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• W2049635672 abstract "PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK½, p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals. PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK½, p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals. Vascular endothelial cells (ECs) 1The abbreviations used are: EC, endothelial cells; PECAM, platelet endothelial cell adhesion molecule; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; KO, knock-out; RC, reconstituted. line the luminal surface of blood vessels and are constantly exposed to hemodynamic forces of blood flow such as fluid shear stress, cyclic strain, and blood pressure. These mechanical forces induce morphological, physiological, and biochemical changes of ECs (1Azuma N. Duzgun S.A. Ikeda M. Kito H. Akasaka N. Sasajima T. Sumpio B.E. J. Vasc. Surg. 2000; 32: 789-794Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 2Sumpio B.E. Hemodynamic Forces and Vascular Cell Biology. R. G. Landes Company, Austin, TX1993Google Scholar). Although mechanisms for mechanosensing and subsequent signaling events in ECs have not yet been well characterized, activation of trimeric G proteins (3Cohen C.R. Mills I. Du W. Kamal K. Sumpio B.E. Exp. Cell Res. 1997; 231: 184-189Crossref PubMed Scopus (27) Google Scholar, 4Gudi S.R. Clark C.B. Frangos J.A. Circ. Res. 1996; 79: 834-839Crossref PubMed Scopus (226) Google Scholar, 5Kuchan M.J. Jo H. Frangos J. Am. J. Physiol. 1994; 267: C753-C758Crossref PubMed Google Scholar, 6Ohno M. Gibbons G.H. Dzau V.J. Cooke J.P. Circulation. 1993; 88: 193-197Crossref PubMed Scopus (235) Google Scholar), Ca2+ mobilization (7Rosales O.R. Isales C.M. Barrett P.Q. Brophy C. Sumpio B.E. Biochem. J. 1997; 326: 385-392Crossref PubMed Scopus (45) Google Scholar, 8Ando J. Komatsuda T. Kamiya A. In Vitro Cell Dev. Biol. 1988; 24: 871-877Crossref PubMed Scopus (202) Google Scholar, 9Mo M. Eskin S. Schilling W. Am. J. Physiol. 1991; 260: H1698-H1707PubMed Google Scholar, 10Shen J. Luscinskas F.W. Connolly A. Dewey Jr., C.F. Gimbrone Jr., M.A. Am. J. Physiol. 1992; 262: C384-C390Crossref PubMed Google Scholar), and increased K+ efflux (11Olesen S.P. Clapham D.E. Davies P.F. Nature. 1988; 331: 168-170Crossref PubMed Scopus (743) Google Scholar, 12Schilling W.P. Mo M. Eskin S.G. Exp. Cell Res. 1992; 198: 31-35Crossref PubMed Scopus (35) Google Scholar) are known as early signaling events. Other investigators have recently demonstrated that a glycoprotein, platelet endothelial cell adhesion molecule-1 (PECAM-1) is rapidly tyrosine-phosphorylated in bovine ECs (BAEC) exposed to fluid flow; this is thought to play important roles in transducing chemical stimuli received on the cell surface to the cytoplasmic aspects of the cell (13Osawa M. Masuda M. Harada N. Lopes R.B. Fujiwara K. Eur. J. Cell Biol. 1997; 72: 229-237PubMed Google Scholar, 14Fujiwara K. Masuda M. Osawa M. Kano Y. Katoh K. Cell Struct. Funct. 2001; 26: 11-17Crossref PubMed Scopus (52) Google Scholar). PECAM-1, also known as CD31 or endoCAM (endothelial cell adhesion molecule), is a 130-kDa transmembrane glycoprotein that is expressed on ECs, platelets, monocytes, and leukocytes (15Newman P. J. Clin. Investig. 1997; 99: 3-8Crossref PubMed Scopus (434) Google Scholar). Specifically, PECAM-1 has been found to concentrate at the junctions of ECs. PECAM-1 is a member of immunoglobulin (Ig) superfamily and has a large extracellular domain containing six Ig homology units, a single membrane-spanning sequence, and a cytoplasmic tail. PECAM-1 has a relatively long (118 amino acids) cytoplasmic tail encoded by seven separate exons (16Newman P.J. Berndt M.C. Gorski J. White 2nd, G.C. Lyman S. Paddock C. Muller W.A. Science. 1990; 247: 1219-1222Crossref PubMed Scopus (827) Google Scholar, 17Xie Y. Muller W.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5569-5573Crossref PubMed Scopus (73) Google Scholar, 18Kirschbaum N.E. Gumina R.J. Newman P.J. Blood. 