- Email: [email protected]
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Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation 97: 1708–1715.
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Matrisian LM: 1990. Metalloproteinases and their inhibitors in matrix remodeling. Trends in Genetics 6:121–125. Montfort I, Perez-Tamayo R: 1975. The distribution of collagenase in normal rat tissues. J Histochem Cytochem 23:910–920. Ohuchi E, Imai K, Fujii Y, et al.: 1997. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem 272:2446–2451. Peterson JT, Hallak H, Johnson L, et al.: 2001. Matrix metalloproteinase inhibition attenuates left ventriclar remodeling and dysfunction in a rat model of progressive heart function. Circulation 103:2303–2309. Peterson JT, Li H, Dillon L, Bryant JW: 2000. Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat. Cardiovascular Res 46:307–315.
Uitto J, Perejda AJ: 1987. Molecular pathology of the extracellular matrix. New York, Marcel Dekker. Vincenti MP, Coon CI, Mengshol JA, et al.: 1998. Cloning of the gene for interstitial collagenase-3 (matrix metalloproteinase-13) from rabbit synovial fibroblasts: differential expression with collagenase-1 (matrix metalloproteinase-1). Biochem J 331: 341–346. Weber KT: 1989. Cardiac interstitium in health and disease: The fibrillar collagen network. J Am Coll Cardiol 13:1637–1652.
Weber KT, Janicki JS, Shroff SG, et al.: 1988b. Collagen remodeling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res 62: 757–765. Weber K, Pick R, Janicki JS, et al.: 1988a. Inadequate collagen tethers in dilated cardiopathy. Am Heart J 116:1641–1646. Weber KT, Pick R, Silver MA, et al.: 1990. Fibrillar collagen and remodeling of dilated canine left ventricle. Circulation 82:1387–1401. Woodwiss AJ, Tsotetsi OJ, Sprott S, et al.: 2001. Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circ 103:155–160. PII S1050-1738(01)00160-8
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Nina L. Tsakadze, Zhendong Zhao, and Stanley E. D’Souza*
Rohde LE, et al.: 1999. Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation 99:3063–3070.
The binding of plasma protein fibrinogen (Fg) to intercellular adhesion molecule-1 (ICAM-1) on endothelial cells mediates the attachment of leukocytes and platelets that may result in vascular occlusion. Fg:ICAM-1 interactions elicit an array of effects that could have implications in vascular pathology and inflammation. ICAM-1 expression is regulated during inflammation and upon Fg binding. The mechanistic model presented provides a framework to delineate the consequences of Fg binding to ICAM-1. (Trends Cardiovasc Med 2002;12:101–108) © 2002, Elsevier Science Inc.
Spinale F, Coker ML, Heung LJ, et al.: 1999. Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res 85:364–376. Spinale F, Coker ML, Heung LJ, et al.: 2000. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation 102:1944–1949. Spinale FG, Zellner JL, Johnson WS, et al.: 1996. Cellular and extracellular remodeling with the development and recovery from tachycardia-induced cardiomyopathy: changes in fibrillar collagen, myocyte adhesion capacity and proteoglycans. J Mol Cell Cardiol 28:1591–1608. Takahashi S, Barry AC, Factor SM: 1990. Collagen degradation in ischaemic rat hearts. Biochem J 265:233–241. Thomas CV, Coker ML, Zellner JL, et al.: 1998.
