Fluid Shear Stress Activation of Focal Adhesion Kinase

Abstract

Shear stress, the tangential component of hemodynamic forces, activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signal transduction pathways in cultured vascular endothelial cells to induce the transcriptional activation of many immediate early genes. It appears that integrins, protein-tyrosine kinases, and the structural integrity of actin are important factors involved in these shear stress-induced responses. The underlying molecular events were investigated by the application of a shear stress of 12 dyn/cm2 on bovine aortic endothelial cells (BAEC). We found that such a shear stress increased the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK) and its association with growth factor receptor binding protein 2 (Grb2) in a rapid and transient manner, suggesting that FAK may be linked to these mitogen-activated protein kinase signaling pathways through a Grb2·Son of sevenless (Sos) complex. FAK(F397Y), which encodes a dominant negative mutant of FAK, attenuated the shear stress-induced kinase activity of Myc epitope-tagged ERK2 and hemagglutinin epitope-tagged JNK1. ΔmSos1, encoding a dominant negative mutant of Sos in which the guanine nucleotide exchange domain has been deleted, also attenuated shear stress activation of Myc-ERK2 and hemagglutinin-JNK1. Pretreating the confluent BAEC monolayers with a blocking type anti-vitronectin receptor monoclonal antibody had similar inhibitory effects in these shear stress-activated ERKs and JNKs. Confocal microscopic observation further demonstrated that FAK tended to cluster with vitronectin receptor near the abluminal side of the sheared BAEC. These results demonstrate that FAK signaling is critical in the shear stress-induced dual activation of ERK and JNK. Shear stress, the tangential component of hemodynamic forces, activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signal transduction pathways in cultured vascular endothelial cells to induce the transcriptional activation of many immediate early genes. It appears that integrins, protein-tyrosine kinases, and the structural integrity of actin are important factors involved in these shear stress-induced responses. The underlying molecular events were investigated by the application of a shear stress of 12 dyn/cm2 on bovine aortic endothelial cells (BAEC). We found that such a shear stress increased the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK) and its association with growth factor receptor binding protein 2 (Grb2) in a rapid and transient manner, suggesting that FAK may be linked to these mitogen-activated protein kinase signaling pathways through a Grb2·Son of sevenless (Sos) complex. FAK(F397Y), which encodes a dominant negative mutant of FAK, attenuated the shear stress-induced kinase activity of Myc epitope-tagged ERK2 and hemagglutinin epitope-tagged JNK1. ΔmSos1, encoding a dominant negative mutant of Sos in which the guanine nucleotide exchange domain has been deleted, also attenuated shear stress activation of Myc-ERK2 and hemagglutinin-JNK1. Pretreating the confluent BAEC monolayers with a blocking type anti-vitronectin receptor monoclonal antibody had similar inhibitory effects in these shear stress-activated ERKs and JNKs. Confocal microscopic observation further demonstrated that FAK tended to cluster with vitronectin receptor near the abluminal side of the sheared BAEC. These results demonstrate that FAK signaling is critical in the shear stress-induced dual activation of ERK and JNK. Hemodynamic forces play important roles in maintaining cardiovascular homeostasis, but they can also be pathophysiological factors in conditions such as atherosclerosis. The involvement of hemodynamic forces in atherogenesis is manifested by the focal distribution of atherosclerotic lesions in the bifurcations and curved regions of the arterial tree where blood flow is disturbed with flow separation. Fluid shear stress, the tangential component of the hemodynamic forces, is low and unsteady in these lesion-prone areas (1Glagov S. Zarins C. Giddens D.P. Ku D.N. Arch. Pathol. Lab. Med. 1988; 112: 1018-1031PubMed Google Scholar,2Nerem R.M. Harrison D.G. Taylor W.R. Alexander R.W. J. Cardiovasc. Pharmacol. 