doi: 10.1161/CIRCRESAHA.108.180836
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© 2008 American Heart Association, Inc.
Editorials |
Choosing Sides in Polarized Endothelial Adaptation to Shear Stress
From the Department of Biomedical Engineering and Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville.
Correspondence to Brian P. Helmke, PhD, Department of Biomedical Engineering, University of Virginia, P.O. Box 800759, Charlottesville, VA 22908. E-mail helmke{at}virginia.edu
See related article, pages 177–185
Key Words: mechanotransduction • planar cell polarity • integrin • Rac • protein kinase A (PKA)
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Introduction |
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 Top Introduction Spatial Organization During...An Integrin Whose Function...Harnessing Mechano-Polarization...References |
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The endothelium is a primary integrator of biophysical and chemical cues that guide vascular wall physiology and pathology. Normally, arterial endothelial cells appear elongated longitudinally and rest on a basement membrane of collagen type IV and laminin. In atherosclerosis, lesions form primarily near arterial bifurcations and along the inner curvature of the aorta where complex spatiotemporal profiles of hemodynamic forces exist and where endothelial cells exhibit a nonpolarized structure and upregulate expression of a provisional matrix enriched in fibronectin and fibrinogen. The regional heterogeneity in endothelial phenotype and matrix expression suggests that lesion progression requires transduction of mechanical cues associated with hemodynamic wall shear stress and artery wall stretch into biochemical signals for inflammation. Integrins have been proposed as candidate mechanotransducers capable of differentiating both physical cues and matrix composition, but an integrin-mediated mechanism that confers directionality in response to shear stress has remained elusive. In this issue of Circulation Research, Goldfinger et al1 report that shear stress activates protein kinase A (PKA) to phosphorylate
4 integrin locally at the downstream edge of endothelial cells, and phosphorylated 4 releases inhibition of the GTPase Rac1 to direct polarized reorganization of the cytoskeleton. The proposed mechanism is important not only because it improves understanding of intracellular spatial organization in mechanotransduction mechanisms but also because it suggests new avenues for engineering a healthy endothelium after bypass grafting or vascular stent procedures. |
Spatial Organization During Endothelial Mechanotransduction |
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TopIntroductionSpatial Organization During...An Integrin Whose Function...Harnessing Mechano-Polarization...References |
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Endothelial cells associated with an atheroprotective phenotype exhibit planar polarity characteristics that include elongated shape, actin stress fibers oriented parallel to the shear stress direction, and microtubule organizing centers (MTOCs) located downstream of the nucleus. Goldfinger et al propose that phosphorylated
4 integrin is localized preferentially near the downstream edge of the cell and serves as an early polarizing signal that is required for these adaptations to occur. What transmits the direction of shear stress to locations in the cell that drive these processes? One possibility involves the apical plasma membrane itself. The lateral mobility of lipids in the plasma membrane is increased in regions downstream of the nucleus after onset of shear stress,2 perhaps enabling increased activation of G protein–coupled receptors.3 It is tempting to propose that this mechanism would also enhance transport rates of 4 integrins to enable spatial concentration near the downstream edge, but this hypothesis would require the unlikely assumption that 4 mobility is independent of interactions with the cytoskeleton. A second possibility for transmitting directional cues involves intracellular "decentralization" of force by transmission through the cytoskeleton from the apical surface to locations where signaling is initiated.4,5 This idea is supported by measurements of strain focusing in the cytoskeleton near adhesions and junctions6 and by intracellular stress tomography after onset of shear stress.7 For example, shear stress onset induces coordinated displacement of stress fiber termini, adhesion sites, and extracellular matrix fibrils in the downstream direction,8 reflecting a coordinated redistribution of intracellular tension. It is likely that redistribution of cytoskeletal tension in response to shear stress contributes to spatially polarized phosphorylation of ligated 4 integrins, as has been demonstrated for other integrins in nascent focal adhesions.
Following integrin activation in this manner, spatial polarization of downstream signaling is required for endothelial cell adaptation to unidirectional shear stress. Shear stress onset induces conformational activation and new ligation of Vβ3 integrins near the cell periphery, leading to transient downregulation of the GTPase RhoA, and adaptive alignment of endothelial cell shape and stress fibers does not occur if any of these events is inhibited.9 Activation of Rac1 locally near the downstream edges of endothelial cells is also required for shear stress–induced alignment.10 Polarized Rac activity promotes actin polymerization associated with leading edge lamellipodia, and endothelial cells in subconfluent layers or at wound edges migrate parallel to shear stress in a process termed mechanotaxis.11 However, a plausible link that translates shear stress–induced integrin activation into spatially polarized signaling has not been proposed until now.
