andby recruitment of intracellular tyrosine kinases (Berk etal., 1995). A Ca2+-independent, focal adhesion protein-tyrosine kinase (FAK) (Figure 6) localized at focal adhe-sions is autophosphorylated after activation of integrinsby an unknown mechanism (Berk et al., 1995; Ishida etal., 1996). FAK phosphorylation recruits intracellularCa2+-independent protein kinases such as src. The srckinases belong to a family of nonreceptor tyrosine ki-nases, whose members are membrane/cytoskeleton-as-sociated tyrosine-specific protein kinases. Activation ofFAK and src leads to phosphorylation of other focaladhesion proteins such as paxillin, which interacts withthe cytoskeleton (Girard and Nerem, 1993; Plopper andIngber, 1993). Tyrosine phosphorylation of focal adhesionproteins can induce changes in the assembly/disassemblydynamics of both microfilament and microtubule net-works and can cause remodeling of focal adhesions andsubsequent cell shape change (Malek and Izumo, 1996).In endothelial cells, FAK tyrosine phosphorylation wasincreased, paxillin changed its alignment, and src ty-rosine kinase was activated in response to flow-inducedshear stress, indicating that integrin-mediated events are


important in flow-induced signal transduction (Berk etal., 1995; Takahashi and Berk, 1996).There is recent evidence that signal transduction

through cell-matrix interactions is linked to phospho-lipid metabolism. This link is mediated through a smallGTP-binding protein, rho (Chong et al., 1994). Rhobelongs to a family of monomeric GTPases, which cyclebetween two conformational states: active when GTP isbound and inactive when GDP is bound. These mono-meric GTPases contain a covalently attached prenylgroup that helps anchor them to the membrane (Macaraet al., 1996). Rho, through a cascade of events, regulatesa PIP 5-kinase that phosphorylates 4-PIP to yield PIP2.PIP2 is the substrate for PLC and can yield IP3 and DAG.Although the Ca2+ dependence of the focal adhesionremodeling is not known, recent studies showed thatchelation of intracellular Ca2+ abolished the tyrosinephosphorylation of many proteins in endothelial cells(Malek and Izumo, 1996).There is evidence that the shear-stress-induced matrix-

integrin interactions can also stimulate a member of theMAPK family, extracellular signal-regulated kinase 1/2(ERK1/2), through src kinase activation (Clark andHynes, 1996; Schlaepfer et al., 1994; Takahashi andBerk, 1996). It has been proposed that src activation,through FAK, induces the tyrosine phosphorylation of alinker protein shr (Takahashi and Berk, 1996). Phos-phorylated shr can recruit the Grb2/Sos complex to themembrane, where Sos can activate ras. Ras, similarlyto rho, belongs to a superfamily of monomeric GTPases(Macara et al., 1996). Through a cascade of events, rasactivates ERK1/2. Although integrin activation can

activate the ras-ERK1/2 pathway, this pathway is notnecessary for integrin-dependent events, such as rear-rangement of focal adhesions (Clark and Hynes, 1996).Recent studies indicated that shear-stress-induced FAKphosphorylation, which was probably due to integrinactivation, only partially accounted for the flow-stimu-lated responses of endothelial cells (Ishida et al., 1996).The above findings indicate that there are synergistic

or additive interactions between integrins, G-protein-coupled receptors, and the ras-ERK1/2 signaling pathwayupon exposure to fluid shear stress.Protein Kinases. The initiation of signal transduc-

tion events by “shear-stress-sensitive receptors” leads toa cascade of downstream signaling events many of whichare mediated by sequentially activated protein kinases.Full activation of protein kinases requires phosphoryla-tion of both a threonine and a tyrosine, which is morestable than tyrosine phosphorylations alone. Once thekinases are activated they relay signals downstream byphosphorylating other protein kinases and transcriptionfactors. These kinases are turned off by activation ofspecific phosphatases. MAPK are the most well studiedkinases in response to hemodynamic forces, and theywere first identified as microtubule associated kinases,due to their involvement with the cytoskeleton (Berk etal., 1995).Shear stress can activate MAPK (in particular the

MAPK subtypes ERK1/2) in endothelial cells (Figure 7)(Berk et al., 1995; Ishida et al., 1996; Takahashi andBerk, 1996). Receptor (or receptor-associated) tyrosinekinases are probably involved in ERK1/2 activation (Berket al., 1995; Davies, 1995; Pelech and Sanghera, 1994).