1994; 84: 4028-4037Crossref PubMed Google Scholar). An ITAM (immunoreceptor tyrosine-based activation motif) domain, encoded by exons 12, 13, and 14, has been identified in the cytoplasmic tail of PECAM-1 (19Lu T.T. Barreuther M. Davis S. Madri J.A. J. Biol. Chem. 1997; 272: 14442-14446Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). PECAM-1 has been noted to be phosphorylated by fluid flow and osmotic shock and also to associate with adapter and signaling molecules in an ITAM-independent manner, as was recently shown for β-catenin (20Ilan N. Mahooti S. Rimm D.L. Madri J.A. J. Cell Sci. 1999; 112: 3005-3014Crossref PubMed Google Scholar), γ-catenin (21Ilan N. Cheung L. Pinter E. Madri J.A. J. Biol. Chem. 2000; 275: 21435-21443Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and STAT (signal transducers and activators of transcription) (22Ilan N. Madri J.A. Curr. Opin. Cell Biol. 2003; 15: 515-524Crossref PubMed Scopus (209) Google Scholar) family members. Functionally, PECAM-1 tyrosine phosphorylation has been shown to play a role in endothelial cell migration and angiogenesis (23Lu T.T. Yan L.G. Madri J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11808-11813Crossref PubMed Scopus (97) Google Scholar, 24Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, whether PECAM-1 tyrosine phosphorylation is involved in the signaling pathway activated by shear stress in ECs is poorly understood. The mitogen-activated protein kinase (MAPK) family participates in transmitting extracellular signals to cytoplasmic and nuclear pathways. We and other laboratories have reported that fluid shear stress activates several members of the MAPK family including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK in cultured bovine ECs (1Azuma N. Duzgun S.A. Ikeda M. Kito H. Akasaka N. Sasajima T. Sumpio B.E. J. Vasc. Surg. 2000; 32: 789-794Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 25Azuma N. Akasaka N. Kito H. Ikeda M. Gahtan V. Sasajima T. Sumpio B.E. Am. J. Physiol. 2001; 280: H189-H197Crossref PubMed Google Scholar). In addition, another distinct but important cell signaling pathway, phosphatidylinositol 3-kinase-AKT, has been shown to be activated by fluid shear stress and other hemodynamic forces (26Li W. Sumpio B.E. Am. J. Physiol. 2005; (in press)Google Scholar, 27Haga M. Chen A. Gortler D. Dardik A. Sumpio B.E. Endothelium. 2003; 10: 149-157Crossref PubMed Google Scholar). In this study, we measured the phosphorylation and enzyme activity of MAPK and AKT to identify the signal events involved in the response of vascular ECs to shear stress. We also addressed the question of whether PECAM-1 functions as a mechanoreceptor of shear stress, activating MAPK and AKT in vascular ECs. We show here the magnitude and time course of PECAM-1, MAPK, and AKT activation induced by shear stress in murine knock-out (KO) and reconstituted (RC) PECAM-1 EC cell lines as well as in human umbilical vein endothelial cells (HUVEC) transfected with antisense or scrambled PECAM-1 oligonucleotides. We also compare the results from confluent cultured and sparsely cultured BAEC and HUVEC. Murine Microvascular EC—The PECAM-KO endothelioma cell line luEND.PECAM-1.1, isolated from a PECAM-1-KO mouse lung, was established by retroviral transduction of the primary endothelial cell culture with the polyoma virus middle T-oncogene. PECAM-1-KO endothelial cells were then retrovirally transduced with full-length murine PECAM-1 cDNA as described previously (28Wong C.W. Wiedle G. Ballestrem C. Wehrle-Haller B. Etteldorf S. Bruckner M. Engelhardt B. Gisler R.H. Imhof B.A. Mol. Biol. Cell. 2000; 11: 3109-3121Crossref PubMed Scopus (85) Google Scholar, 29Graesser D. Solowiej A. Bruckner M. Osterweil E. Juedes A. Davis S. Ruddle N.H. Engelhardt B. Madri J.A. J. Clin. Investig. 