1993; 21: S6-S10Crossref PubMed Scopus (101) Google Scholar). Vascular endothelial cells (ECs), 1The abbreviations used are: EC, vascular endothelial cell; BAEC, bovine aortic endothelial cell(s); ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase(s); FITC, fluorescein isothiocyanate; Grb2, growth factor receptor-2; GST, glutathione S-transferase; JNK, c-Jun NH2-terminal kinase; MAPKs, mitogen-activated protein kinases; MBP, myelin basic protein; PTKs, protein-tyrosine kinases; SH2 and SH3 domain, Src homology-2 and -3 domain, respectively; Sos, Son of sevenless; VNR, vitronectin receptor(s); mAb, monoclonal antibody; HA, hemagglutinin. serving as a barrier between the vessel and blood, are exposed to shear stress under physiological and pathophysiological conditions. To unravel the roles played by hemodynamic forces in atherogenesis, flow channels have been used as in vitro systems to study functional changes of ECs in response to shear stress (for a review, see Ref. 3Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2389) Google Scholar). These studies indicate that fluid shear stress induces a rapid induction of immediate early genes. c-Src and Ras are important mediators of shear stress activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs), also known as stress-activated protein kinases, to activate these immediate early genes (4Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (270) Google Scholar, 5Li Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar, 6Takahashi M. Berk B.C. J. Clin. Invest. 1997; 98: 2623-2631Crossref Scopus (191) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 8Jalali S. Usami S. Shyy J.Y-J. Chien S. FASEB J. 1997; 11: A18Google Scholar). Although many mechanisms have been proposed to explain the EC response to shear stress, including those via G-proteins, cytoskeletal structure, membrane-associated K+ channels, and integrins (for a review, see Ref. 9Berk B.C. Corson M.A. Peterson T.E. Tseng H. J. Biomechanics. 1995; 28: 1439-1450Crossref PubMed Scopus (140) Google Scholar), there is little experimental evidence indicating where and how the mechanotransduction occurs to activate the downstream signaling events, e.g. the activation of the MAPKs. In response to shear stress, concomitant with the elongation of ECs and the alignment of stress fibers with the direction of flow, the focal adhesions on the abluminal side of ECs undergo dynamic, local reorientation without a noticeable change in the total attachment area (10Davies P.F. Robotewskyj A. Griem M.L. J. Clin. Invest. 1994; 93: 2031-2038Crossref PubMed Scopus (297) Google Scholar). At the molecular level, such a dynamic rearrangement of focal adhesions may be related to spatial and temporal responses of the associated proteins, e.g. focal adhesion kinase (FAK), paxillin, tensin, and Src family protein-tyrosine kinases (PTKs) (for a review, see Ref. 11Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2824) Google Scholar). Indeed, it was recently shown that shear stress causes a rapid and transient activation of c-Src in ECs (6Takahashi M. Berk B.C. J. Clin. Invest. 1997; 98: 2623-2631Crossref Scopus (191) Google Scholar). Both chemical stimulation, e.g. the treatment of monocytes with monocyte/macrophage colony-stimulating factor, and the adhesion of NIH3T3 fibroblasts on fibronectin promote the interaction of growth factor receptor-binding protein 2 (Grb2) with FAK (12Kharbanda S. Saleem A. Yuan Z. Emoto Y. Prasad K.V. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6132-6136Crossref PubMed Scopus (73) Google Scholar, 13Schlaepfer D.D. 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Biol. 1994; 14: PubMed Scopus Google Scholar). Grb2 has a that Src homology-2 domain by SH3 K. M. A. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Grb2 is to Son of sevenless a guanine nucleotide exchange factor, via its SH3 The activates Ras by the to the Nature. 1993; PubMed Scopus Google Scholar, P. van J. D. Science. 1993; PubMed Scopus Google Scholar). Grb2 also with receptor tyrosine kinases such as the growth factor receptor and monocyte/macrophage colony-stimulating factor through the interaction of its SH2 with the of these receptor tyrosine kinases Li B. R. A. D. J. Cell. Full Text PDF PubMed Scopus Google Scholar, T. M. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar, der Geer P. Hunter T. J. 1993; PubMed Scopus Google Scholar). The association of with receptor tyrosine kinases Sos to the of the where it can with membrane-associated Ras to exchange A. R. P. D. B. J. Nature. 