Figure 6. Integrin activation by shear stress as a possible mode of endothelial cell signal transduction. Activation of this pathwayincludes the tyrosine kinases FAK and src, which phosphorylate the cytoskeletal protein paxillin. Src activation also induces ERK1/2phosphorylation while rho regulates adhesion-dependent formation of 4,5-PIP2.


Ligand binding causes receptor dimerization and tyrosinephosphorylation. A growth factor receptor-binding pro-tein 2 (Grb2) binds to the phosphorylated receptor,probably through interactions with PKC, and activatesSos, which subsequently activates ras. Ras recruits andactivates raf-1 kinase (also referred to as MAP-kinase-kinase-kinase or MAPKKK) at the plasma membrane,which then phosphorylates MEK (also referred to asMAP-kinase-kinase or MAPKK). Active MEK can thenactivate ERK1/2 by phosphorylation (Berk et al., 1995).G-protein-coupled receptors can also feed into the ERK1/2pathway, probably (a) by activation of receptor-associatedtyrosine kinases, such as src, which phosphorylate thelinker protein shr, which then interacts with the Grb2/Sos complex, or (b) by G protein âγ subunit binding tothe Grb2/Sos complex (Chen et al., 1994; Takahashi andBerk, 1996). ERK1/2 activates transcription factors (suchas c-fos, c-myc) and proteins involved in protein synthesis(such as ribosomal S6 protein) (Berk et al., 1995).Although the pathways that lead from ligand binding

to ERK1/2 activation have been elucidated, little is knownabout the shear-stress-induced activation of ERK1/2. Li,Y.-S., et al. showed that ras has an important role in theflow-induced response of vascular endothelial cells andthat its amount is increased during shear stress exposure(Li, Y.-S., et al., 1996). ERK1/2, activated by shear stress,required G-protein and PKC activation but was indepen-dent of Ca2+ mobilization (Tseng et al., 1995). Interest-ingly, no shear-stress-sensitive phosphatases have beenidentified to date.Another mitogen-activated protein kinase pathway

recently found to be involved in shear-induced geneexpression is the ras-JNK (Jo et al., 1997; Li, Y.-S., etal., 1996). Activation of ras, by a variety of cellular

stresses, can activate the MEK kinase (MEKK) to triggerdownstream kinases, such as c-Jun NH2-terminal kinases(JNK), p38MAPK, and less preferentially MAPK (Berket al., 1995; Li, S., et al., 1996; Li, Y.-S., et al., 1996).JNK activates c-jun by phosphorylation (Li, Y.-S., et al.,1996), while p38MAPK activates MAP kinase-activatedprotein kinase-2 (MAPKAP kinase-2), which then phos-phorylates small heat shock proteins Hsp25/Hsp27 (Li,S., et al., 1996). It has been shown that shear stresspreferentially activated the Ras-JNK pathway to theMAPK pathway, and the activation of this pathway isprimarily responsible for AP-1/TRE-mediated gene ex-pression (Li, Y.-S., et al., 1996). Another report providedevidence that shear stress (1) differentially activated bothERK1/2 (rapid biphasic time course and shear stressdependence) and JNK (slow, prolonged time course andmaximum activation by a threshold level of shear stress)and (2) activated the JNK pathway which required theactivation of G-proteins, ras, and tyrosine kinases (Jo etal., 1997). The same investigators proposed that activa-tion of JNK or ERK1/2 pathway may regulate theexpression of shear-stress-sensitive genes differentiallyand that loss of the balance between JNK and ERK1/2may contribute to the progression of cardiovasculardiseases such as atherosclerosis.Furthermore, Li, S., et al. have shown that shear stress

caused prolonged phosphorylation of HSP27/25 in vas-cular endothelial cells. Activation of HSP27/25 has beenassociated with changes in microfilament organizationand may be associated with signal transduction eventsthat lead to reorganization of the cytoskeleton and changeof cell shape upon exposure to shear stress (Li, S., et al.,1996). Prolonged phosphorylation events allow the signal

Figure 7. Stimulation of various protein kinases by fluid shear stress. Activation of hypothetical mechanoreceptors leads to activationof a cascade of protein kinases which might subsequently signal downstream events. The major protein kinases families activatedby shear stress are ERK1/2, JNK, and p38MAPK/RK.


transmission events to continue for longer periods of time,independent of surface receptor desensitization.Shear stress thus appears to activate multiple signal-

ing pathways in endothelial cells, due to activation ofdifferent mechanoreceptors, that form a highly intercon-nected signaling network.Transcription Factors, Immediate Early Genes.

