Effects of Mechanical Forces on Signal Transduction and Gene Expression in Endothelial Cells
Fluid shear stress and circumferential stretch play important roles in maintaining the homeostasis of the blood vessel, and they can also be pathophysiological factors in cardiovascular diseases such as atherosclerosis and hypertension. The uses of flow channels and stretch devices as in vitro models have helped to elucidate the mechanisms of signal transduction and gene expression in cultured endothelial cells in response to shear stress, which is a function of blood flow and vascular geometry, or mechanical strain, which is a function of transmural pressure and the mechanical properties and geometry of the vessel. Shear stress has been found to increase the activities of a number of kinases to modulate the phosphorylation of many signaling proteins in endothelial cells, eg, the proteins in focal adhesion sites and the proteins in the mitogen-activated protein kinase pathways. Downstream to such signaling cascades, multiple transcription factors such as AP-1, NF-κB, Sp-1, and Egr-1 are activated. The actions of these transcription factors on the corresponding cis-elements result in the induction of genes encoding for vasoactivators, adhesion molecules, monocyte chemoattractants, and growth factors in endothelial cells, thus modulating vascular structure and function. Some of the effects of mechanical strain on endothelial cells are similar to those by shear stress, eg, the signaling pathways and the genes activated, but there are differences, eg, the time course of the responses. Studies on the effects of mechanical forces on signal transduction and gene expression provide insights into the molecular mechanisms by which hemodynamic factors regulate vascular physiology and pathophysiology.
Vascular endothelial cells (ECs), in addition to providing a barrier between the blood and vessel wall, mediate many physiological and pathological processes by expressing proteins and factors which function as vasodilators (eg, NO), vasoconstrictors (eg, ET-1), growth factors (eg, PDGF), growth inhibitors (eg, heparin), adhesion molecules (eg, ICAM-1), and chemoattractants (eg, MCP-1). In vivo, ECs are constantly exposed to hemodynamic forces, which include the shear stress, the tangential force due to blood flow, and the circumferential stress, the normal force due to transmural pressure. To better control the chemical and mechanical environment of the cell, in vitro experiments have been performed to investigate the effects of shear stress and mechanical strain on the structure and functions of cultured ECs using devices such as flow channels and stretch apparatus. Results from these in vitro studies demonstrate that shear stress and/or mechanical strain can modulate the expression of genes, which are critical in endothelial physiology and pathophysiology. This article provides a brief summary of the signal transduction mechanisms and the attendant gene expression in the EC in response to shear stress and mechanical strain. The results provide some insights into the sequence of events by which the signaling molecules mediate the gene expression in response to mechanical forces.
Shear Stress Induction of Gene Expression in ECs
Shear stress plays a major role in modulating endothelial functions.1 The modulation is mediated in part by the regulation of genes encoding for many proteins including vasoactive substances, growth factors, adhesion molecules, chemotactic molecules, coagulation factors, and proto-oncogenes (Table).
|Factors||Level†||Shear Stress, dyn/cm2‡||Shear Stress-Inducible Elements in the Promoter||References|
|ET-1||↓||⇓||L(8–25), P(12–18), T(15)||5–9|
|PDGF-A||↑||⇒||L(6–51), P(12–18)||Egr-1 binding site||14–16|
|PDGF-B||↑||⇒||L(6–51), P(12–18)||NF-κB binding site||14,15,17,18|
|bFGF||↑||⇒||L(15–36), P(12–18), T(15)||19|
|MCP-1||↑||⇓||L(16)||AP-1 binding site||27,28|
|TF||↑||⇒||L(12)||Sp1 binding site||29|
|Thrombomodulin||↑||⇑||L(15–36), P(12–18), T(15)||31,32|
Shear stress increases the mRNA level of NOS and the production of NO.2–4 The transient burst of NO is followed by a sustained upregulation, which causes the relaxation of vessels constantly exposed to shear stress. Shear stress also augments the production of prostacyclin, another potent vasodilator,10,11 which has been suggested to be dependent on the NO signaling.36 The NO production in the EC monolayer preconditioned with shear stress is accompanied by a decrease in monocyte adhesion.37 Together, these results suggest that the shear stress induction of NO is critical in maintaining the vessels in a dilated state, which may be antiatherogenic. Consistent with this notion, the vasoconstrictor ET-1 and its mRNA levels in ECs decrease after exposure to arterial levels of shear stress.5–7,9 However, low shear stress (>5 dyn/cm2) may increase the mRNA level and the release of ET-1.8,9
Adhesion molecules, eg, vascular cell adhesion molecule-1, ICAM-1, and E-selection, mediate leukocyte adhesion and rolling. It has been shown that laminar shear stress downregulates the expression of vascular cell adhesion molecule, which accounts for the decrease of lymphocyte adhesion to ECs.25,26 In contrast, the expression of E-selectin is not sensitive to shear stress and that of ICAM-1 is upregulated.22–24 The initial increase of ICAM-1 expression can be correlated to the increase in leukocyte adhesion to ECs,38 suggesting that ICAM-1 is involved in the inflammatory response to acute changes of shear stress, eg, in reperfusion injury. MCP-1 mRNA is regulated by shear stress in a transient manner with a rapid increase followed by a suppression,27 indicating that the monocyte chemoattractant MCP-1 is also involved in the responses to a sudden change of shear stress.