2002; 109: 383-392Crossref PubMed Scopus (271) Google Scholar), generating a PECAM-1-RC cell line expressing PECAM-1 at a level similar to that of wild type EC similarly immortalized (Fig. 1). The endothelioma cell lines retained surface expression of VE-cadherin by fluorescence-activated cell sorter analysis and showed contact inhibition on confluence. Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 10 mm HEPES, pH 7.4, 1% l-glutamine, 1% nonessential amino acids, 1% pyruvate, 10,000 units/ml penicillin/streptomycin, and 105m 2-mercaptoethanol (Invitrogen) and were incubated at 37 °C in 5% CO2. Selection of PECAM-1 expression on RC cells was maintained with 1 mg/ml puromycin. Bovine Aortic EC—BAEC were obtained by scraping the intimal surface of aorta obtained from freshly killed calves at a local slaughterhouse (30Awolesi M.A. Sessa W.C. Sumpio B.E. J. Clin. Investig. 1995; 96: 1449-1454Crossref PubMed Scopus (281) Google Scholar). For all experiments, ECs were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT) and antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml, and amphotericin B 250 ng/ml, (Invitrogen) in a humidified atmosphere of 5% CO2 on type I collagen-coated 35-mm 6-well culture dishes at 37 °C. Serum-containing medium was replaced by serum-free medium 24 h prior to each experiment. Cells were synchronized by serum starvation for 24 h. PECAM-1 expression was confirmed in BAEC (Fig. 1) Human Umbilical Vein EC—HUVEC were purchased from the BCMM cell culture core (Boyer Center for Molecular Medicine, Yale University Medical School) and cultured on gelatin in HUVEC media (M199 medium, 20% fetal bovine serum, 50 mg/ml endothelial cell growth supplement, 10 mm HEPES, 2 mm l-glutamine, 1 mm sodium pyruvate, and antibiotics). HUVEC were used between passages 3 and 4. Antisense oligonucleotide 5′-TCCTTCCAGGG ATGTGATC-3′ for human PECAM-1 and control scrambled oligonucleotide 5′-TTCTACCTCGCGCGATTTAC-3′ were provided by F. Bennett and T. Condon at Isis™ Pharmaceuticals (Carlsbad, CA). HUVEC at 70–80% confluence were transfected using Lipofectin as described (31Gratzinger D. Barreuther M. Madri J.A. Biochem. Biophys. Res. Commun. 2003; 301: 243-249Crossref PubMed Scopus (51) Google Scholar). After 48 h of transfection, cells were subjected to serum-free medium during 60 min for synchronization, previous to exposure to shear stress. Pilot studies have confirmed that the expression of PECAM-1 in PECAM-1 scrambled HUVEC was notably stronger compared with the PECAM-1 antisense HUVEC and comparable with the wild type HUVEC (Fig. 1). A transfection efficiency of >90% was observed by fluorescence microscopy when cells were transfected with fluorescently labeled oligonucleotides. Fluid flow was applied to confluent cultures with an orbital shaker (Lab-Line, Melrose Park, IL) (32Yun S. Dardik A. Haga M. Yamashita A. Yamaguchi S. Koh Y. Madri J.A. Sumpio B.E. J. Biol. Chem. 2002; 277: 34808-34814Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 33Kraiss L.W. Weyrich A.S. Alto N.M. Dixon D.A. Ennis T.M. Modur V. McIntyre T.M. Prescott S.M. Zimmerman G.A. Am. J. Physiol. 2000; 278: H1537-H1544PubMed Google Scholar, 34Dardik A. Chen L. Frattini J. Asasda H. Aziz F. Kudo F. Sumpio B. J. Vasc. Surg. 2005; (in press)Google Scholar). Although this technique does not result in uniform application of laminar shear stress across the entire monolayer, the majority of the cells are exposed to near maximal shear stress (τmax), which can be calculated as τmax=aρη(2πf)3(Eq. 1) where a is the radius of orbital rotation (1.4 cm), ρ is the density of the culture medium (1.0 g/ml), η is the viscosity of the medium (0.0075 poise measured by viscometer), and f is the frequency of rotation (rotations/s). This equation yields shear stress of 14 dynes/cm2, normal arterial level, at shaking frequency of 270 rpm for 0, 5, 10, 30 or 60 min. ECs were washed three times with cold phosphate-buffered saline