Laminar Shear Stress
Mechanisms by Which Endothelial Cells Transduce an Atheroprotective Force
- Shear Stress and Endothelial Cell Biology: Relevance to Atherosclerosis
- NO: A Critical Factor in Shear Stress–Mediated Atheroprotection
- Mitogen-Activated Protein Kinases: Likely Signaling Molecules in the Transduction of Shear Stress
- Upstream Effectors of ERK1/2 Activity
- Potential Shear Stress Receptors
- Selected Abbreviations and Acronyms
- Figures & Tables
- Info & Metrics
Abstract—Mechanical forces are important modulators of cellular function in many tissues and are particularly important in the cardiovascular system. The endothelium, by virtue of its unique location in the vessel wall, responds rapidly and sensitively to the mechanical conditions created by blood flow and the cardiac cycle. In this study, we examine data which suggest that steady laminar shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. We explore the ability of shear stress to modulate atherogenesis via its effects on endothelial-mediated alterations in coagulation, leukocyte and monocyte migration, smooth muscle growth, lipoprotein uptake and metabolism, and endothelial cell survival. We also propose a model of signal transduction for the endothelial cell response to shear stress including possible mechanotransducers (integrins, caveolae, ion channels, and G proteins), intermediate signaling molecules (c-Src, ras, Raf, protein kinase C) and the mitogen activated protein kinases (ERK1/2, JNK, p38, BMK-1), and effector molecules (nitric oxide). The endothelial cell response to shear stress may also provide a mechanism by which risk factors such as hypertension, diabetes, hypercholesterolemia, and sedentary lifestyle act to promote atherosclerosis.
- Received July 14, 1997.
- Accepted December 3, 1997.
Numerous studies suggest that normal functioning of the endothelium is critical in limiting the development of atherosclerosis, as illustrated by the correlation between risk factors for atherosclerosis (smoking, high cholesterol, high homocysteine, decreased estrogen, increasing age, and hypertension) and endothelial dysfunction.1 Endothelial cells play a critical role in vascular homeostasis by performing many functions. They sense and integrate hemodynamic and hormonal stimuli and effect alterations in vascular function through the secretion of various mediator proteins and molecules.2 As a result of these properties, endothelial cells modulate biological processes related to the blood vessel wall, including regulation of the permeability of plasma lipoproteins, adhesion of leukocytes, and release of prothrombotic and antithrombotic factors, growth factors, and vasoactive substances.3 Impairment of these endothelial cell–mediated processes has been postulated to play a central role in the pathogenesis of atherosclerosis.1
Just as other tissues have developed mechanisms to detect changes in the physiological conditions to which they are exposed, endothelial cells respond not only to humoral factors in the circulation but also to the mechanical conditions created by blood flow and the cardiac cycle.4 As a result of their unique location, endothelial cells experience three primary mechanical forces: pressure, created by the hydrostatic forces of blood within the blood vessel; circumferential stretch or tension, created as a result of defined intercellular connections between the endothelial cells that exert longitudinal forces on the cell during vasomotion; and shear stress, the dragging frictional force created by blood flow. Of these forces, shear stress appears to be a particularly important hemodynamic force because it stimulates the release of vasoactive substances and changes gene expression, cell metabolism, and cell morphology.4
The nature and magnitude of shear stress plays an important role in long-term maintenance of the structure and function of the blood vessel. The nature of shear stress experienced by endothelial cells is a function of blood flow patterns throughout the vasculature generated by the cardiac cycle. In “linear” areas of the vasculature, blood flows in ordered laminar patterns in a pulsatile fashion dependent on the cardiac cycle, and endothelial cells experience pulsatile shear stress with fluctuations in magnitude that yields a mean positive shear stress. This flow pattern should be distinguished from the flow pattern that is often used in experimental preparations and that generates a steady positive shear stress, being temporally and spatially uniform. While steady shear stress generally stimulates many of the same endothelial cell responses as pulsatile stress, there are some qualitative and quantitative differences.5 6 7 Cells exposed to positive shear stress undergo reorientation, with their longitudinal axis parallel to the direction of blood flow.8 9 This reorientation streamlines the endothelial cell, decreasing the effective resistance and lowering shear stress,10 a phenomenon which may or may not be important in terms of adaptation or filtering of shear stress stimuli.4 At areas of abrupt curvatures in the vasculature, as in the carotid bifurcation, the laminar flow of blood is disrupted and separated flow patterns result. Specifically, the medial wall of the carotid bulb experiences higher shear stress, while the lateral wall experiences recirculation vortexes that vary with the cardiac cycle, resulting in flow reversal.11 Thus, the lateral area of the carotid bulb experiences oscillatory shear stress (periodic flow reversal with time-averaged shear stress approaching zero) and low mean shear stress. As a result of the low magnitude of the time-averaged shear stress, these cells do not reorient12 and may be exposed to high shear gradients (differences in shear stress magnitude on a cell scale)4 because their lack of streamlining yields a membrane topology in opposition to the mean shear vector. Several investigators have demonstrated that endothelial cells may actually be sensitive to the magnitude of the shear gradient.12 13 14 Whether these shear gradients or the time-averaged mean shear stress is more critical in terms of atherogenesis remains to be defined. Nevertheless, the significance of these flow patterns is demonstrated by studies that correlate development of atherosclerotic lesions (fatty streaks and small plaques) with areas of the carotid that experience these flow reversals with low time-averaged shear stress.11 15 Regions of the carotid bifurcation that experience pulsatile and mean positive shear stress as the result of laminar blood flow patterns, however, were relatively protected from atherosclerosis. Other investigators have confirmed these observations throughout the vasculature.16 The mechanism by which the physical force generated by fluid shear stress is transduced into biological signals remains unclear.