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Nina L. Tsakadze, Zhendong Zhao, and Stanley E. D’Souza are from the Department of Physiology & Biophysics, University of Louisville, Louisville, Kentucky, USA. * Address correspondence to: Stanley E. D’Souza, Department of Physiology & Biophysics, A-1101 Health Sciences Center, University of Louisville, Louisville, KY 40292, USA. Tel.: (11) 502-852-3194; fax: (11) 502-8526239; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
Several reviews deal with the structural and functional aspects of intercellular adhesion molecule-1 (ICAM-1) and their role in immune and adhesive interactions (Carlos and Harlan 1994, Springer 1994, Van de Stolpe and Van der Saag 1996). This article focuses on the binding of ICAM-1 to plasma protein fibrinogen (Fg) and its derivatives. The intracellular signals, generated through this interaction, and their functional effect
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on the vascular endothelium is discussed. On this subject, the review by Altieri (1999) is highly informative. • Structural Features and Expression of ICAM-1 ICAM-1 (CD 54)—a 90- to 110-kDa cell surface glycoprotein expressed on monocytes, T and B lymphocytes, macrophage and fibroblasts, epithelial cells, and endothelial cells, plays an important role in immune-mediated cell–cell adhesive interactions (Springer 1994). ICAM-1 is an integral membrane protein (Figure 1) with an extracellular region composed of five immunoglobulin (Ig)-like domains, a transmembrane region, and a short cytoplasmic tail (Staunton et al. 1988). The Ig domains create a rigid structural framework, while a flexible bend (“hinge”) appearing between the second and third domains provides molecular flexibility, maximizing the accessibility of domains 1 and 2 (Wang and Springer 1998). Although low under basal conditions, the expression of ICAM-1 can be induced to higher levels by inflammatory mediators, such as tumor necrosis factor a (TNFa), interleukin (IL)-1b, IL-4, and interferon g, as well as by shear stress. Glucocorticoids have an inhibitory effect on ICAM-1 expression. The induced cell surface expression is the result of de novo mRNA transcription and translation, but mobilizable intracellular pools of ICAM-1 have been identified in monocytes. ICAM-1 can be detected 4 hours after the induction, becomes maximal within 16–24 hours, and remains high for about 48 hours. • Functional Role of ICAM-1 ICAM-1 is a ligand for leukocyte integrins lymphocyte function-associated antigen-1 (LFA-1; aLb2) and Mac-1 (aMb2),
and provides a mechanism for leukocyte–endothelial interaction and for the migration of leukocytes to the sites of inflammation. LFA-1 binds to the first and second, and Mac-1 to the third, domain of ICAM-1. Human ICAM-1 also is a receptor for the rhinovirus, coxsackie A13 virus, and Plasmodium falciparum malaria-infected erythrocytes (Springer 1994, Van de Stolpe and Van der Saag 1996), and thus facilitates viral and parasite invasion. Ligand binding avidity toward ICAM-1 depends on the presence of the divalent cations and is probably mediated through the conformational changes of the molecule. ICAM-1 is heavily glycosylated and variability in carbohydrate content of ICAM-1 of different cell types suggests glycosylation as a possible mechanism for affinity regulation, presumably by shielding the ligand-binding epitope. The main function of ICAM-1 is to promote intercellular communication through cell adhesion. The ICAM-1– dependent adhesion plays an important role in both afferent and efferent immune responses, including antigen presentation, T-cell–dependent antibody responses, T-cell proliferation and cellmediated cytotoxicity, and leukocyte attachment and migration through endothelial cell monolayers. ICAM-1 plays a costimulatory role on antigen-presenting cells in major histocompatibility complex (MHC) class II restricted T-cell activation and in cell-mediated cytotoxicity by activation of non-MHC– restricted killer cells and MHC class I restricted T-lymphocytes. ICAM-1– deficient mice are viable, but display an impaired inflammatory reaction; however, ICAM-1 deficiency has been demonstrated to be protective against lethal septic shock and ischemia/reperfusion injury (Sligh et al. 1993).
Figure 1. The structural boundaries of the extracellular, transmembrane, and cytoplasmic domains of ICAM-1.
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• ICAM-1 Expression in Diseases Increased levels of ICAM-1 have been reported in a variety of conditions characterized by local or generalized inflammation—including atherosclerosis, ischemia and reperfusion, stroke, malignancies, and autoimmune disorders—and suggest a role for ICAM-1 in the clinical evolution of disease. Low expression of ICAM-1 on tumor cells is associated with a reduced in vitro sensitivity to lymphocyte cytotoxicity, correlating with more aggressive clinical course, and a higher risk of metastasis. Inflammatory reactions in animal models have been down-regulated by the monoclonal antibodies directed against ICAM-1.
• Soluble ICAM-1 The ICAM-1 ectodomain undergoes proteolytic cleavage releasing soluble ICAM-1 from the cell surface. Matrix metalloproteinases (MMP) are likely to be involved in the cleavage process, as synthetic inhibitors of MMP-reduced soluble ICAM-1 in cell culture medium. Elevated levels of soluble ICAM-1 occur in vascular disease and appear to parallel the severity of the disease process. Soluble ICAM-1 may compete with membrane-bound form for the binding to its ligands and thus may be capable of modulating ICAM-1–mediated inflammatory responses. In this respect, it is interesting to note that gene polymorphism at residue 469 in the fifth Ig-like domain has been linked to inflammatory bowel diseases (Yang 1997), probably interfering with the rate of ICAM-1 ectodomain cleavage.