and scraped into 500 μl of lysis buffer (50 mm Tris-HCl, pH 8.0, at 4 °C, 150 mm NaCl, 2 mm EDTA, 50 mm NaF, 1% Triton X-100, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 1 μg of leupeptin/ml). The cells were incubated at 4 °C for 30 min with rotation. After centrifugation for 15 min, the supernatant was transferred to a fresh microcentrifuge tube, and the extract was precleared with 20 μl of protein G Plus-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h with rotation. The beads were pelleted, the supernatant was transferred to a new tube, and 20 μl of protein G Plus-agarose beads conjugated to PECAM-1-specific antibody (M20, Santa Cruz Biotechnology) was added. Immunoprecipitation was performed overnight at 4 °C with rotation, after which the immunoprecipitated proteins were washed four times with lysis buffer. After the last wash, the beads were resuspended in 12.5 μl of 2× Laemmeli sample buffer and boiled for 5 min. The samples were separated by 10% SDS-polyacrylamide gel, transferred on to nitrocellulose membrane, and probed with anti-phosphotyrosine-specific antibody (Santa Cruz Biotechnology). Tyrosine-phosphorylated PECAM-1 was detected using an enhanced chemiluminescence system. Activation of the MAPKs and AKT was assessed by determining phosphorylation of ERK½, p38 MAPK, and AKT with immunoblotting using phosphospecific antibodies (Cell Signaling) as described previously (25Azuma N. Akasaka N. Kito H. Ikeda M. Gahtan V. Sasajima T. Sumpio B.E. Am. J. Physiol. 2001; 280: H189-H197Crossref PubMed Google Scholar, 26Li W. Sumpio B.E. Am. J. Physiol. 2005; (in press)Google Scholar, 35Ikeda M. Takei T. Mills I. Kito H. Sumpio B.E. Am. J. Physiol. 1999; 276: H614-H622PubMed Google Scholar). In brief, after exposure to the shear stress, ECs were lysed in 50 mm HEPES, 150 mm NaCl, 10% glycerol, 1 mm EDTA, 100 mm NaF, 10 mm sodium pyrophosphate, 1% Triton X-100, 1.5 mm MgCl2, 1 mm Na3VO4, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. The extracts were heat-denatured in Laemmeli sample buffer, size-fractionated in a 10% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane (Amersham Biosciences). After blocking for 1 h with Tris-buffered saline containing 0.1% Tween 20 (TTBS) and 5% nonfat dry milk, the blots were exposed to polyclonal antibodies. After rinsing three times for 15 min in TTBS, the blot was incubated with a 1:2,000 dilution of anti-rabbit IgGs and anti-mouse IgGs for 1 h. After three additional 15-min rinses in TTBS, the resulting protein-antibody complexes were detected by enhanced chemiluminescence (ECL, Amersham Biosciences). Data are presented as mean ± S.E. Student's t test was used for analyzing densitometric data for each experiment compared with the respective static control (time, 0 min). A p value of <0.05 was considered significant. Effect of Cell-Cell Contact of BAEC or HUVEC Exposed to Fluid Shear Stress on PECAM-1 Tyrosine Phosphorylation and ERK1/2, p38 MAPK, and AKT Activation—PECAM-1 protein is predominantly localized at lateral cell-cell adhesion sites in confluent EC but not in sparsely cultured cells. We investigated whether the formation of cell-cell contact was necessary for PECAM-1, ERK½, p38, and AKT phosphorylation induced by shear stress in BAEC and HUVEC. As shown in Fig. 2, 14 dynes/cm2 shear stress applied to 100% confluent BAEC resulted in PECAM-1 tyrosine phosphorylation in a time-dependent manner that peaked 10 min after initiation of the stimulus. However, in 50% confluent BAEC, PECAM-1 was not phosphorylated, suggesting that PECAM-1 engagement through cell-cell contact is necessary for its tyrosine phosphorylation as a response to shear stress. In both confluent and subconfluent BAEC, ERK½, p38 MAPK, and AKT were activated by shear stress in a time-dependent manner. Activated levels of ERK½ phosphorylation peaked at 5 min, and p38 MAPK phosphorylation peaked after 10 min. AKT phosphorylation