Below we will briefly review the atheroprotective effects of the endothelium that are influenced by shear stress and then discuss several signal-transduction mechanisms by which shear stress exerts its beneficial effects on endothelial function.
Shear Stress and Endothelial Cell Biology: Relevance to Atherosclerosis
The hypothesis that physical injury to the endothelium might precipitate the atherosclerotic process was introduced over two decades ago. More recently this concept has been modified to include biochemical and cellular alterations in endothelial cell function. There is a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow reversal (Fig 1⇓). Manifestations of dysfunctional endothelium can be readily observed in certain areas of the arterial tree, such as branch points, which experience low mean shear stress and flow reversal.11 These sites demonstrate increased uptake of lipoproteins, appearance of leukocyte adhesion molecules on the surface of the endothelial cells, and leukocyte transmigration. Secretion of chemotactic factors and growth factors causes proliferation of resident monocyte/macrophages, as well as smooth muscle cells. Smooth muscle cells synthesize a connective tissue matrix comprised of elastic fibers, proteins, collagen, and proteoglycans, and the accumulation of lipids and free and esterified cholesterol follows.17 Recent data suggest that low shear stress and, more importantly, oscillatory flow and flow reversal are permissive or even causative in this pathogenic process.4 19
In areas downstream of vessel bifurcations, laminar shear stress predominates, and the endothelial cells experience pulsatile flow, with shear stress on the order of 10 to 30 dyne/cm2.4 The endothelium in these regions maintains circulatory and blood vessel integrity through its ability to regulate several different processes: coagulation, growth of underlying smooth muscle, leukocyte adhesion to and transmigration into the blood vessel wall, and lipoprotein uptake and metabolism.
Coagulation stimulates the release of powerful antithrombotic agents from endothelial cells. Prostacyclin was the first inhibitor of platelet aggregation shown to be released from endothelial cells on exposure to shear stress.7 18 Secretion of prostacyclin from endothelial cells is enhanced when the shear stress is pulsatile compared with steady.7 Numerous investigators have demonstrated that shear stress is one of the most powerful stimuli for release of the vasodilator NO,2 20 21 which also possesses strong anti–platelet aggregation properties.22 Shear stress can also stimulate release of factors that inactivate the clotting cascade.5 Recent studies have shown that shear stress regulates generation of thrombomodulin, which interacts with protein C and protein S to inactivate certain clotting factors. Malek et al23 reported that steady shear resulted in a small transient increase in thrombomodulin expression but continued exposure to shear resulted in decreased thrombomodulin expression in bovine aortic endothelial cells. However, two other laboratories reported that steady shear stress results in sustained increased thrombomodulin expression in human umbilical vein endothelial cells.24 25 The reason for this disparity in species response is unclear. In addition to the potential effect on thrombomodulin, fluid shear stress has also been shown to stimulate expression of tissue plasminogen activator23 24 26 and reduce secretion of plasminogen activator inhibitor type-1.24 Importantly, endothelial cells exposed to turbulent flow failed to show increases in thrombomodulin and tissue plasminogen activator.
Leukocyte Adhesion and Migration
Endothelial cells regulate leukocyte adhesion and migration of monocytes and leukocytes into the blood vessel wall by secretion of chemotactic factors and expression of cell-surface molecules. ICAM-1 binds β2-integrins on various white blood cell derivatives, while VCAM-1 mediates adhesion of monocytes to the endothelium.27 VCAM-1 is one of the earliest markers for fatty streaks and is upregulated in areas of the endothelium surrounding atherosclerotic plaques.27 Several investigators have demonstrated an inverse relationship between VCAM-1 expression and shear stress,28 29 30 31 32 33 which suggests that leukocyte binding should be decreased under conditions of high shear stress. However, other investigators have demonstrated that ICAM-1 expression is upregulated by high shear stress34 35 36 and that leukocyte binding after exposure to shear stress is increased.35 36
The leukocyte binding experiments described above used static cultures of endothelial cells that were exposed to shear stress prior to the binding of leukocytes. However, these experimental conditions do not accurately simulate the physiological conditions of leukocyte binding and the complex interplay between expression of ICAM-1, VCAM-1, and the physical disruption of the leukocyte-endothelial cell interaction by high levels of shear stress.37 38 A study performed by Walpola et al28 is helpful in analyzing which of these parameters may be of physiological importance. In this study, shear stress in rabbit carotid arteries was altered and expression of endothelial cell adhesion molecules, as well as leukocyte binding, was measured. Low shear stress resulted in VCAM-1 expression 30 times greater than that of control vessels, while ICAM-1 expression fell to approximately 30% of control vessels. High levels of shear stress also increased VCAM-1 expression (to 3.5 times that of control vessels), while ICAM-1 expression levels increased (to 1.6 times that of control). Importantly, extensive monocyte adhesion was noted under low shear stress, which colocalized to areas of VCAM-1 expression, while no monocyte adhesion was noted at high shear conditions, indicating that low mean shear stress promotes leukocyte binding in vivo compared with higher shear stress.