• Fg: A ligand for ICAM-1 More recently, the plasma protein Fg has been demonstrated to serve as a ligand for ICAM-1. Fg:ICAM-1 interaction was initially shown to promote bridging of monocytic cells to endothelium (Languino et al. 1993). Fg (340 kDa), a plasma glycoprotein composed of three pairs of disulfide-bonded polypeptide chains (Aa, Bb, and g; Figure 2), plays a key role in blood coagulation, inflammation, and tissue repair (Doolittle 1984). Fg binds to integrin aIIbb3 on activated platelets, promoting platelet adhesion and aggregation, thus playing a central role in primary hemoTCM Vol. 12, No. 3, 2002
Figure 2. Structure of fibrinogen. FpA and FpB, fibrinopeptides A and B.
stasis. Fg is spatially arranged into three globular domains: a central E domain and two peripheral D domains. Under the action of thrombin, fibrinopeptides A and B are cleaved from the amino-terminal of chains Aa and Bb of Fg, with a resultant polymerization into fibrin. Release of FpA and FpB exposes the A and B polymerization sites, respectively, that function cooperatively in the self-assembly process. The human polymerization site located at the aminoterminal end of the fibrin a chain (Aa 17–20) contains the sequence gly-proarg-val, while that located in the aminoterminal region of the fibrinogen Bb chain, in the close proximity to position 14, contains the sequence gly-his-argpro. The synthetic peptide gly-pro-argpro, which encompasses both these sequences, indeed binds to both the sites and prevents polymerization of fibrin monomer (Mosesson 1990). During fibrinolysis, fibrin(ogen) is proteolytically cleaved by plasmin, releasing fibrinogen degradation products (fragments D and E). Generation of fibrin and Fg-dependent formation of platelet plugs are essential components of both normal hemostasis and occlusive vascular diseases such as atherosclerosis and thrombosis. Accumulating evidence, however, suggests much broader physiological and pathophysiological roles for Fg than simply triggering blood coagulation and thrombosis. • Effect of Fg and Its Derivatives on the Vessel Wall Function Fg accumulation, at the site of tissue injury, occurs in a variety of pathologic processes, including inflammation, ischemia, trauma, and neoplasm. As an acute phase reactant, Fg readily responds to inflammatory stimuli. Plasma Fg level TCM Vol. 12, No. 3, 2002
is a strong and consistent predictor of cardiovascular events in individuals with or without clinically manifested vascular disease. Increased plasma Fg is associated with a significantly increased risk of myocardial infarction, stroke, peripheral arterial disease, atrial fibrillation, heart failure, and restenosis following angioplasty (Ernst and Resch 1993, Lowe 1997). There are several interactive, plausible, potential biological mechanisms by which Fg might be involved in cardiovascular disease pathogenesis, apart from its effects on blood coagulation and blood viscosity. Fibrin(ogen) and its degradation products can display mitogenic (Levesque et al. 1986) and angiogenic (Thompson et al. 1992) effects on endothelial cells, and increase endothelial cell permeability and detachment (Ge et al. 1992). It can stimulate the release of growth factors (Lorenzet et al. 1992), tissue plasminogen activator (t-PA), prostacycline (Kaplan et al. 1989), urokinase plasminogen activator (uPA) and von Willebrand factor (Ribes et al. 1989). • Structural Basis of the Fg:ICAM-1 Recognition The recognition sequences located on the (chain of Fg and within the first Ig-like domain of the ICAM-1 mediate Fg:ICAM-1 interaction. Altieri et al. (1995), utilizing synthetic peptides, identified amino-acid sequence 117–133 on the g chain of Fg as the region involved in binding to ICAM-1. Peptide g (117– 133) (117NQKIVNLKEKVAQLEA133), designated g3, blocked Fg-mediated adhesion of leukocytes to ICAM-1–expressing cells. Amino-acids encompassing sequence 8–21 within the first Ig domain of the ICAM-1 (8KVILPRGGSVLVTC21) interacts with intact Fg and with fragment D (fragment D contains the Fgg(117–133) sequence). ICAM-1(8–21) inhibited adhesion of Fg to ICAM-1– expressing lymphoid cells (D’Souza et al. 1996). Duperray et al. (1997) verified the location of the Fg binding epitope within the first domain of ICAM-1 using mutational analyses and a panel of inhibitory anti–ICAM-1 antibodies. In this analysis, however, aspartic acid (D26) and proline (P70) residues within ICAM-1 were determined to be critical for Fg binding. Therefore, there is a discrepancy between the sites identified by