1993; PubMed Scopus Google Scholar). the study demonstrate that shear stress induces the activation of FAK and the of in signaling leads to the activation of ERK and JNK. the integrins by with FAK, may be involved in the mechanotransduction that fluid shear stress were aortic endothelial cells were bovine with by with a BAEC were cultured in with 2 and of and were in a at were with to and the were used to BAEC in to the application of shear flow was used to fluid shear stress on cultured ECs as Science. PubMed Scopus Google Scholar). In a was with a confluent of BAEC. was between the and to a flow in in and in with and the cultured BAEC to fluid shear flow the was by a and a During the flow the was at and with with hemagglutinin and were D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (401) Google Scholar, B. M. T. Su B. T. Karin M. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, M. D. M. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). The were BAEC at the the cells were with and in to the BAEC was 12 and to fluid shear stress as The used in and were (14Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1463) Google Scholar), antibody and cells were the were and the were with and protein at The were and used kinase activity the in were on in the were to a The was with by with the antibody in and bovine The were by a a and the To ERK BAEC were in a kinase and 2 ERK2 was with and protein To kinase the were in the and in a kinase and 2 of myelin basic protein and of in of kinase with were to kinase at The were by and the were In the epitope-tagged Myc-ERK2 were with encoding the negative BAEC. shear stress the cells were in the kinase Myc-ERK2 was with and protein by Myc-ERK2 kinase activity the as The kinase of the and epitope-tagged were by the as those ERK, that glutathione protein was used as the FAK kinase activity cells were in a and The cell were with by kinase activity in a kinase 2 of and monolayers of BAEC were in at The cells were in a at a of and at The were in and with a fluorescein and a of FAK and was under a with a and a was at a of and a between and was at and a between and We and have shown that shear stress activates many transcriptional factors in including c-Jun and which to the induction of genes such as the J.Y-J. J. Y. M. Chien S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, M.A. T. J. Clin. Invest. 1995; PubMed Scopus Google Scholar, P.F. Res. 1994; PubMed Scopus Google Scholar). of transcriptional activation is at in to the activation of MAPKs, including ERK and (4Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (270) Google Scholar, 5Li Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar, 6Takahashi M. Berk B.C. J. Clin. Invest. 1997; 98: 2623-2631Crossref Scopus (191) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). To the functional roles of PTKs and actin involved in shear stress activation of ERK and JNK, confluent monolayers of BAEC cultured on were with by to fluid shear stress (for ERK2 (for these are the at which the are the kinases Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar). that shear stress increased the kinase of ERK and in cells that were with as and in and of BAEC with had little on the ERK2 activity but activity in the as with the the shear stress activation of ERK2 and JNK1. These results that the shear stress activation of the signaling as as the integrity of the actin To tyrosine phosphorylation in ECs in response to shear stress, confluent monolayers of BAEC were to a shear stress of 12 dyn/cm2 of and cell sheared BAEC were with with molecular of and were on in the sheared cells To the protein to FAK, FAK was these cell and by with shown in shear stress a rapid and transient tyrosine phosphorylation of FAK with a molecular of was which was 2 that the kinase activity of FAK was also increased by shear a the kinase activity of FAK increased by by FAK The shear stress activation of FAK was also actin of the cells with attenuated such The activation of ERK and in response to shear stress is by Ras Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), which is in many growth factor by the complex. shear stress the association of Grb2 with FAK in BAEC by the used in 2 with a shown in there was in the of Grb2 with FAK in ECs to shear stress The increased association of Grb2 with FAK at and to a similar to that in the In a cell with a and with the a of association in and sheared cells that Grb2 is associated with the results in 2 and that the is by shear stress, which may the signaling to activate the downstream ERK and pathways in We used dominant negative of FAK and Sos to the that the is critical shear stress activation of ERK and JNK. of FAK is the autophosphorylation which Src family D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (401) Google Scholar, 17Schaller M.D. Hildebrand J.D. Shannon J.D. Parsons J.T. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). encodes a encodes a in which has been by that the binding of Src family and Grb2 to FAK D.D. Hunter T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). We with the epitope-tagged Myc-ERK2 the epitope-tagged to the functional roles of the of FAK in shear stress activation of ERK and JNK. The cells were as to a treatment of shear stress (for Myc-ERK2 (for by kinase protein as the shown in the of in cells with the shear stress increased the kinase activity of as demonstrated by the In of the shear stress activation of with of that the Myc-ERK2 was by the of the shear stress also the phosphorylation of in cells with the with the phosphorylation by The phosphorylation of was to the activity that of the kinase that the FAK the by The of also that the was by the of the were by to the effects of functional blocking of on the shear stress activation of ERK and JNK. In ΔmSos1, the between and that to the guanine nucleotide exchange domain in the Sos has been the protein as a dominant negative mutant of Sos M. D. M. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). shown in and of phosphorylation of and in response to shear stress, indicating a of Myc-ERK2 and in these The results in and indicate that shear stress activates in which is a functional signaling of ERK and JNK. The adhesion of fibroblasts to the interaction of Grb2 with FAK (14Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1463) Google Scholar). of in results in in the Ras C. P. J. S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). response of cells is fibronectin and vitronectin K. J. Clin. Invest. 1995; PubMed Scopus (270) Google Scholar). These with the that actin is important the shear stress activation of ERK and to the roles of integrins in the shear stress activation of ERK and JNK. We in study to its in EC and its in the atherosclerotic M. Circ. Res. 1995; 77: PubMed Scopus Google Scholar). monolayers of BAEC were 2 with antibody that has been shown to endothelial P. M. M.A. J. Sci. 1993; PubMed Google and to in S. R. D. J. Clin. Invest. 1995; PubMed Scopus Google Scholar). a the was at focal adhesion cell and by with indicating that such of the antibody to the abluminal side of shear stress of 12 dyn/cm2 was to these cells (for the ERK2 activity (for the activity shown in BAEC with attenuated the shear stress activation of ERK2 and as by the phosphorylation of and These results that is involved in the mechanotransduction that the shear stress activation of ERK and We also investigated the distribution of FAK in EC, its spatial to VNR, and dynamic in response to shear BAEC were to a shear stress of 12 with a and was microscopic The focal of FAK and were found at the abluminal side of the cells and but at the side of the cells BAEC of FAK and at the of the abluminal side of the cells and with and with that there was in the of FAK and FAK were in these may be of the linked to the shear stress-activated FAK to the signaling Fluid shear stress is a physiological of forces, and it causes a dual activation of ERK and in may have on vascular ERK is involved in the of cell and may be in the stress responses and cell (for a review, see Ref. B. Karin M. 1996; PubMed Scopus Google Scholar). The inhibitory effects of that PTKs are in the signaling that leads to dual PTKs are also critical in the shear stress of endothelial cell and stress fibers S. J. Sci. 1996; PubMed Google Scholar). PTKs can be receptor tyrosine kinases and PTKs FAK and that have kinase but have extracellular through its association with FAK is in focal adhesion and is in response to cell adhesion as as the by a of growth factors growth and and (for a review, see Ref. J.T. Schaller M.D. Hildebrand J. Leu T.H. A. C. J. Sci. 1994; Google Scholar). Although FAK has been shown to be in ECs in response to shear stress T. Peterson T.E. Berk B.C. Circ. Res. 1996; PubMed Scopus Google Scholar), the functional in and evidence to that FAK can be of the activation of ERK and JNK, which is at in by The phosphorylation of is critical in these signaling the dominant negative mutant attenuated the shear stress activation of ERK and JNK. The of ERK but of by that the shear stress-activated ERK is but there are also pathways to the activation of JNK. signaling be involved in such the shear stress activation of study that the in the FAK in cells S. J. Science. 