Another key factor in monocyte recruitment is the chemoattractant peptide MCP-1.1 Shyy et al39 showed that MCP-1 expression was transiently increased in human umbilical vein endothelial cells on exposure to shear stress. However, MCP-1 gene expression then decreased to basal levels at 4 hours, and once gene expression was fully suppressed, it remained so even after static incubation, leading the authors to suggest that MCP-1 expression is likely suppressed in endothelial cells exposed to steady pulsatile shear stress. Thus, it seems probable that for conduit vessels, high shear stress inhibits leukocyte binding and chemoattractant protein expression while low shear stress and flow reversal promote leukocyte binding and transmigration.
Smooth muscle proliferation is increased in atherosclerotic lesions1 and is likely stimulated by endothelial cell factors that are regulated by shear stress. Kraiss et al40 showed that endothelial platelet-derived growth factor-A mRNA levels and smooth muscle proliferation were increased in areas that experience low blood flow in an arteriovenous fistula model of altered flow in baboons. Endothelin-1, a smooth muscle mitogen that acts synergistically with platelet-derived growth factor, was shown to be dramatically reduced by exposure to 25 dyne/cm2.41 NO42 and transforming growth factor-β,43 both inhibitors of vascular smooth muscle cell growth, are secreted by endothelial cells in response to shear stress. Angiotensin II is an important growth factor for vascular smooth muscle and may also be antiapoptotic.44 Shear stress regulates tissue levels of angiotensin II by virtue of changes in angiotensin-converting enzyme expression. Rieder et al45 recently demonstrated that prolonged exposure to shear stress significantly reduced angiotensin-converting enzyme mRNA and activity. With its ability to regulate these disparate smooth muscle cell growth factors, it seems likely that shear stress plays a role in the increased proliferation of smooth muscle seen at areas of low shear stress and flow reversal.
Unlike the effects of shear stress on growth factor secretion, the role of shear stress in lipoprotein transport and LDL metabolism is less well defined. Deng et al46 reported that the concentration of LDL at the surface of canine carotid arteries was inversely related to wall shear stress rate and suggested that increased surface LDL concentration results in an increased rate of lipid infiltration into the blood vessel. This hypothesis is complemented by studies which demonstrate that areas exposed to flow reversal are relatively permeable to macromolecules including LDL and that LDL accumulation within the vascular wall is preferentially localized to these areas of disturbed flow. Berceli et al47 reported that the LDL incorporation in the rabbit aorta–iliac bifurcation was elevated in the lateral region that experiences flow reversal versus the medial regions that experience higher steady shear, while no differences were present in the transitional or unidirectional zone that experiences relatively steady shear. Other investigators have confirmed these findings in different areas of the vasculature for both rabbits48 and pigs.49 Mechanistically, it appears that compromised endothelial cell integrity, and hence increased macromolecule permeability, results from high shear gradients that are present in low shear stress/flow reversal conditions (See Weinbaum and Chien50 for review). A study by Sprague et al51 showed that 125I-LDL internalization increased in bovine aortic endothelial cells exposed to steady stress conditions for 24 hours; however, this likely reflects an increased metabolic need for LDL under steady shear conditions rather than increased LDL incorporation into the arterial wall.52 Additional studies to define the mechanistic details by which LDL accumulation is linked to low shear stress and flow reversal conditions are warranted.
Endothelial Cell Survival
Finally, shear stress may be critical for endothelial cell survival. Early studies performed by Davies et al12 demonstrated increased endothelial cell turnover in areas that experience turbulent shear stress conditions, suggesting compromised endothelial cell integrity under these conditions. Several recent studies report that the lack of shear stress triggers apoptosis in endothelial cells.53 54 Other investigators have demonstrated that shear stress is required for optimal regeneration of an injured endothelium. Vyalov et al55 reported that under low shear stress conditions, endothelial cells on the border of a wound edge failed to maintain contact with neighboring cells and were oriented randomly. Further, the cells spread and migrated into wound sites more slowly. While steady shear seems to be necessary for endothelial cell integrity, several investigators have demonstrated that steady shear inhibits proliferation of cultured endothelial cells.56 Thus, it appears that shear stress acts as an endothelial cell “survival” factor rather than as a “growth” factor.