D’Souza et al. (1996) and Duperray et al. (1997), even though they lie within the first domain of ICAM-1. It is quite likely that the critical residues in the sites reported are proximal to each other. Nevertheless, this discrepancy warrants further clarification. It is quite possible, that conformational changes of the Fg molecule may be required in order to expose g3 sequence for Fgg(117– 133):ICAM-1(8–21) interaction to occur. Altieri et al. (1995) proposed that the exposure of the g3 site may occur upon Fg binding to Mac-1 subsequent to Mac-1 activation by inflammatory stimuli. On the other hand, exposure of ICAM-1(8–21) may be regulated by initial low-affinity Fg binding sites on endothelial cells. These may be regulatory mechanisms limiting the Fg:ICAM-1 pathway activation under physiologic conditions. • Cellular Bridging through ICAM-1:Fg Increased levels of plasma Fg often coexist with an upregulated ICAM-1 expression in a number of cardiovascular disorders and are essential components of the inflammatory reaction. The mechanism for Fg:ICAM-1 interaction and their mutually potentiating interrelationship is now emerging. Fg functions as a coupling molecule between Mac-1 on leukocytes and ICAM-1 on endothelial cells, thus providing an alternative mechanism for their interaction and enhancing adhesion and migration of leukocytes (Languino et al. 1993 and 1995). Sriramarao et al. (1996), using intravital microscopy in rabbit mesentery, demonstrated that Fg induced a dose-dependent increase in the adhesion of monocytes to the vessel wall. The specificity of this cellular interaction was demonstrated by blockage in the presence of ICAM-1 monoclonal antibodies or Fgg(117–133) peptide. Interestingly, the adhesion was dependent on shear forces and occurred in venules, but not in arterioles or capillaries. These studies were the initial indication that cellular bridging mediated through Fg:ICAM-1 may be physiologically applicable. Sans et al. (2001), using Chinese hamster ovary cells expressing various constructs of ICAM-1, demonstrated that ICAM-1, through its interactions with Fg, is independently capable of mediating leukocyte transmigration in Transwell assays. Given these obser-
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vations, in experiments using intravital microscopy, one could speculate that ICAM-1–deficient mice may completely lack the capacity for Fg-dependent monocyte migration. Fg:ICAM-1 bridging is implicated in the adhesion of activated platelets to endothelial cells (Bombeli et al. 1998). In these studies, platelet adhesion to endothelial cells was markedly reduced in the presence of monoclonal antibodies to ICAM-1, aVb3, and adhesive proteins, including Fg. Fg deposition on endothelium has been demonstrated by intravital microscopy in a model of intestinal ischemia/reperfusion in mice (Massberg et al. 1999). Deposition of Fg induced platelet recruitment and adhesion to postischemic endothelium that was attenuated in the presence of antiFg antibodies and demonstrated to be integrin aIIb/b3 mediated. Fg deposition in postischemic reperfusion was markedly decreased in ICAM-1–deficient mice. • Other Functions Implicating Fg:ICAM-1 Interaction Fg ligation to ICAM-1 elicits vasomotor responses in the human saphenous veins (vasodilatation followed by vasoconstriction at higher concentrations), perhaps through the production of vasoactive mediators (Hicks et al. 1996). Fg-mediated relaxation was inhibited in the presence of anti–ICAM-1 antibodies and K1 channel blockers. Indomethacin and Nv-nitro-L-arginine methyl ester, inhibitors of endothelium-derived vasodilators (prostacycline and nitric oxide, respectively), did not significantly affect Fg-mediated relaxation. The vasomotor response was increased after exposure of the vessel to the arterial flow and correlated with the enhanced expression of ICAM-1 under conditions of high shear stress. Fg is known to induce mitogenesis (Levesque et al. 1986). Fg was found to induce proliferation of B-lymphoid Raji cells mediated through Fg:ICAM-1 interaction (Gardiner and D’Souza 1997). Similar mitogenic response was elicited by Fgg(117–133) and was blocked by ICAM-1(8–22) peptide. The phosphorylation of pp60Src and extracellular signalregulated kinase (ERK-1/2) appear to regulate mitogenic signals (Gardiner and D’Souza 1999). These signals also mediate endothelial cell survival and prevent