could be detected at 10 min and peaked at 30 min. Fig. 3 demonstrates that confluent and subconfluent layers of HUVEC had a similar response to fluid shear stress. PECAM-1 was phosphorylated by 10 min after initiation of the stimulus in confluent but not in subconfluent HUVEC. In both confluent and subconfluent HUVEC, ERK½, p38 MAPK, and AKT were activated by shear stress in a time-dependent manner. These findings are consistent with the concept that MAPK and AKT activation are both independent of PECAM-1 tyrosine phosphorylation in BAEC and HUVEC exposed to fluid shear stress. Effect of Fluid Shear Stress on PECAM-1 Antisense Transfected HUVEC—Fig. 4 summarizes the effects of shear stress on PECAM-1 scrambled and PECAM-1 antisense oligonucleotide-transfected HUVEC. Shear stress activated PECAM-1 phosphorylation after 10 min of stimulus in confluent PECAM-1 antisense and scrambled oligonucleotide-transfected HUVEC, although, as expected, the total PECAM-1 expression and its phosphorylation in the PECAM-1 antisense oligonucleotide-transfected HUVEC was significantly weaker when compared with the PECAM-1 scrambled oligonucleotide-transfected HUVEC. PECAM-1 was not phosphorylated by shear stress in sparse PECAM-1 antisense or scrambled oligonucleotide-transfected cells. These findings are again consistent with the concept that cell-cell PECAM-1 engagement is necessary for its tyrosine phosphorylation following shear stress. Furthermore, in PECAM-1 antisense and scrambled oligonucleotide-transfected HUVEC, activation of ERK½, p38 MAPK, and AKT induced by shear stress was present both in confluent and sparse culture conditions. These findings indicate a minimal role, if any, of PECAM-1 on the activation of MAPKs ERK½ and p38 and AKT intracellular signaling following shear stress in the conditions examined. PECAM-1, ERK1/2, p38 MAPK, and AKT Expression Are Activated after Shear Stress in PECAM-1-KO and -RC Murine Microvascular EC—As shown in Fig. 5, 14 dynes/cm2 shear stress induced tyrosine phosphorylation of PECAM-1 in murine PECAM-1 RC in a time-dependent manner. The time for peak activation was between 10 and 30 min, and the phospho-activation returned to static levels after 60 min. The PECAM-1-KO cells were absent any PECAM-1. In the PECAM-1-RC and -KO cells, shear stress activated ERK½ very rapidly, reaching a peak as early as 5 min after the initiation of the stimulus. There was also a similar time-dependent phosphorylation of p38 MAPK in the cells, with a significant increase after 30 min compared with control. AKT was also significantly phosphorylated after 30 min of exposure to shear. These findings suggest that neither the expression nor the tyrosine phosphorylation of PECAM-1 had any major modulatory effect on the activation of ERK½, p38 MAPK, or AKT intracellular signaling following shear stress in murine microvascular EC. The vascular endothelium is consistently exposed to hemodynamic forces of blood flow. In vivo and in vitro studies have revealed that flow induces a number of morphological, physiological, and biological changes in ECs (2Sumpio B.E. Hemodynamic Forces and Vascular Cell Biology. R. G. Landes Company, Austin, TX1993Google Scholar), and disturbed flow is observed in the areas of high incidence of atherosclerosis and intimal hyperplasia. In such areas there is disturbed flow, and specifically there is reduced shear stress compared with unaffected portions of the vessel. Thus, it is widely accepted that the reduced level of fluid shear stress in large arteries is proatherogenic and is also thought to be involved in the arterialization and development of anastomotic intimal hyperplasia of vein grafts (2Sumpio B.E. Hemodynamic Forces and Vascular Cell Biology. R. G. Landes Company, Austin, TX1993Google Scholar). There are most likely many different mechanoreceptors in ECs to detect the different hemodynamic forces or alternative coupling pathways from single receptors (36Davies P.