NO: A Critical Factor in Shear Stress–Mediated Atheroprotection
NO appears to be a key mediator of the atheroprotective effects of shear stress on the blood vessel wall. NO has been reported to play a role in platelet aggregation and leukocyte binding to the endothelium, in inhibition of vascular smooth muscle tone and growth, and in alteration of lipoprotein metabolism.2 The ability of shear stress to regulate these processes is abrogated by inhibitors of NO production, suggesting that shear stress exerts its effects through the release of NO. Further, it has been postulated that the beneficial effects of regular aerobic training, including its antiatherogenic properties, may be mediated through shear-induced increases in NO secretion.57
NO is produced by a unique enzyme present in the endothelium, termed ecNOS.58 59 60 Shear stress is the most potent physiological stimulus for NO production in endothelial cells. Rapid increases in NO production are due to posttranslational activation of ecNOS, while chronic alterations in ecNOS expression are due to changes in gene expression. Experiments by our laboratory and others61 62 indicate that two distinct signaling pathways (a Ca2+-dependent and a Ca2+-independent pathway) seem to be involved in rapid shear-mediated increases in NO production.63 We compared NO production in response to the Ca2+ ionophore A23187 with shear stress. While A23187 increased NO production by 3-fold to 6-fold, shear stress stimulated NO production by 10-fold to 30-fold above static levels. The initial rapid increase in NO required Ca2+, while the sustained increase in NO production was independent of changes in intracellular Ca2+.64 Further experiments by our laboratory have demonstrated that ecNOS was phosphorylated in response to shear.64 Although the relationship between ecNOS phosphorylation and NO production is unclear, phosphorylation may regulate the activity of ecNOS. To better understand how shear stress influences ecNOS activity and expression, it will be necessary to identify upstream mediators of ecNOS function that are activated by shear stress, such as protein kinases.
Mitogen-Activated Protein Kinases: Likely Signaling Molecules in the Transduction of Shear Stress
Several features of the endothelial cell response to shear stress are analogous to receptor-mediated signaling: dependence on G proteins, increase in intracellular Ca2+, and changes in gene expression. The family of kinases termed MAP kinases are potential candidates to mediate some of the effects of shear stress on endothelial cells. MAP kinases are ubiquitously expressed serine/threonine protein kinases that are activated in response to a variety of extracellular stimuli involved in cell growth, transformation, and differentiation (Fig 2⇓). The extracellular signal–regulated kinases (ERK1/2), members of the MAP kinase family, have many potential substrates, including other protein kinases (p90rsk, MAPKAP, Raf-1, MEK), transcription factors (c-myc, c-jun, c-fos, p62TCF), enzymes (cPLA2), and cell-surface proteins (EGF receptor), and thus have many effects on cellular physiology and gene expression.63
The pathway for ERK1/2 activation in response to growth factors has been well characterized (Fig 2⇑). The MAP and ERK kinase (MEK-1) is a dual-specificity kinase that phosphorylates ERK1/2 on T-E-Y. MEK-1 is itself regulated by a MAP kinase kinase kinase, one of which has been identified as Raf-1. Raf-1 is activated by translocation to the membrane and association with the small GTP-binding protein, ras. The GTPase activity of ras is regulated by a complex involving Grb2 and mSOS which are recruited and activated by a tyrosine kinase receptor.65
We have recently reported that ERK1/2 is activated by shear stress in endothelial cells in a time- and force-dependent manner.66 Shear stress stimulation of ERK1/2 was unaffected by treatment with the Ca2+ chelator BAPTA-AM, indicating the response was Ca2+ independent. These data, combined with observations that ecNOS contains multiple consensus sites for phosphorylation by a variety of kinases including ERK1/2,63 make this pathway a likely candidate to participate in the stimulation of sustained NO production in response to shear stress. Additionally, several shear stress–responsive genes contain elements (eg, AP-1)39 67 that may be influenced by ERK1/2-mediated phosphorylation of transcription factors,63 such as c-fos, c-jun, and c-myc.
Another member of the MAP kinase family shown to be regulated by shear stress is the stress-activated protein kinase JNK/SAPK. Two laboratories have shown increases in JNK activity by shear stress, although with varying kinetics.68 69 Preliminary results in our laboratory show that shear stress inhibits tumor necrosis factor–stimulated JNK activity in endothelial cells,69A a finding consistent with the recently reported ability of shear stress to inhibit endothelial cell apoptosis.53 54 Additional experiments by our laboratory indicate that other members of the MAP kinase family, p38 and BMK-1 (ERK5), are also activated by shear stress in endothelial cells.69B Experiments to determine the roles of individual MAP kinase family members in endothelial gene expression will be important areas for future research.