Shear stress can modulate the endothelial functions in thrombosis and fibrinolysis. It has been shown that shear stress upregulates cyclooxygenase-2, which is an antithrombotic enzyme, and thrombomodulin, which is a potent activator of protein C anticoagulant pathway.31–33 In addition, shear stress causes a sustained induction of tissue plasminogen activator30 and a transient induction of TF, an initiator of the coagulation cascade.29 Thus, shear stress can modulate the thrombotic and fibrinolytic activities of ECs through an intricate balance among different coagulation factors.
The shear stress-induced expression of IE genes and genes encoding for growth factors are also transient. IE genes such as c-fos and c-jun are activated within minutes of shear.15 Transient activation by shear stress has been shown for PDGF-B, basic fibroblast growth factor, and heparin-binding epidermal growth factor-like growth factor,14,19,21 whereas the shear stress-induced activation of transforming growth factor β-1, an inhibitor for smooth muscle cell growth, is sustained.20 These results suggest that while a sudden increase of shear stress may result in a transient induction of mitogenic responses, chronic exposure of ECs to shear stress, which is the physiological condition in vivo, may be antiproliferative.
Effects of Flow Pattern and the Magnitude of Shear Stress on Gene Expression
Various types of flow channels have been used to apply different forms of shear stress to ECs by generating laminar steady flow, laminar pulstile flow, and turbulent flow. Pulsatile laminar flow has been shown to have effects qualitatively similar to those of steady laminar flow in modulating genes encoding for ET-1, PDGF-B, basic fibroblast growth factor, thrombomodulin, c-Fos, and c-Jun.7,15,19,31 Turbulent flow has been shown to induce ET-1, basic fibroblast growth factor, and thrombomodulin.7,20,31 It is intriguing that laminar flow, but not turbulent flow, induces a sustained induction of NOS, cyclooxygenase-2 (antithrombotic), and Mn superoxide dismutase (antioxidant), suggesting the protective effects of shear stress on ECs by expressing these antiatherogenic genes.33
Under laminar flow conditions, the response of EC gene expression is a function of the magnitude of the shear stress. The expression of c-fos, ICAM-1, and C-type natriuretic peptide is correlated with the magnitude of shear stress.13,15,22 The expression of tissue plasminogen activator and thrombomodulin is increased only by shear stresses higher than 5 dyn/cm.2 30,31 In contrast, ET-1 secretion is increased at low shear stresses (>5 dyn/cm2) but decreased by moderate and high shear stresses.9 Because the magnitude of shear stress varies at different parts of the vascular network, these findings suggest that some genes can be differently regulated as a function of topographical locations in the vascular tree.