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TNFa-induced apoptosis (Pluskota and D’Souza 2000). We have observed, however, that proliferative response in endothelial cells is mediated mainly though Fg interaction with aVb3 integrin. Fg:ICAM-1 interaction plays only a minor role, even under conditions wherein ICAM-1 was upregulated in the presence of TNFa. This is likely due to the apoptotic effect of TNFa that may counteract the proliferative effect of Fg:ICAM-1 ligation. It has been suggested that release of vasoactive mediators coupled with the mitogenic response promoted through Fg may have a role in the causation of vascular diseases (Hicks et al. 1996). Interestingly, apart from the proinflammatory cytokine-induced ICAM-1 expression, fibrin and Fg itself have also been shown to upregulate ICAM-1 on the endothelial cells and human synovial fibroblasts (Harley et al. 2000, Liu and Piela-Smith 2000, Qi et al. 1997) perhaps due to FgBb15–42 ligation to the endothelial cadherin (VE-cadherin; Harley et al. 2000). VE-cadherin mediates homotypic cell adhesion and formation of the adherens junctions (Dejana et al. 1999). VE-cadherin has been identified as a receptor for the Bb15–42 sequence on the N-terminus of fibrin (Bach et al. 1998). For this interaction to occur, Bb15–42 needs to be exposed as a result of proteolytic degradation of fibrin(ogen) and cleavage of fibrinopeptide B (Bb1–14) by thrombin, uPA, or tPA. Interestingly, exposure of the FgBb15–42 sequence has a mitogenic effect on endothelial cells (Sporn et al. 1995). In synovial fibroblasts, fibrin(ogen)-induced ICAM-1 upregulation was regulated through the transcription factor nuclear factor-kB (NF-kB) that also caused an increase in IL-8 production. The increases in IL-8 and ICAM-1 may result in the recruitment of lymphocytes and in sustaining ongoing inflammation (Liu and Piela-Smith 2000). Interestingly, when incubated with endothelial cells, activated platelets (but not resting platelets) also promoted ICAM-1 expression, secretion of monocyte chemotactic protein-1, and activation of the transcription factor NF-kB (Gawaz et al. 1998). Other sequences within Fg or fibrin molecules exposed during fibrinogenesis and/or fibrin polymerization may be capable of increasing ICAM-1 expression (Liu and Piela-Smith 2000). This at-
tributes a regulatory role to fibrin(ogen), which may expose neo-epitopes and trigger different molecular events depending on the environmental stimuli. Taken together, these studies suggest that Fg plays a myriad role—directly or through cellular bridging—in generation and maintenance of inflammatory processes, and provides a biochemical basis for the long-recognized link between activation of coagulation and inflammation. • Signaling through ICAM-1 Several reports indicate the participation of ICAM-1 in signal transduction. ICAM-1 crosslinking with anti–ICAM-1 antibodies results in an oxidative burst in mononuclear leukocytes in the presence of the bacterial proinflammatory peptide N-formyl-met-leu-phe (Rothlein et al. 1994), plays a co-stimulatory role in the expression of IL-2 receptors in B-cells (Poudrier and Owens 1994), induces an increase in intracellular calcium in Burkitt lymphoma (van Horssen et al. 1995), and activates transcription factor AP-1 and the transcription of the IL-1b gene in a rheumatoid synovial cell line (Koyama et al. 1996). Interestingly, ICAM-1 ligation also results in upregulation of adhesion molecules. ICAM-1 crosslinking, on human umbilical vein endothelial cells, caused expression of vascular cell adhesion molecule-1 (VCAM1) with activation of ERK-1 and transcription factor complex, but without any increase in NF-kB activity (Lawson et al. 1999). Clayton et al. (1998) reported de novo synthesis of ICAM-1 and VCAM-1 mRNA upon ICAM-1 and VCAM-1 crosslinking with a concomitant elevation of the cytosolic free Ca21, with a resultant increase of leukocyte adhesion to fibroblasts and endothelial cells. It is quite plausible that ICAM-1 ligation on one cell may result in transactivating adjacent cells, thereby contributing to the maintenance of the inflammatory response. The transactivation of endothelial cells through stimulated platelets has been likewise reported to cause an increase in levels of ICAM-1 and monocyte chemotactic protein-1 (Gawaz et al. 1998). • Tyrosine Phosphorylation Mediated through ICAM-1 Tyrosine phosphorylation of intracellular proteins is a key event for signal TCM Vol. 12, No. 3, 2002
transduction (Neel and Tonks 1997, Aplin et al. 1999). Fg:ICAM-1 ligation causes phosphorylation of two of the three tyrosines (tyrosines 474 and 485) within the cytoplasmic sequence of ICAM-1 (Pluskota et al. 2000, Pluskota and D’Souza 2002). ICAM-1 ligation in brain microvessel endothelial cells, mediated through antibody crosslinking, induced phosphorylation of the cytoskeletonassociated proteins cortactin (a substrate of Src kinase), focal adhesion kinase, paxillin, and the adaptor protein p130Cas (Durieu-Trautman et al. 1994, Etienne et al. 1998). The activation of the small GTP-binding protein, Rho, resulted in the facilitation of lymphocyte migration through Transwells (Adamson et al. 1999). The Rho-dependent leukocyte migration was abrogated when ICAM-1 cytoplasmic sequence was deleted (Sans et al. 2001). A direct in vivo evidence for this premise, however, is yet to be obtained. Tyrosine phosphorylation of cortactin, an actin-binding protein, occurs in endothelial cells upon Fg:ICAM-1 ligation (Harley and Powell 2000, Pluskota et al. 2000). For the transmigration of leukocytes through the endothelium, Rho activation and cortactin phosphorylation may act in concert to regulate cytoskeletal reorganization— that occurs upon engagement of the extracellular domain of ICAM-1—that is very likely dependent on the phosphorylation status of the cytoplasmic sequence of ICAM-1. The mitogen-activated protein kinase (MAPK)/ERK pathway plays a pivotal role in cell proliferation, migration, differentiation, and gene induction, and ERK1/2 phosphorylation is involved in a variety of cellular activities. Fg:ICAM-1 ligation, in Raji B-cells and in endothelial cells, resulted in ERK1/2 and pp60src phosphorylation, indicating activation of the classical MAPK cascade (Gardiner and D’Souza 1999, Pluskota and D’Souza 2000). Studies by Holland and Owens (1997) have reported the activation of the Src-related kinase Lyn in B-cells. ERK1/2 phosphorylation occurred within the first 5 min of ligation and persisted up to 60 min. By 120 min, ERK1/2 was completely dephosphorylated. This activation was mimicked upon Fg-g(117– 133) ligation to ICAM-1 and ICAM-1(8– 21) peptide blocked ERK1/2 activation. Fg:ICAM-1 ligation promotes endothelial cell survival and ERK1/2 activation TCM Vol. 12, No. 3, 2002
appears to be essential because survival was dramatically reduced in cells transfected with dominant-negative mutations within ERK-1/2. MAPK kinase (MEK) inhibitor PD98059 and pp60src inhibitors geldanamycin and herbimycin abrogated the survival and proliferative response, indicating involvement of these kinases in the Fg:ICAM-1 functions (Gardiner and D’Souza 1999). Moreover, studies indicate Fg:ICAM-1 ligation promotes endothelial cell survival and prevents TNFa-induced apoptosis through activation of the Bcl family of the survival factors, A1 (Pluskota and D’Souza 2000). In contrast to the positive survival signals through ICAM-1 in endothelial cells, crosslinking of ICAM-1 induced transient tyrosine phosphorylation and inactivation of cdc2 kinase on Tcells (Chirathaworn et al. 1995) that may be related to apoptosis through arrest of cell cycle. • Proteins Directly Associating with ICAM-1 Cytoplasmic Sequence The emerging paradigm is that ICAM-1 interacts