Upstream Effectors of ERK1/2 Activity
PKC is required for ERK1/2 activation in response to shear stress because the ERK1/2 activation by shear stress reported by our group was attenuated when endothelial cells were pretreated with either phorbol ester for 24 hours or with staurosporine for 30 minutes66 (Fig 3⇓). PKCs are well characterized serine/threonine kinases that are activated by a variety of stimuli.70 A classification system for the PKC family separates the different isoforms into distinct classes: the “classical” PKC isoforms, (PKC-α, -βI, -βII, -γ) are Ca2+ independent and phorbol ester responsive; the “novel” PKC isoforms (PKC-δ, -ε, -θ, -η) are Ca2+ independent and phorbol ester responsive; and the “atypical” PKC isoforms (including ζ, λ/ι), are Ca2+ independent and phorbol ester unresponsive. Human umbilical vein and bovine aortic endothelial cells express primarily three PKC isoforms: PKC-α, PKC-ε, and PKC-ζ.71 Through experiments using antisense oligonucleotides to inhibit expression of the various PKC isoforms in endothelial cells, we identified PKC-ε as the isoform necessary for the ERK1/2 stimulation by shear stress.71
A recent study by Cai et al72 provides evidence for the mechanism by which PKC activates ERK1/2. These investigators demonstrated that activation of the MAP kinase kinase kinase, Raf-1, a key activator of ERK1/2, is dependent on several criteria: (1) recruitment of Raf-1 to the plasma membrane; (2) activation of Raf-1 by the GTP-bound form of ras; (3) tyrosine phosphorylation of Raf-1, presumably by c-Src or a c-Src-family kinase; and (4) phosphorylation of Raf-1 on serine and threonine residues. Both PKC-α and PKC-ε were directly responsible for phosphorylation of serine and threonine residues on Raf-1 in response to various stimuli. This redundancy in PKC signaling to Raf-1 would allow for signaling regardless of a rise in intracellular Ca2+, as PKC-ε is Ca2+ independent. Thus, it seems likely that activation of PKC isoforms, particularly PKC-ε, by shear stress results in stimulation of ERK1/2 through their action on Raf-1, regardless of intracellular Ca2+ concentration.
Potential Shear Stress Receptors
A question of great importance in the field of mechanotransduction pertains to the identity of the primary mechanoreceptor(s) responsible for initiating signal transduction. Transduction of mechanical forces in anchorage-dependent cells is due to a combination of force transmission via the cytoskeletal elements and transduction of the physical forces to biochemical signals at mechanotransducer sites.63 Based on the data presented above, the candidate mechanotransducer molecules should be responsive to shear stress over the physiological range and result in the activation of a tyrosine kinase (eg, c-Src), PKC, and ERK1/2. Due to their interaction with specific signaling molecules already implicated in signal transduction, four candidates have been proposed as likely mechanotransducers: integrin-matrix interactions, specialized membrane microdomains, ion channels, and G proteins.
To sense and transduce signals in response to shear stress, endothelial cells must be anchored to their matrix.73 Integrins are ubiquitous α/β heterodimeric transmembrane glycoproteins that act as adhesion receptors involved in the interaction between cells and extracellular matrix. Integrins play an important role in biological processes, including cell adhesion, cell migration, cell growth, tissue organization, blood clotting, inflammation, target recognition by leukocytes, and cell differentiation.74 Studies performed by Wang et al75 and Ingber76 using magnetic torsion have demonstrated that integrins are capable of transducing mechanical stimuli to biochemical signals. A recent study by Muller et al77 showed that flow-induced vasodilation in coronary arteries, which is mediated by NO release, could be blocked with RGD peptides, which compete with the matrix for integrin interactions. Similar attenuation of flow-induced vasodilation was obtained if a blocking antibody against the β3 integrin was employed, supporting the hypothesis that integrins are involved in the mechanotransduction of shear stress. Integrins are also a particularly attractive candidate in that they have been reported to associate with PKC71 and c-Src–family tyrosine kinases.78 Through the use of antisense oligonucleotides against the various PKC isoforms, our laboratory has shown that adhesion-mediated activation of ERK1/2 in human umbilical vein endothelial cells is dependent on PKC-α and PKC-ε,71 a finding that parallels the data from Cai’s group. Other studies by our laboratory have demonstrated that activation of β1 integrins (the predominant β isoform on endothelial cells) with an activating antibody also stimulated ERK1/2, although at levels less than that observed with shear stress.79 Further, human endothelial cells showed adhesion-mediated ERK1/2 activation when plated on a matrix of fibronectin, which engages β1 integrins, but showed no ERK1/2 activation when they adhered to matrix consisting of poly-l-lysine.73 The relatively small magnitude of ERK1/2 stimulation by integrin activation does not preclude a key role for integrins in shear-mediated ERK1/2 activation; based on the importance of shear stress to endothelial cell function and integrity, it is likely that redundant pathways with different mechanotransducer molecules mediate the full ERK/12 response to shear stress.
Another possible candidate for the transduction of shear stress into biochemical signals are caveolae, specialized domains of the plasma membrane that are rich in cholesterol. Because of their high cholesterol content, caveolae are more rigid than other portions of the plasma membrane. Caveolae are abundant in endothelial cells and have been implicated in transcytosis, ion movement across the membrane, and signal transduction.80 The principal component of caveolae is a 21- to 24-kD integral membrane protein called caveolin. Caveolin seems to function as a scaffold for the recruitment and sequestration of signaling molecules. Among signaling molecules known to associate with caveolae are G proteins, c-Src–family tyrosine kinases, ras, PKC, ecNOS,81 shc, Grb2, mSOS, Raf-1, and ERK1/2 (see Reference 8282 ) Caveolae represent an attractive site for mechanotransduction on the basis of their biophysical characteristics and interactions with signaling molecules. Experiments to determine the significance of caveolae and what effect changes in caveolae number and properties may have in shear-mediated signaling should prove an exciting area for future research.