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1755-1757Crossref PubMed Scopus (33) Google Scholar). Recent studies have demonstrated that rapid change of flow shear stress activates trimeric G protein (4Gudi S.R. Clark C.B. Frangos J.A. Circ. Res. 1996; 79: 834-839Crossref PubMed Scopus (226) Google Scholar, 5Kuchan M.J. Jo H. Frangos J. Am. J. Physiol. 1994; 267: C753-C758Crossref PubMed Google Scholar, 6Ohno M. Gibbons G.H. Dzau V.J. Cooke J.P. Circulation. 1993; 88: 193-197Crossref PubMed Scopus (235) Google Scholar), induces Ca2+ mobilization (8Ando J. Komatsuda T. Kamiya A. In Vitro Cell Dev. Biol. 1988; 24: 871-877Crossref PubMed Scopus (202) Google Scholar, 9Mo M. Eskin S. Schilling W. Am. J. Physiol. 1991; 260: H1698-H1707PubMed Google Scholar, 10Shen J. Luscinskas F.W. Connolly A. Dewey Jr., C.F. Gimbrone Jr., M.A. Am. J. Physiol. 1992; 262: C384-C390Crossref PubMed Google Scholar), which may be caused by phosphoinositide turnover, and increases K+ conductance (11Olesen S.P. Clapham D.E. Davies P.F. Nature. 1988; 331: 168-170Crossref PubMed Scopus (743) Google Scholar, 12Schilling W.P. Mo M. Eskin S.G. Exp. Cell Res. 1992; 198: 31-35Crossref PubMed Scopus (35) Google Scholar), which may be caused by K+-channel activation in ECs. These early signaling events occur at the cell surface. Tyrosine phosphorylation of receptors and regulatory proteins of various signal transduction pathways have been shown to play important roles in transducing chemical and mechanical stimuli received on the cell surface (14Fujiwara K. Masuda M. Osawa M. Kano Y. Katoh K. Cell Struct. Funct. 2001; 26: 11-17Crossref PubMed Scopus (52) Google Scholar, 36Davies P.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1755-1757Crossref PubMed Scopus (33) Google Scholar, 37Yano Y. Geibel J. Sumpio B.E. Am. J. Physiol. 1996; 271: C635-C649Crossref PubMed Google Scholar). PECAM-1, also known as CD31 or endoCAM, is a 130 kDa transmembrane glycoprotein belonging the Ig superfamily of cell adhesion molecules and is most abundantly expressed on ECs, making it a useful and commonly used marker of EC, platelets, monocytes, neutrophils, and certain subsets of T-cells (15Newman P. J. Clin. Investig. 1997; 99: 3-8Crossref PubMed Scopus (434) Google Scholar, 16Newman P.J. Berndt M.C. Gorski J. White 2nd, G.C. Lyman S. Paddock C. Muller W.A. Science. 1990; 247: 1219-1222Crossref PubMed Scopus (827) Google Scholar). PECAM-1 has important intracellular signaling/scaffolding capacities that function to control the activation and cell survival (22Ilan N. Madri J.A. Curr. Opin. Cell Biol. 2003; 15: 515-524Crossref PubMed Scopus (209) Google Scholar, 38Newman P.J. Newman D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 953-964Crossref PubMed Scopus (339) Google Scholar). The best described signaling property of PECAM-1 is the phosphorylation of the two ITAM tyrosine residues at positions 663 and 686 of the PECAM-1 cytoplasmic domain, which occur in response to exposure of cells to a number of different chemical and mechanical stimuli (13Osawa M. Masuda M. Harada N. Lopes R.B. Fujiwara K. Eur. J. Cell Biol. 1997; 72: 229-237PubMed Google Scholar, 19Lu T.T. Barreuther M. Davis S. Madri J.A. J. Biol. Chem. 1997; 272: 14442-14446Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38Newman P.J. Newman D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 953-964Crossref PubMed Scopus (339) Google Scholar, 39Maas M. Wang R. Paddock C. Kotamraju S. Kalyanaraman B. Newman P.J. Newman D.K. Am. J. Physiol. 