1996; PubMed Scopus Google Scholar). the of in BAEC and in endothelial PTKs that with FAK such as c-Src may also be involved in the shear stress activation of MAPKs. adhesion on activates c-Src and its association with FAK (for a review, see Ref. J.T. Biol. 1996; PubMed Scopus Google Scholar). of FAK is in such by serving as a binding site the SH2 domain of Src family PTKs D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (401) Google Scholar, 16Cobb B.S. Schaller M.D. Leu T.H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (496) Google Scholar, 17Schaller M.D. Hildebrand J.D. Shannon J.D. Parsons J.T. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). The phosphorylation of of FAK the of Grb2 to FAK D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (401) Google Scholar). and Berk have recently shown that shear stress activates c-Src in ECs (6Takahashi M. Berk B.C. J. Clin. Invest. 1997; 98: 2623-2631Crossref Scopus (191) Google Scholar). c-Src and Src family may in with FAK to the downstream MAPKs. The dual activation of ERK and in response to shear stress is by Ras Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). We have shown that of and the negative of Ras and in BAEC the shear stress activation of and the c-Jun transcriptional activity Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar). In to such as and have also been shown to be of in and NIH3T3 cells A. A. A. Karin M. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). We found that the shear stress activation of ERK and JNK, as as factor protein was attenuated by negative of and Li and J. and may with Ras to shear stress-induced associated with a in the protein was recently and shown to the of and J.D. Taylor J.M. Parsons J.T. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). Although is in that signaling pathways may also FAK to in response to shear in the pathways has and induction by shear stress is those to the FAK, Grb2, Sos, and the activation by fluid shear stress is a of The is are the activation of ERK Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), which is a kinase involved in In activation of occurs at the application of shear stress Y.-S. Shyy J.Y.-J. Li S. Lee J.D. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (208) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The between the temporal activation of ERK and is with the that pathways are involved in the shear stress activation of JNK. The of of the kinases and may be important factors in the temporal of the of in the transient of the FAK to the signaling may of In the shear stress-induced signaling it that and pathways can be the of ERK and activation also be to a and is by a H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (241) Google that shear stress activation of ERK is that of is studies have shown that fluid shear stress the focal adhesion with cell elongation and the alignment of stress fibers with the direction of flow, there was a of and at the of the sheared ECs (10Davies P.F. Robotewskyj A. Griem M.L. J. Clin. Invest. 1994; 93: 2031-2038Crossref PubMed Scopus (297) Google Scholar, R.M. J. Cell. Physiol. 1995; PubMed Scopus Google Scholar). We found that FAK and VNR, in focal adhesion at the abluminal side of BAEC, association in the sheared cells In to FAK, c-Src is also by shear stress (6Takahashi M. Berk B.C. J. Clin. Invest. 1997; 98: 2623-2631Crossref Scopus (191) Google and can be to these focal adhesion the shear stress activation of ERK, JNK, and FAK suggesting that the activation of pathways in response to shear stress is the integrity of the actin which is the signaling by with and the of FAK and Src family PTKs, with signaling such as Grb2, Sos, and MAPKs, and ERK S. S.K. Science. 1995; PubMed Scopus Google Scholar, K. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar, S. H. J.S. S.K. J. Biol. 1995; PubMed Scopus Google Scholar). The focal of these at the and tyrosine phosphorylation are by S. H. J.S. S.K. J. Biol. 1995; PubMed Scopus Google Scholar). The between these and in the study that signaling is involved in shear stress activation of In to the of shear stress on ECs and adhesion on ECM, vascular cells also signaling K. J. Clin. 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