Recent data reported by Gudi et al83 indicate that G proteins may act as primary mechanosensors in endothelial cells. This laboratory showed that treatment of endothelial cells with antisense Gαq oligonucleotides inhibited shear stress–induced ras-GTPase activity, while scrambled oligonucleotide treatment had no effect. Another study reported that treatment of endothelial cells with pertussis toxin prevented shear stress–mediated activation of ERK1/2,69 also suggesting that G proteins are activated in response to shear stress. Further, Gudi et al demonstrated that G proteins reconstituted in liposomes, in the absence of protein receptors, showed an increase in activity in response to shear stress.84 This shear stress–mediated increase in G protein activity could be attenuated if the lipid bilayer was made more rigid by the addition of cholesterol, a significant finding in the context of caveolae as shear stress signaling domains.
A common mechanism that has evolved to sense changes in mechanical stimuli are the mechanosensitive ion channels. These channels are widely distributed in tissues and participate in processes such as hearing, balance, and reflex contraction of both smooth and skeletal muscle. Endothelial cells exhibit ion channel responses to mechanical forces that are likely to participate in the signaling response to shear stress. Several different mechanosensitive ion channels are present in endothelial cells, including a shear-responsive K+ channel and a stretch-activated Ca2+ channel.4 Studies have shown that blockade of mechanosensitive K+ channels with barium chloride or tetraethylammonium blocked shear-mediated increases in NO production85 and transforming growth factor-β release,43 suggesting that transmembrane ion flux and intracellular ion homeostasis are important mediators of the endothelial cell response to shear stress. However, efforts to clone the mechanosensitive K+ channel from the endothelial cell have not yet been successful.
Based on the demonstrated importance of shear stress to endothelial cell function and integrity, it is likely that each of these putative mechanoreceptors activates intracellular signaling pathways to effect the complete endothelial response to shear stress. Differential coupling of signaling mechanisms and subsequent endothelial cell response to the individual shear stress receptor “subtypes” may provide a flexibility to the endothelial cells in terms of responding to varying types and degrees of shear stress that they may encounter.
We have reviewed data showing that shear stress has direct influences on the pathogenesis of atherosclerosis via regulation of endothelial cell function and integrity. Shear stress influences many of the processes relevant to development of the atherosclerotic lesion, including secretion of growth factors, regulation of coagulation, and transmigration of leukocytes. Regulation of these processes (Fig 3⇑) is proposed to occur via shear-activated endothelial cell signal-transduction pathways that involve primary mechanotransducers, resulting in the activation of ERK1/2, and possibly ecNOS, through signaling molecules such as nonreceptor tyrosine kinases, ras, and PKC.
While hemodynamic considerations are important in atherogenesis, it is unlikely that fluid mechanical forces are the sole positive or negative atherogenic stimuli. The potential influence of local and systemic biochemical factors and their interplay with mechanical factors must also be considered.86 87 Apart from the direct effects of shear stress on endothelial cell function, flow reversal results in alterations in mass transport, increasing the probability of leukocyte localization and altering delivery of biochemical factors such as inflammatory mediators that may contribute to the local atherogenic state. The conclusion that the hemodynamic force of fluid shear stress plays an important role in the pathogenesis of atherosclerosis, however, does provide a framework by which independent risk factors for atherosclerosis may be understood. Studies suggest that hypertension,88 diabetes,89 and hypercholesterolemia90 promote atherosclerosis by disrupting the ability of the endothelium to respond to shear stress, while regular aerobic exercise exerts atheroprotective effects through shear-mediated increases in NO.57 Further elucidation of the mechanisms of shear stress–mediated signal transduction and its alteration with these risk factors will greatly advance our understanding of atherosclerosis.
Selected Abbreviations and Acronyms
|ecNOS||=||endothelial constitutive NO synthase|
|ICAM-1||=||intracellular adhesion molecule-1|
|MCP-1||=||monocyte chemoattractant protein-1|
|PKC||=||protein kinase C|
|VCAM-1||=||vascular cell adhesion molecule-1|
This work was supported by a Medical Scientist Training Program grant (NIGM-GM07266) and Poncin Fellowship to O.T., an AHA National Grant-in-Aid (94014290) to B.C.B., and NIH PO1 Hl18645. B.C.B. is an Established Investigator of the American Heart Association
Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519–560.
Helmlinger G, Berk BC, Nerem RM. The calcium responses of endothelial cell monolayers subjected to pulsatile and steady laminar flow differ. Am J Physiol. 1995;269:C367–C375.
Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science. 1985;227:1477–1479.
Flaherty JT, Pierce JE, Ferrans VJ, Patel DJ, Tucker WK, Fry DL. Endothelial nuclear patterns in the canine arterial tree with particular reference to hemodynamic events. Circ Res. 1972;30:23–33.
Barbee KA, Mundel T, Lal R, Davies PF. Subcellular distribution of shear stress at the surface of flow-aligned and nonaligned endothelial monolayers. Am J Physiol. 1995;268:H1765–H1772.
Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis. 1985;5:293–302.
Davies PF, Remuzzi A, Gordon EJ, Dewey CF Jr, Gimbrone MA Jr. Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro. Proc Natl Acad Sci U S A. 1986;83:2114–2117.
DePaola N, Gimbrone MA Jr, Davies PF, Dewey CF Jr. Vascular endothelium responds to fluid shear stress gradients. Arterioscler Thromb. 1992;12:1254–1257.
Barbee KA, Davies PF, Lal R. Shear stress–induced reorganization of the surface topography of living endothelial cells imaged by atomic force microscopy. Circ Res. 1994;74:163–171.
Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990;66:1045–1066.
Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996;93:10417–10422.
Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol. 1986;19:H1145–H1149.
Busse R, Pohl U, Luckhoff A. Mechanisms controlling the production of endothelial autacoids. Z Kardiol. 1989;6:64–69.
Stamler J, Mendelsohn ME, Amarante P, Smick D, Andon N, Davies PF, Cooke JP, Loscalzo J. N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor. Circ Res. 1989;65:789–795.
Malek AM, Jackman R, Rosenberg RD, Izumo S. Endothelial expression of thrombomodulin is reversibly regulated by fluid shear stress. Circ Res. 1994;74:852–860.
Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science. 1991;251:788–791.
Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL. Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. Arterioscler Thromb Vasc Biol. 1995;15:2–10. Erratum. Arterioscler Thromb Vasc Biol. 1995;15:429.
Ando J, Tsuboi H, Korenaga R, Takada Y, Toyama S-N, Miyasaka M, Kamiya A. Shear stress inhibits adhesion of cultured mouse endothelial cells to lymphocytes by downregulating VCAM-1 expression. Am J Physiol. 1994;267:C679–C687.
Tsao PS, Lewis NP, Alpert S, Cooke JP. Exposure to shear stress alters endothelial adhesiveness: role of nitric oxide. Circulation. 1995;92:3513–3519.
Morigi M, Zoja C, Figliuzzi M, Foppolo M, Micheletti G, Bontempelli M, Saronni M, Remuzzi G, Remuzzi A. Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells. Blood. 1995;85:1696–1703.
Nagel T, Resnick N, Atkinson WJ, Dewey CF Jr, Gimbrone MA Jr. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest. 1994;94:885–891.
Lawrence MB, Smith CW, Eskin SG, McIntire LV. Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium. Blood. 1990;75:227–237.
Schmid-Schoenbein GW, Fung YC, Zweifach BW. Vascular endothelium-leukocyte interaction: sticking shear force in venules. Circ Res. 1975;36:173–184.
Shyy JY, Lin MC, Han J, Lu Y, Petrime M, Chien S. The cis-acting phorbol ester “12-O-tetradecanoylphorbol 13-acetate”–responsive element is involved in shear stress–induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci U S A. 1995;92:8069–8073.
Kraiss LW, Raines EW, Wilcox JN, Seifert RA, Barrett TB, Kirkman TR, Hart CE, Bowen PDF, Ross R, Clowes AW. Regional expression of the platelet-derived growth factor and its receptors in a primate graft model of vessel wall assembly. J Clin Invest. 1993;92:338–348.
Buga GM, Gold ME, Fukuto JM, Ignarro LJ. Shear stress–induced release of nitric oxide from endothelial cells grown on beads. Hypertension. 1991;17:187–193.
Ohno M, Cooke JP, Dzau VJ, Gibbons GH. Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production: modulation by potassium channel blockade. J Clin Invest. 1995;95:1363–1369.
Berk BC, Duff JL, Marrero MB, Bernstein KE. Angiotensin II signal transduction in vascular smooth muscle. In: Sowers JR, ed. Endocrinology of the Vasculature. Totowa, New Jersey: Humana Press; 1996:187–204.
Rieder MJ, Carmona R, Krieger JE, Pritchard KA Jr, Greene AS. Suppression of angiotensin-converting enzyme expression and activity by shear stress. Circ Res. 1997;80:312–319.
Berceli SA, Warty VS, Sheppeck RA, Mandarino WA, Tanksale SK, Borovetz HS. Hemodynamics and low density lipoprotein metabolism: rates of low density lipoprotein incorporation and degradation along medial and lateral walls of the rabbit aortoiliac bifurcation. Arteriosclerosis. 1990;10:686–694.
Schwenke DC, Carew TE. Quantification in vivo of increased LDL content and rate of LDL degradation in normal rabbit aorta occurring at sites susceptible to early atherosclerotic lesions. Circ Res. 1988;62:699–710.
Fry DL, Herderick EE, Johnson DK. Local intimal-medial uptakes of 125I-albumin, 125I-LDL, and parenteral Evans blue dye protein complex along the aortas of normocholesterolemic minipigs as predictors of subsequent hypercholesterolemic atherogenesis. Arterioscler Thromb. 1993;13:1193–1204.
Sprague EA, Steinbach BL, Nerem RM, Schwartz CJ. Influence of a laminar steady-state fluid-imposed wall shear stress on the binding, internalization, and degradation of low-density lipoproteins by cultured arterial endothelium. Circulation. 1987;76:648–656.