2003; 285: H2336-H2344Crossref PubMed Scopus (21) Google Scholar). Functionally, PECAM-1 tyrosine phosphorylation has been shown to play a role in the migration of EC, neutrophil transmigration, and angiogenesis (23Lu T.T. Yan L.G. Madri J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11808-11813Crossref PubMed Scopus (97) Google Scholar, 24Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 38Newman P.J. Newman D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 953-964Crossref PubMed Scopus (339) Google Scholar) In cultured endothelial cells, PECAM-1 is distributed diffusely in the plasma membrane of solitary cells, but once a cell-cell contact is made, it accumulates at lateral contact sites and is thought to play a role in the formation and maintenance of monolayer integrity and in platelet and leukocyte interactions with endothelium (29Graesser D. Solowiej A. Bruckner M. Osterweil E. Juedes A. Davis S. Ruddle N.H. Engelhardt B. Madri J.A. J. Clin. Investig. 2002; 109: 383-392Crossref PubMed Scopus (271) Google Scholar, 40Albelda S.M. Oliver P.D. Romer L.H. Buck C.A. J. Cell Biol. 1990; 110: 1227-1237Crossref PubMed Scopus (334) Google Scholar, 41Duncan G.S. Andrew D.P. Takimoto H. Kaufman S.A. Yoshida H. Spellberg J. Luis de la Pompa J. Elia A. Wakeham A. Karan-Tamir B. Muller W.A. Senaldi G. Zukowski M.M. Mak T.W. J. Immunol. 1999; 162: 3022-3030PubMed Google Scholar). In EC forming a 100% confluent monolayer, PECAM-1 is predominantly localized to the cell-cell borders in culture and in tissue, and its localization does not change before and after tyrosine phosphorylation initiated by mechanical stress (14Fujiwara K. Masuda M. Osawa M. Kano Y. Katoh K. Cell Struct. Funct. 2001; 26: 11-17Crossref PubMed Scopus (52) Google Scholar). Recent reports indicate that in confluent EC, phosphorylation of PECAM-1 does not depend on membrane localization of the protein or on cell-cell binding. Furthermore, the transmembrane and extracellular domains of PECAM-1 do not appear to be necessary for responsiveness of EC to shear stress (42Kaufman D.A. Albelda S.M. Sun J. Davies P.F. Biochem. Biophys. Res. Commun. 2004; 320: 1076-1081Crossref PubMed Scopus (27) Google Scholar). In contrast, we observed that in all of the EC populations studied cell-cell contact was required for shear stress-induced PECAM-1 tyrosine phosphorylation. These differences may be ascribed to differences in the derivations of the endothelial cell populations and/or the substrate used in the studies (gelatin versus native type I collagen) and the effects it may have on cell responsiveness. Previous studies have shown that Tyr-686 is phosphorylated in EC that are stimulated by fluid shear stress of more than 5 dynes/cm2 or hyper- and hypoosmotic shocks (13Osawa M. Masuda M. Harada N. Lopes R.B. Fujiwara K. Eur. J. Cell Biol. 1997; 72: 229-237PubMed Google Scholar). Such PECAM-1 tyrosine phosphorylation in mechanically stimulated EC is a rapid event, detectable within 30 s of stimulation (13Osawa M. Masuda M. Harada N. Lopes R.B. Fujiwara K. Eur. J. Cell Biol. 1997; 72: 229-237PubMed Google Scholar, 43Harada N. Masuda M. Fujiwara K. Biochem. Biophys. Res. Commun. 1995; 214: 69-74Crossref PubMed Scopus (50) Google Scholar). Other studies revealed that mechanically induced tyrosine phosphorylation of PECAM-1 is not a downstream event of Ca2+ mobilization, K+ channel activation, stretch-activated cation channel activity, or protein kinase C activation, which are all known as the earliest flow-dependent cellular events; this PECAM-1 phosphorylation also initiates a signaling cascade leading to ERK activation. Therefore, although these data are not conclusive, these results suggest that tyrosine phosphorylation of PECAM-1 induced by fluid shear stress can be a mechanosensing event in ECs (14Fujiwara K. Masuda M. Osawa M. Kano Y. Katoh K. Cell Struct. Funct. 2001; 26: 11-17Crossref PubMed Scopus (52) Google Scholar). Current thinking also holds that the activation of PECAM-1 is a prerequisite for the expression of subsequent MAPKs in ECs. The MAPK family is a class of signal transduction proteins capable of transmitting extracellular signals to cytoplasmic and nuclear pathways (1Azuma N. Duzgun S.A. Ikeda M. Kito H. Akasaka N. Sasajima T. Sumpio B.E. J. Vasc. Surg. 2000; 32: 789-794Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 35Ikeda M. Takei T. Mills I. Kito H. Sumpio B.E. Am. J. Physiol. 