Shear Stress-Inducible Elements
Modulation of gene expression results from the binding of specific transcription factors to their target cis-element in the promoter region of the gene. To identify the transcription factors involved in shear stress-induced gene expression, many laboratories, including ours, have used reporter systems to investigate the shear stress-inducible cis-elements in the promoter of various genes known to be regulated by shear stress. The shear stress-responsive element in the PDGF-B promoter is the first identified cis-element, which is inducible by shear stress. Transfection of a series of deletion mutants of PDGF-B promoter fused to chloramphenicol acetyltransferase has led to the characterization of the shear stress-responsive element with the deoxynucleotide sequence of GAGACC.17 It has further been demonstrated that nuclear factor NF-κB/p50-p65 heterodimer binds to the shear stress-responsive element.18 This is in concert with the finding that shear stress increases the binding activities of NF-κB to their target sequences demonstrated by electrophoretic mobility shift assays.39
AP-1, which binds to the TRE, is one of the transcription factors implicated in the MAPK-mediated gene regulation induced by ultraviolet irradiation and osmotic stress.40,41 AP-1 binding activity to EC nuclear extract is increased by shear stress.39 To search for the cis-elements in the promoter region of the MCP-1 gene responsible for shear stress induction, our laboratory has found that a divergent TRE with the sequence of TGACTCC is critical for shear inducibility.28 Deletion or mutation of this element in the chimeric constructs abolishes their responses to shear stress. At the upstream, the phosphorylation of c-Jun is critical for the activation of AP-1 and its induction of TRE-mediated gene expression in response to shear stress.42 Another shear stress-inducible element is the Sp 1 sites in the promoter of the TF gene.29 Functional analysis of the promoter region of the TF gene indicates that a GC-rich region containing three copies each of the Egr-1 and Sp1 sites is required for TF gene induction by shear stress. Mutation of the Sp1 sites, but not the Egr-1 sites, attenuates the response of the TF promoter to shear stress, implying that Sp-1 is critical for shear inducibility of the TF gene. In contrast, for the PDGF-A gene, the Egr-1 sites in its promoter, rather than the Sp1, is responsible for its shear stress inducibility.16
There are several other instances in which the shear stress-inducible element for one gene may be present in the promoter in another gene but is not responsible for shear inducibility. Such examples include the shear stress-responsive element in the promoter of the MCP-1 gene and the TRE and κB in the promoter of the TF gene. Therefore, different genes may use different sets of cis-elements in their responses to shear stress, and there is not a single shear-inducible cis-element. For a given gene that possesses multiple cis-elements, not all of them are responsible for shear inducibility. It is possible that the apparently nonresponsible cis-elements may play a role in fine tuning the shear inducibility through their interactions with the primary shear-inducible cis-element.
Shear Stress Activation of Signal Transduction in ECs
The activation of transcription factors to induce gene expression requires first the transduction of the mechanical stimuli to chemical signals. This mechanochemical transduction involves the activation at the cell membrane surface and the signaling pathways inside the cytoplasm. It has been reported that shear stress modulates many signaling molecules in ECs, including the membrane K+ channel,43 G proteins,44 intracellular Ca2+,45 cAMP,46 cGMP,47 inositol trisphosphate,48 protein kinase C,9 MAPKs such as ERKs and JNKs,42,49 small GTPases such as Ras,42 and PTKs such as FAK.50,51The modulation of these signaling molecules has been linked to the regulation of the expression of many genes in ECs in response to shear stress. For example, it has been shown that the K+ channel is involved in the shear stress induction of NOS3; protein kinase C regulates the induction of heparin-binding epidermal growth factor-like growth factor;21 whereas G proteins and protein kinase C may be involved in the expression of PDGF.52
It appears that in contrast to the specific activation of membrane receptors by chemical ligands, mechanical forces such as shear stress activate simultaneously many membrane proteins, including receptors and ion channels. Thus, instead of specific conformation changes induced by ligand binding, mechanical forces may induce conformational changes or clustering of membrane proteins in a relatively nonspecific manner, leading to the initiation of signal transduction. Molecular dynamics such as lateral mobility in the plane of the cell membrane may play an important role in the initial step of mechanochemical transduction. It is interesting to note that changes in lipid fluidity alter the activation of G proteins in liposomes by shear stress.53 In addition, the reorganization of the focal adhesion complexes at the abluminal side of the EC and the integrity of cytoskeleton, especially actin structure, may also play important roles in the initiation of the signaling cascades. Shear stress can induce the rearrangement of microfilaments and focal adhesions in ECs, and the force may be transduced by microfilaments to focal adhesion complexes.51,54,55 Disruption of actin filaments attenuates shear stress-induced MAPK activation and TRE transcriptional activity,51 but enhances shear stress-induced ET-1 gene expression,8 indicating that cytoskeleton has differential roles in the expression of different genes. The NOS gene has been shown to be regulated by membrane initiated signaling, but not cytoskeleton-related signaling,56 suggesting that membrane- and cytoskeleton-regulated signaling could be independent.