Wiklund O, Carew TE, Steinberg D. Role of the low density lipoprotein receptor in penetration of low density lipoprotein into rabbit aortic wall. Arteriosclerosis. 1985;5:135–141.
Langille BL, Graham JJ, Kim D, Gotlieb AI. Dynamics of shear-induced redistribution of F-actin in endothelial cells in vivo. Arterioscler Thromb. 1991;11:1814–1820.
Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, Murphy TJ. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest. 1992;90:2092–2096.
Janssens SP, Shimouchi A, Quertermous T, Bloch DB, Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem. 1992;267:14519–14522. Erratum. J Biol Chem. 1992;267:22694.
Sessa WC, Harrison JK, Barber CM, Zeng D, Durieux ME, D’Angelo DD, Lynch KR, Peach MJ. Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase. J Biol Chem. 1992;267:15274–15276.
Kuchan MJ, Frangos JA. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am J Physiol. 1994;266:C628–C636.
Corson MA, James NL, Latta SE, Nerem RM, Berk BC, Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res. 1996;79:984–991.
Pelech SL, Sanghera JS. MAP kinases: charting the regulatory pathways. Science. 1992;257:1355–1356.
Tseng H, Peterson T, Berk BC. Fluid shear stress stimulates mitogen-activated protein kinases in bovine aortic endothelial cells. Circ Res. 1995;77:869–878.
Uematsu M, Navas JP, Nishida K, Ohara Y, Murphy TJ, Alexander RW, Nerem RM, Harrison DG. Mechanisms of endothelial cell NO synthase induction by shear stress. Circulation. 1993;88(suppl I):I-184. Abstract.
Yi-Shuan L, John Y-J, Shyy S. The cytoplasmic kinase pathways are involved in the shear stress–induced gene expression. Circulation. 1995;92(suppl I):I-1. Abstract.
Jo H, Sipos K, Go Y-M, Law R, Rong J, McDonald JM. Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. J Biol Chem. 1997;272:1395–1401.
Surapisitchat J, Berk BC. TNF-alpha activation of JNK but not ERK 1/2 is inhibited in human umbilical cord endothelial cells by fluid shear stress. FASEB J. In press.
Yan C, Takahashi M, Lu JD, Berk BC. Flow activates big MAP kinase in endothelial cells: role of reactive oxygen species, calcium, and tyrosine kinases. FASEB J. In press.
Traub O, Monia BP, Dean NM, Berk BC. PKC-ε is required for mechano-sensitive activation of ERK1/2 in endothelial cells. J Biol Chem. 1997; 272:31251–31257.
Cai H, Smola U, Wixler V, Eisenmann T-I, Diaz M-MT, Moscat J, Rapp U, Cooper GM. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase. Mol Cell Biol. 1997;17:732–741.
Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science. 1993;260:1124–1127.
Muller JM, Chilian WM, Davis MJ. Integrin signaling transduces shear stress–dependent vasodilation of coronary arterioles. Circ Res. 1996;80:320–326.
Ishida T, Peterson T, Kovach N, Berk B. Integrins modulate fluid shear stress signal transduction in endothelial cells. Circulation. 1995;92(suppl I):I-629. Abstract.
Schnitzer JE, Liu J, Oh P. Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J Biol Chem. 1995;270:14399–14404.
Garcia-Cardena G, Oh P, Liu J, Schnitzer JE, Sessa WC. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci U S A. 1996;93:6448–6453.
Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem. 1997;272:6525–6533.
Gudi SRP, Huver IV, Taliana AP, Boss GR, Frangos JA. Fluid flow-induced ras activation is mediated by Gaq in human vascular endothelial cells. FASEB J. 1997;11:A223. Abstract.
Frangos JA, Gudi SRP. Shear stress activates reconstituted G proteins in the absence of protein receptors by changes in membrane fluidity. FASEB J. 1997;11:A521. Abstract.
Uematsu M, Ohara Y, Navas JP, Nishida K, Murphy TJ, Alexander RW, Nerem RM, Harrison DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol. 1995;269:C1371–C1378.
Koller A, Huang A. Shear stress–induced dilation is attenuated in skeletal muscle arterioles of hypertensive rats. Hypertension. 1995;25:758–763.
Corson MA, MacKellar SL. Fibronectin advanced glycation end products (FN-AGE) impair flow-dependent endothelial cell (EC) mechanotransduction. Circulation . 1996;94(suppl I):I-703. Abstract.
Smart EJ, Ying YS, Conrad PA, Anderson RG. Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J Cell Biol. 1994;127:1185–1197.
- Shear Stress and Endothelial Cell Biology: Relevance to Atherosclerosis
- NO: A Critical Factor in Shear Stress–Mediated Atheroprotection
- Mitogen-Activated Protein Kinases: Likely Signaling Molecules in the Transduction of Shear Stress
- Upstream Effectors of ERK1/2 Activity
- Potential Shear Stress Receptors
- Selected Abbreviations and Acronyms
- Figures & Tables
- Info & Metrics