1999; 276: H614-H622PubMed Google Scholar, 44Kito H. Chen E.L. Wang X. Ikeda M. Azuma N. Nakajima N. Gahtan V. Sumpio B.E. J. Appl. Physiol. 2000; 89: 2391-2400Crossref PubMed Scopus (56) Google Scholar). The activation of MAPK induces a number of cellular changes including cell proliferation, morphologic changes, and increased transcription of immediate early genes, leading to altered vasoactive protein production. Thus, activation of mechanoreceptors on the cell surface must be necessary to transmit the extracellular signals to the nucleus through MAPK activation. In our studies, shear stress tyrosine-phosphorylated PECAM-1 in PECAM-reconstituted microvascular ECs and BAECs, with peak activation at 10 min. We also demonstrated that shear stress activated ERK½ in microvascular ECs and BAECs. However, the latter activation occurred almost immediately, as early as 5 min after the initiation of shear stress, and was sustained for at least 60 min. Thus, the temporal pattern of PECAM-1 activation was significantly different from that of ERK½ activation in ECs exposed to shear stress, suggesting that other mechanoreceptors existing on the cell surface, other than PECAM-1, may be activated by shear stress and may cause a much more rapid induction of MAPK activity. Although we have studied the downstream pathways and subsequent nuclear events modulated by shear stress, additional studies still need to be performed to elucidate the upstream mechanisms of the mechanosensing signaling pathway(s). PECAM-1 has been shown to be an important scaffolding molecule involved in several signaling pathways (22Ilan N. Madri J.A. Curr. Opin. Cell Biol. 2003; 15: 515-524Crossref PubMed Scopus (209) Google Scholar, 38Newman P.J. Newman D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 953-964Crossref PubMed Scopus (339) Google Scholar). However, the data presented in this report are consistent with the concept that PECAM-1 is not a major mechanosensor in the EC populations studied. Our studies differ from those performed previously in that we performed all our studies on a type I collagen coating compared with poly-l-lysine or gelatin coating, and EC populations were utilized exclusively throughout the studies (14Fujiwara K. Masuda M. Osawa M. Kano Y. Katoh K. Cell Struct. Funct. 2001; 26: 11-17Crossref PubMed Scopus (52) Google Scholar, 42Kaufman D.A. Albelda S.M. Sun J. Davies P.F. Biochem. Biophys. Res. Commun. 2004; 320: 1076-1081Crossref PubMed Scopus (27) Google Scholar). As cell-matrix adhesion is known to be a major determinant of cellular responsiveness to a wide variety of mechanical and chemical stimuli, we suggest that in our in vitro studies (and possibly in vivo as well) the dynamic adhesion and interaction of EC with physiologic substrata likely play major roles in modulating the responsiveness of EC to shear forces (45Bershadsky A.D. Balaban N.Q. Geiger B. Annu. Rev. Cell Dev. Biol. 2003; 19: 677-695Crossref PubMed Scopus (711) Google Scholar). Thus, further studies are obviously needed to definitively identify and characterize the many other potential mechanoreceptors likely responsible for transducing shear stimuli in EC (42Kaufman D.A. Albelda S.M. Sun J. Davies P.F. Biochem. Biophys. Res. Commun. 2004; 320: 1076-1081Crossref PubMed Scopus (27) Google Scholar, 45Bershadsky A.D. Balaban N.Q. Geiger B. Annu. Rev. Cell Dev. Biol. 2003; 19: 677-695Crossref PubMed Scopus (711) Google Scholar)." @default.
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• W2049635672 title "MAPKs (ERK½, p38) and AKT Can Be Phosphorylated by Shear Stress Independently of Platelet Endothelial Cell Adhesion Molecule-1 (CD31) in Vascular Endothelial Cells" @default.
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