Transcription factors or “third messengers” are proteinstypically located in the cytosol or in the plasma mem-brane (Davies, 1995). These readily available elementsare activated post-translationally by “second messen-gers”, and they can rapidly regulate the expression ofselective immediate early genes (IEG) (Davies, 1995;Nollert et al., 1992). Shortly after the cell is stimulated,IEG are transcribed and code for “immediate early”proteins, which play a regulatory role in the transcriptionof other “late response” proteins. Endothelial IEG’stransiently upregulated by shear stress are c-myc, c-fos,and c-jun (Hsieh et al., 1993). c-fos and c-jun encodeproteins that belong to nuclear activator protein 1 (AP-1) family of transcription factors, while c-myc protein canassociate with other proteins to form a “putative” tran-scription factor. Two transcription factor families presentin endothelial cells are stimulated by a number of definedstimuli as well as shear stress (Figure 8) (Lan et al.,1994). These families are (1) AP-1 and (2) Rel-relatednuclear factor kappa B (NFκB).

AP-1 complex is formed from the dimerization of Fos/Jun families of proteins through a “leucine zipper”(Nollert et al., 1992). AP-1 can bind to cis-acting ele-ments, TRE (tumor-promoting agent response element)and CRE (cAMP response element), which are found oncertain gene promoters. The consensus sequences ofthese cis-acting elements are shown in Table 2. The c-fos/c-jun heterodimers exhibit higher binding affinity for theTRE site compared to c-jun/c-jun homodimers, whereasthe c-fos/c-fos homodimers are unable to efficiently bind.Despite the higher stability of the heterodimer complex,recent studies provided evidence that the c-jun/c-junhomodimer rather than the c-fos/c-jun heterodimer is theactivator of the downstream AP-1/TRE in endothelialcells exposed to arterial levels of shear stress (Li, Y.-S.,et al., 1996). Through the ras-MEKK-JNK pathway, thelatent c-jun is activated and translocated to the nucleus,where c-jun/c-jun complexes are formed and interact withTRE to activate the target gene (Li, Y.-S., et al., 1996).Furthermore, Lan et al. showed that rapidly upon

Figure 8. Proposed model for the shear stress regulation of gene expression. Shear stress alters the transcriptional activity ofvarious genes via the activation of transcription factors which bind to certain regulatory cis-elements in the promoter region of thegene.

Table 2. Transcription Factors Involved inFlow-Induced Gene Regulation and Their BindingSequences

DNA binding factor DNA binding sequence

AP-1 (c-fos/c-jun or c-jun/c-jun) TRE: TGACTCACRE: TGACGTCA

NFκB (p50/p65) NFκB: GGAAGATCCCTNFkB, others (p50/p65, others) SSRE: GAGACC


exposure to shear stress there was a biphasic inductionof AP-1/DNA binding compared to very little bindingunder stationary conditions (Lan et al., 1994). Genesthat contain AP-1 binding sites (TRE or CRE) in their 5′promoter region are c-fos, collagenase, human metal-lothionein IIa, monocyte chemotactic protein 1 (MCP-1),intracellular adhesion molecule 1 (ICAM-1), platelet-derived growth factor A/B (PDGF-A/B), tissue plasmino-gen activator (tPA), plasminogen activator inhibitor 1(PAI-1), and endothelin 1 (ET-1) TGF-â1 (Malek andIzumo, 1995; Shyy et al., 1995b). To date, of all the abovegenes, only MCP-1 has been shown to respond to shearstress through AP-1/DNA binding in endothelial cells(Shyy et al., 1995b).NFκB is a pluripotent transcription factor, activated

by a variety of stimuli, which is found in the cytosol ofinactivated cells as a heterodimer of a p50 and a p65protein, bound to an inhibitory, cytoplasmic IκB protein(Lan et al., 1994; Read et al., 1994). Rapidly uponactivation, the p50/p65 protein complex dissociates fromIκB, allowing transport of free NFκB to the nucleus whereit can bind to specific NFκB consensus sites (Table 2).The cytoplasmic form of NFκB (bound to IκB) can notbind to DNA or to translocate to the nucleus, and thefree IκB is rapidly degraded (Read et al., 1994). It hasbeen suggested that NFκB is activated by protein kinases(such as PKC) since dissociation from IκB was precededby phosphorylation events (Hsieh et al., 1992; Li et al.,1993). The DNA binding activity of NFκB steadilyincreases during the first 1 h of flow exposure in endot-helial cells (Lan et al., 1994). NFκB/DNA binding sitesare found primarily on inducible pathophysiologicallyrelevant genes in vascular cells, such as VCAM-1 (vas-cular cell adhesion molecule 1), E-selectin, tissue factor(TF), IL-1, IL-6, IL-8, and c-myc, (Read et al., 1994). Inthe TF promoter, both the NFκB enhancer element anda GC-rich region were involved in the shear-stress-induced expression of the TF gene (Lin et al., 1995).Recently, it has been proposed that physical forces such