The following paragraphs provide a discussion of the shear stress-induced responses of MAPKs, small GTPases and heterotrimer G proteins, and PTKs, which are among the molecules in the signaling pathways that play important roles in the mechanotransduction in ECs in response to shear stress.
MAPKs are a group of Ser/Thr kinases that are activated in response to extracellular stimuli through dual-phosphorylation at conserved threonine and tyrosine residues.41 To date, several MAPKs, including JNK, ERK, and p38, have been characterized. MAPKs are activated by MAPK kinases, which are in turn activated by MAPK kinase kinases. Shear stress has been found to activate ERK and JNK in ECs in a rapid and transient manner (Fig 1).42 ERK activation may lead to c-fos gene expression, and JNK activation may induce c-Jun phosphorylation which further increases AP-1/TRE transcriptional activity.42,57 The dual activation of ERK and JNK in ECs may have significant implications in vascular biology since ERK is involved in the mediation of cell growth and JNK may be engaged in programmed cell death.41 Upstream to MAPK activation, small GTPases such as Ras are involved.42,58
Small GTPases and Heterotrimer G Proteins
Small GTPases, eg, Ras and Rho family proteins, are guanine nucleotide-binding proteins with an intrinsic GTPase activity. The Rho family GTPases Cdc42, Rac, and Rho regulate actin-based cytoskeletal structure: Cdc42 has been shown to modulate the microspike formation induced by bradykinin, and Rac regulates the growth factor-induced membrane ruffling; whereas Rho increases cell contractility and regulates the formation of focal adhesions and actin stress fibers in response to growth factors and lysophosphatidic acid.59 Cdc42 and and Rac have also been shown to regulate the JNK pathway.60 Ras is an important mediator for cell proliferation and cellular responses to extracellular stimuli. We have found that shear stress induces a transient and rapid activation of Ras in ECs. RasN17, a dominant negative mutant of Ha-Ras, attenuates the shear-activation of JNK and ERK,42,58 indicating that Ras is upstream to MAPKs in response to shear stress. Sos is a guanine nucleotide exchange factor that activates Ras by converting it from the GDP-bound inactive state to the GTP-bound active state.61 The negative mutant of Sos attenuates the shear stress induction of c-Jun transcriptional activity, suggesting that Sos is an upstream molecule in regulating the shear-activated Ras signaling.42 Our recent work has shown that Cdc42 and Rac are parallel to Ras in regulating JNK in ECs in response to shear stress, and this is different from the hierarchical relationship among Rho family GTPases (eg, Cdc42→Rac) in the modulation of focal adhesion formation.62 Disruption of actin filaments with cytochalasin B does not affect the JNK-activation by Cdc42 and Rac (S.C., et al, 1997, unpublished data), suggesting that the small GTPases regulate actin structure and MAPKs signaling through different pathways. Studies on the effects of chemical stimuli in other cell types demonstrate that heterotrimer G protein-linked receptors and PTKs could lead to the activation of Rho family GTPases.63,64
Heterotrimer G proteins Gq and Gi3 have been shown to be activated by shear stress in ECs,44 and antisense Gαq inhibits shear stress-induced Ras activation.65 Expression of the carboxy terminus of β-adrenergic receptor kinase (βARK-ct), a Gβ/γ scavenger, inhibits the shear stress-activation of HAJNK, whereas blockade of Gα12 with the mutant α12(G203) or antisense Gα12 prevents shear-dependent activation of HAERK.58 These results suggest that ERK and JNK can be regulated through different G protein-dependent mechanisms and that more than one type of G protein subunit can be activated by shear stress.