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An Integrin Whose Function Is Not Adhesion Strengthening? |
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TopIntroductionSpatial Organization During...An Integrin Whose Function...Harnessing Mechano-Polarization...References |
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Most work in integrin mechanosignaling has focused on explaining adhesion strengthening and cytoskeletal reinforcement or stiffening under an external applied stress.12–14 In these models,
5β1 or Vβ3 integrins interact with "synergy" and "cell-binding" domains in type III repeats 9 and 10, respectively, of matrix fibronectin. Although adhesion strengthening occurs locally where forces are applied with micrometer scale probes, evidence for spatial polarity in response to a force gradient at the cell length scale (as might be the case for shear stress) is lacking. The CS-1 domain of fibronectin is a variably spliced segment containing the LDV (leucine-aspartate-valine) consensus sequence of amino acids that serves as a ligand for 4β1 and 4β7 integrins. Goldfinger et al adhered endothelial cells on CS-1 fragment to limit ligated integrin to 4 only. This strategy revealed a role for 4 in sensing shear stress direction that may be distinct from the functions of 5 and V in modulating mechanotransmission and cytoskeletal reinforcement.
How does 4 transmit the direction of shear stress? Previous work on cell migration suggests a mechanism.15 4 is phosphorylated on Serine-988 by PKA, preventing binding of paxillin. Along the sides and trailing edges of migrating cells where 4 is not phosphorylated, paxillin binds and recruits a GTPase-activating protein (GAP) for ADP-ribosylation factor (Arf). The Arf-GAP, known as GIT1, decreases Arf activity, causing local inhibition of Rac1 activity. The resulting spatial polarization of activated Rac leads to stabilization of a directional lamellipodium. Goldfinger et al now suggest a similar role for PKA-mediated 4 phosphorylation in shear stress–induced directional Rac activation, lamellipodium stabilization, and cell migration. PKA was responsible for phosphorylating 4, because PKA inhibitors blocked 4 phosphorylation at the leading edge, Rac1 activation near the leading edge, and adaptive elongation and alignment of the cells. Thus, a primary role for 4 integrin in establishing planar polarity in response to unidirectional shear stress has been established.
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Harnessing Mechano-Polarization Mechanisms |
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TopIntroductionSpatial Organization During...An Integrin Whose Function...Harnessing Mechano-Polarization...References |
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Several major questions remain to be answered to clear the path for engineering endothelial wound healing after bypass grafting or stent placement. For example, why is
4 phosphorylated by PKA only at the leading edge? In neutrophils, exposure to a spatial gradient of PKA inhibitor is sufficient to stimulate directional migration,16 but it remains unknown whether PKA activation in endothelial cells is spatially localized near the leading edge after shear stress onset. On an in vivo matrix, local activation of PKA may depend on crosstalk with other newly ligated integrins. Shear stress induces activation of PKA and suppression of Vβ3 conformational activation in endothelial cells plated on collagen, whereas PKC is activated and 2β1 is suppressed in cells plated on fibronectin.17 Thus, elucidating the relative roles of interacting integrin and matrix signals remains a hurdle to solving directional mechanosensing.
Alternate 4 phosphorylation sites may also play an important role in directional sensing. For example, overexpression in Chinese hamster ovary (CHO) cells of 4 with Tyrosine-991 mutated to alanine prevents paxillin binding to 4 and promotes leading edge spreading in response to shear stress,18 suggesting that shear stress–induced tyrosine phosphorylation of 4 may counteract directional sensing independently of paxillin binding. Interestingly, wild-type 4 expressed in CHO cells was phosphorylated on Ser-988 both at the leading and trailing edges of cells migrating in response to shear stress, and mutation of Ser-988 inhibited both leading edge extension and trailing edge retraction. Thus, Ser-988 phosphorylation may serve a dual role to enhance directional sensing in some cases.
Spatial polarization of 4-paxillin-GIT1 is not the only mechanism proposed to regulate spatial activation of Rac. Rac is activated in waves propagating from newly formed adhesions in cells on micropatterned fibronectin substrates,19 suggesting that an alternative mechanism for establishing Rac polarity exists that depends on new ligation of Vβ3 or 5β1. However, a role for 4 ligation cannot be ruled out because micropatterns were generated with full-length fibronectin, so a "leading edge" would be determined by the geometry of the micropatterns.
Biomedical engineers seek the ability to harness mechanotransduction mechanisms to design substrates that enhance endothelial wound healing for development of artificial vascular grafts. On substrates coated with CS-1 fragment and exposed to arterial levels of shear stress, retention of some endothelial cell types but not others was improved, probably because of variability in expression levels of 4.20 Even when 4 is exogenously overexpressed, the correlation between 4-paxillin–mediated signaling and adhesion strength is weak.18 However, the ability to control directional migration to enhance wound healing or reendothelialization may represent the real opportunity for improving therapies in patients with advanced atherosclerosis. The mechanism elucidated by Goldfinger et al represents a major step in the right direction.
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Acknowledgments |
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Sources of Funding
The author is supported by NIH grants HL-071958 and HL-080956.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
4 Integrin Phosphorylation Directs Shear Stress–Induced Endothelial Cell Alignment
Circ. Res. 2008 103: 177-185.
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