as fluid shear stress can initiate molecular signalingthrough “stress-sensitive” promoter elements, present onspecific genes (Malek and Izumo, 1995; Patrick et al.,1995; Resnick et al., 1993; Resnick and Gimbrone, 1995).By analyzing the activity of a series of PDGF-B promoter-CAT (chloramphenicol acetyltransferase) deletion mu-tants, Resnick et al. identified a shear stress responseelement (SSRE), shown in Table 2, in the humanPDGF-B promoter, that was necessary for the shear-stress-induced increase in PDGF-B mRNA levels (Resnicket al., 1993). The transcription factor which interactsfunctionally with the SSRE in the promoter of PDGF-Bappears to be the heterodimeric NFκB (Khachigian et al.,1995). Within 1 h of flow exposure, p50 and p65 subunitsaccumulate in the cell nucleus and bind to PDGF-BSSRE. The SSRE is found in a number of shear respon-sive genes including c-fos, c-jun, ICAM-1, tPA, nitric oxidesynthase III (NOS III), MCP-1, and TGF-â1 (Malek andIzumo, 1995; Resnick et al., 1993). Regardless of thepresence of the SSRE element, the activation of theMCP-1 is mediated via the TRE element (Shyy et al.,1995b), and the shear-induced activation of the TGF-â1

was found in promoter constructs that contained noSSRE or complementary sequences (Ohno et al., 1995).It has been found recently that the shear-stress-

induced expression of ET-1 gene is regulated via a ciselement in its promoter in the region between -2.5 and2.9 kb upstream of the transcription initiation site(Patrick et al., 1995). Furthermore, although shearstress facilitated the binding of AP-1 and NFκB to DNA,this was not sufficient to trigger the transcription of allgenes that contained AP-1 and/or NFκB nucleotiderecognition sequences (Khachigian et al., 1995). Thissuggests that AP-1 or NFκB could be cooperating withother transcription factors to stimulate expression ofcertain genes. In addition, there are multiple potentialcis-elements, located at the 5′ promoter of a gene, whichcould be shear sensitive, and their selective interactionsunder different conditions could modulate the responseof a gene to shear stress (Malek and Izumo, 1995; Shyyet al., 1995a).Another way that shear stress can regulate gene

expression, besides transcriptionally, is at the level ofmRNA stability (Malek and Izumo, 1995; Patrick et al.,1995; Ross, 1995). This could be important for genes thatare downregulated by shear stress, such as VCAM-1(Ando et al., 1994; Sampath et al., 1995). In the 3′untranslated region of the human VCAM-1 gene thereare six dispersed AUUUA repeats, which are known toinduce instability in certain genes (Patrick et al., 1995).In contrast, genes whose mRNA levels are increased uponexposure to shear stress, such as tPA and PDGF-B, haveonly one AUUUA sequence, while the constitutivelyexpressed glyceraldehyde phosphate dehydrogenase (GAP-DH) gene has none. However, these AUUUAmotifs mayonly be involved in the transport of mRNA to thecytoplasm, and VCAM-1, or any other gene that isdownregulated by shear stress, might be regulated at thetranscriptional level.

Flow-Induced Responses of Smooth MuscleCells

Although the effects of shear stress on endothelial cellsare well documented, very few studies address the effectsof shear stress on smooth muscle cells. This may notseem relevant, as smooth muscle cells in most arteriesare physically isolated from the lumen by the endothe-lium and internal elastic lamina and thus are distantfrom the primary site at which shear stress is experi-enced. However, there is evidence that smooth musclecells are exposed to wall shear stress which is derivedfrom transmural pressure gradients even in the presenceof the intact endothelium (Wang and Tarbell, 1995).Although the transmural flow is very low, the interstitialspaces in the tissue are small and the resistance providedby the proteoglycan collagen matrix can lead to thinboundary layers and surprisingly high shear stresses (onthe order of several dyn/cm2). Although there is limitedbiological evidence, alterations in blood pressure mightaffect transmural flow and thus the function of smoothmuscle cells in the normal vascular wall. Another waythat smooth muscle cells may be directly exposed to shear