PTKs play an important role in the signaling process that leads to the activation of MAPKs, as indicated by the finding that genistein, a PTK inhibitor, can attenuate the shear stress activation of ERK and JNK.51,58,66 PTKs are also critical in the shear stress-regulation of EC shape and stress fibers,67 as well as in the early phase of flow-dependent NO production.68 Cellular PTKs can be generally divided into two major categories, receptor tyrosine kinases and nonreceptor PTKs. Nonreceptor PTKs (eg, FAK and c-Src) represent cellular enzymes that have intrinsic kinase activities but do not possess extracellular domains. FAK and c-Src are present in focal adhesion sites and are tyrosine-phosphorylated in response to cell adhesion and the stimulation by a number of growth factors.69 Shear stress causes an increase in the tyrosine phosphorylation of FAK and c-Src in ECs.50,51,57,66 Interfering negative mutants of FAK and c-Src attenuate the shear stress activation of ERK and JNK, indicating that these PTKs in the focal adhesion sites are involved in the mechanotransduction.51,57 We have also found that shear stress increases the association of FAK with Grb2 and that the negative mutant of Sos inhibits MAPKs activation, indicating that Grb2/Sos provides a critical link between FAK and MAPKs in response to shear stress.51 The modulation of PTKs in ECs by shear stress demonstrated by the various in vitro studies is supported by an ex vivo experiment in which increased tyrosine phosphorylation has been found in perfused vessels.70
Signal Transduction and Gene Expression in ECs in Response to Mechanical Stretch
The effects of circumferential stress on ECs have been investigated by applying cyclic stretch to ECs cultured on an elastic membrane mounted in a stretch device. Studies from such in vitro experiments demonstrate that cyclic strain increases the expression of NOS,71 MCP-1,72 ET-1,73,74 ICAM-1,75 and plasminogen activator inhibitor-1.76 Many of the signaling events induced by stretch are similar to those by shear stress, eg, the increases of intracellular Ca,2+,77 inositol trisphosphate, and diacylglycerol;78 the activation of protein kinase C and the adenylate cyclase/cAMP/protein kinase A pathway.79-81 Cyclic strain causes in increase in thiobarbituric acid-reactive substances (an index of lipid peroxidation) and H2O2 in porcine aortic ECs, and the augmentation is probably due to an activated NADH/NADPH oxidase.82 Thus, cyclic strain imposes an oxidative stress on ECs. Cyclic strain or treatment with either H2O2 or xanthine oxidase/hypoxanthine has been shown to induce the expression of MCP-1.83 Pretreating ECs with catalase or the antioxidant N-acetylcysteine inhibits the strain- or oxidant-induced MCP-1 mRNA. Functional analysis of the MCP-1 promoter indicates that TRE is sufficient for strain or H2O2 inducibility, as in the case of shear inducibility. In the investigation of the mechanical strain-induced signal transduction pathways, Sumpio and colleagues84,85 reported that cyclic strain increases the tyrosine phosphorylation of FAK and paxillin in ECs with a concurrent cell elongation and the alignment of F-actin, FAK, and paxillin. Tyrphostin A25, a tyrosine kinase inhibitor, and Clostridium botulimum C3 transferase (C3 exoenzyme), a specific inhibitor of Rho small GTPase, inhibit these strain-induced responses. Electrophoretic mobility shift assay shows that the binding activities of transcription factors AP-1, CRE, and NF-κB are increased in human aortic ECs and human umbilical vein ECs exposed to cyclic strain.86 Cyclic strain causes a reorganization of α5, α2, and β1 integrins in a linear pattern in human umbilical vein ECs seeded on fibronectin (a ligand for α5β1) or collagen (a ligand for α2β1),87 demonstrating that α5β1 and α2β1 integrins in human umbilical vein ECs play an important role in the mechanotransduction induced by mechanical strain.