Table 3. Shear-Stress-Mediated Smooth Muscle Cell Responses

effect refs

inhibition of cell proliferation Papadaki et al., 1996; Sterpetti et al., 1993no alignment to the direction of flow Papadaki et al., 1996increased production of PGE2, PGI2 Alshihabi et al., 1996release of bFGF, PDGF Sterpetti et al., 1994increased production of NO, downregulation of thrombin receptormRNA and protein levels, increased production of tPA

Papadaki et al., 1997; Ruef et al., 1997

increases in intracellular pH Stamatas et al., 1997no changes in intracellular Ca2+ Geiger et al., 1992; Stamatas et al., 1997


stress is after cardiovascular interventions. In vivostudies using rat carotid artery balloon catheter injury,in which the endothelium was completely removed, havedemonstrated that areas of low shear stress had signifi-cantly more intimal hyperplasia than high shear stressareas (Kohler et al., 1991; Kraiss et al., 1991). Inaddition, smooth muscle cells indirectly respond tochanges in fluid shear stress in the presence of an intactendothelium under pathophysiological conditions, sincealterations in the local blood flow pattern, in addition tosystemic risk factors, play a role in determining the rateof graft thickening and atherogenesis (Dobrin et al., 1989;Gibson et al., 1993; Kohler et al., 1991; Kraiss et al.,1991).Little is known about the biological effects of fluid

shear stress on cultured smooth muscle cell metabolismand gene regulation (Alshihabi et al., 1996; Papadaki etal., 1996; Sterpetti et al., 1993). Table 3 summarizessome of the recent advances on the flow-induced re-sponses of smooth muscle cells, which indicate thatsmooth muscle cells in addition to endothelial cells aresensitive to alterations in shear stress.

Conclusions

In the last few years, with the recent advances in celland molecular biology, as well as with the developmentof quantitative instrumentation and modeling tech-niques, progress has been made toward the identificationof the molecular mechanisms by which vascular cellssense and respond to changes in flow-induced shearstress. Understanding the complex interplay of shear-stress-induced signal transduction and gene regulationin vascular cells will facilitate our understanding ofatherosclerosis, thrombosis, restenosis, and angiogenesisand will help to develop strategies to manage cardiovas-cular diseases. Gene therapy technologies can be ef-fectively used to differentially turn on/off shear-stress-sensitive promoters to regulate gene expression in differentflow regimes of the vascular wall. Alternatively, use oftransgenic animals with shear-stress-sensitive knockoutgenes may provide insight into the relative contributionof the different flow-responsive genes in the progressionof hyperproliferative vascular diseases.

NotationAA arachidonic acidAC adenylate cyclaseAP-1 activator protein-1ATP adenosine triphosphatecAMP cyclic adenosine monophoshatecGMP cyclic guanosine monophosphateCRE cAMP response elementDAG diacylglycerolERK1/2 extracellular signal-regulated kinase 1/2ET-1 endothelin-1FAK focal adhesion kinasesGAPDH glyceraldehyde phosphate dehydrogenaseGC guanylate cyclaseHUVEC human umbilical vein endothelial cellsICAM-1 intracellular adhesion molecule 1IEG immediate early genesIF intermediate filamentsIP3 inositol 1,4,5-trisphosphateMAPK mitogen-activated protein kinaseMCP-1 monocyte chemotactic protein 1MEK MAP-kinase-kinaseMEKK MEK kinaseMF microfilaments

MT microtubulesNFκB nuclear factor kappa BNO nitric oxidePA phosphatidatePAI-1 plasminogen activator inhibitor 1PDGF-A/B platelet-derived growth factor A/BPGI2 prostacyclinPIP2 phosphatidylinositol 4,5-bisphosphatePKC protein kinase CPLC phospholipase CSSRE shear stress response elementTF tissue factorTGF-â1 transforming growth factor â1tPA tissue plasminogen activatorTRE tumor-promoting agent response elementVCAM-1 vascular cell adhesion molecule 1

Acknowledgment

This work was supported in part by NIH GrantsHL18672 and NS23326, NASA Grant NAGW-5007, WelchFoundation Grant C-0938, and TATP Grant 003604 andby Texas Biotechnology Corp.

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Accepted April 14, 1997.X

BP970029F

X Abstract published in Advance ACS Abstracts,May 15, 1997.


effects of fluid shear stress on gene regulation of vascular cells

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