Discussions and Conclusions
There is increasing evidence that multiple genes can be activated by shear stress, which is modulated by a balance among different signaling pathways, and that the temporal and spatial responses of gene expression and signal transduction to shear stress have fundamental importance in vascular biology.88,89
The various signaling molecules in ECs exhibit different time courses in their responses to the applied mechanical stimuli (Fig 2), probably reflecting the temporal sequence of their activation. Those associated with the cell membrane, eg, Ras and c-Src, respond in a time frame of 1 minute or less to reach their peaks in less than 5 minutes. In the downstream, cytoplasmic kinases are activated with a slower time course to reach their peak activities in 10 to 30 minutes. The transcription factors (eg, AP-1) activated through the protein phosphorylation cascades can then translocate into the nucleus to act on different target cis-elements in different genes. Transcriptional activation of IE genes has a time frames of minutes, with genes such as c-fos and c-jun reaching their peaks in less than 30 minutes and genes such as MCP-1 reaching their peak in 1 to 2 hours (Fig 1). The inductions of such mRNA and gene products are not only transient by are followed by a downregulation, with the levels of mRNA and gene products decreased to below the basal level for long periods of time. In contrast to these IE genes, some of the genes encoding for vasoactive substances (eg, NOS) and antithrombotic factors (eg. cyclooxygenase-2) are persistently activated by the applied laminar shear stress.
It seems that the time course of the activation of genes is tuned to their functional roles. IE genes such as c-fos and MCP-1 are concerned with proliferative responses and monocyte attraction, respectively, and are needed for short-term response to vascular injury. These genes are downregulated by sustained shearing. In the physiological situations in vivo, the shear stress in the straight part of the aorta remains high, and hence these genes are downregulated. At the branch points and curvatures, however, the shear stress undergoes considerable temporal and spatial fluctuations, and the flow streams may change not only magnitude but also direction. Therefore, these are the regions where the IE genes are not downregulated and are more susceptible to activation by mechanical and chemical stimuli. Such regional predilection for the activation of atherogenesis-related genes is in agreement with the distribution of atherosclerotic lesions in the arterial tree and provides a hemodynamic and molecular basis for the focal nature of the disease. Along these lines, high laminar shear stress causes the induction of vasodilators such as NO and decreases the expression of vasoconstrictors such as ET-1; it also causes a sustained induction of the fibrinolytic tissue plasminogen activator and the smooth muscle growth inhibitor transforming growth factor β-1, but only a transient induction of TF and several growth factors (eg, PDGF-B, basic fibroblast growth factor, and heparin-binding epidermal growth factor-like growth factor). Thus, the sustained high levels of laminar shear stress in the straight part of the aorta have a protective effect against atherogenic processes, whereas such protection is least in the branch and curved regions of the aorta where flow is unsteady and undergoes directional changes. The beneficial effects of exercise in protecting atherogenesis may be partially related to the enhanced blood flow and the attendant increase in shear stress extending into the branch and curved regions. thus providing the ECs in these regions a more favorable hemodynamic environment in modulating their gene expression.
The responses of endothelial cells to mechanical and chemical stimuli share many signaling pathways and cis-elements. Thus, endothelial cells and most likely other types of cells also use a few basic mechanisms to regulate their gene expression in response to a variety of stimuli. The intricate interplay among these signaling pathways and cis-elements may play a significant role in orchestrating the gene regulation in different cells under different conditions in health and disease.
Some of the effects of mechanical strain on ECs are similar to those caused by shear stress, eg, the increased activities of tyrosine kinase and protein kinase C, the augmented expression of IE genes, and the enhanced binding activities of AP-1, CRE, and NF-κB to their target cis-elements. There are, however, some differences. For example, the MCP-1 gene activation is transient in response to shear stress.26 but is sustained in response to mechanical strain.72 There is insufficient parallel experiments to allow a systematic comparison of the effects of shear stress and mechanical strain.
Although there have been remarkable advances in the understanding of the molecular mechanisms by which hemodynamic forces modulate signal transduction and gene expression in ECs, there are still many missing links. Some of the key issues that remain to be definitely settled include: the mechanism of the initial event of mechanochemical transduction at the EC membrane, the role of integrins in mechanotransduction, the interaction of different signaling molecules in modulating a variety of mechanoresponsive genes, and the interplays between mechanical and chemical events in ECs. Such information is important for the elucidation of the fundamental mechanism of mechanotransduction and endothelial functions in health and diseases.
This work was supported in part by Grants HL19454. HL43026. HL44147 (S.C.), and HL56707 (J.S.) from the National Heart. Lung, and Blood Institute and a gift from the Cho Chang Tsung Foundation for Education.
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