with the actin cytoskeleton to form structural framework “scaffolds,” allowing efficient signal transduction to occur. In agreement with this concept, inhibitors of microtubule assembly and actin polymerization (nocodazole and cytochalasin, respectively) inhibited ERK1/2 phosphorylation, indicating that intact cytoskeleton architecture is required for ERK1/2 signaling (Pluskota and D’Souza 2000). Physical association of the ICAM-1 cytoplasmic tail with the actin cytoskeleton has been documented (Carpen et al. 1992). In studies by Pluskota et al. (2000), Fg:ICAM-1 ligation on endothelial cells resulted in tyrosine phosphorylation of ICAM-1 and of Src homology domain 2 (SH2) containing phosphatase-2 (SHP-2). In immunoprecipitation experiments, phosphorylated ICAM-1 coprecipitated with phosphorylated SHP-2 and vice versa. SHP-2, a widely expressed cytosolic protein tyrosine phosphatase involved in promoting mitogenic signals, is a mammalian homologue of the gene product of Drosophila corkscrew (Feng 1999, Stein-Gerlach et al. 1998). SHP-2 associates with phosphorylated cytoplasmic regions of several membranebound receptors, through the sequence termed immunoreceptor tyrosine-based inhibition motif (ITIM). SHP-2 is largely
a positive regulator involved also in promoting mitogenic signals. SHP-2 also serves as a scaffolding protein mediating the assembly of Grb2, which is bound to SOS and promotes activation of Ras, initiating the classical Raf-1/ MEK-1/ERK pathway. Activation of these transduction pathways finally results in the activation of the transcription factors and modulation of gene expression. In endothelial cells, SHP-2 phosphorylation follows a time course similar to that seen with ERK-1/2. SHP-2 is phosphorylated at early stages of ICAM-1 ligation (5 to 30 min) and becomes completely dephosphorylated by 2 h. The cytoplasmic tail of ICAM-1 (PKIKK485YRLQ) conforms poorly to the consensus ITIM sequence (I/V/L)XYXX(L/ V). This sequence in ICAM-1, however, binds directly to SHP-2 (Pluskota et al. 2000). In fact, the NH2-terminal SH2 domain of SHP-2 associates with the sequence surrounding phosphotyrosine at 485. Substitution of tyrosine 485 to alanine (Y485A) results in loss of ICAM-1 binding to SHP-2. Moreover, 293 cells expressing ICAM-1 (Y485A) have a reduced survival capacity compared to wildtype ICAM-1 upon adhesion of the cells to Fg. More recently, our results indicate that tyrosine 474 at the membrane proximal region of ICAM-1 also becomes phosphorylated. The sequence surrounding Y474 poorly resembles the consensus ITIM, yet this phosphopeptide is capable of directly associating with SHP-2. Interestingly, Y474 seems to specifically bind to carboxyl SH2 domain in SHP-2. Mutational studies suggest that 293 cells expressing ICAM-1 (Y474A) are also defective in maintaining cellular survival upon Fg ligation (Pluskota and D’Souza 2002; Figure 3). Moreover, mutational studies indicate that tyrosine 476 is either not phosphorylated or weakly phosphorylated upon Fg:ICAM1 ligation, and therefore shows no association with SHP-2 and is not involved in ICAM-1–mediated cellular survival. Several questions remain unanswered in our understanding of intracellular signals from ICAM-1, including the identity of the kinase involved in phosphorylating ICAM-1, the mechanism by which ICAM-1 is dephosphorylated, and if, in fact, the phosphatase SHP-2 is involved in ICAM-1 dephosphorylation. The key question is to relate the kinetics of ICAM-1 phosphorylation and its sig-
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nals in specific function of Fg:ICAM-1 interactions. • Conclusion
Figure 3. Signaling mediated through ICAM-1:Fg interaction. ICAM-1 is tyrosine phosphorylated upon ligation with Fg. Phosphorylated ICAM-1 associates with phosphatase SHP-2, which also becomes phosphorylated. The C-terminal SH2 domain of SHP-2 interacts with ICAM-1 through the ITIM (shown in bold circles) that encompasses phosphotyrosine 474. The N-terminal SH2 domain interacts with ITIM within phosphotyrosine 485, thus initiating signaling cascade, that promotes cell survival. SHP2 phosphorylation is followed by the activation of Ras, Raf-1/Mek/ERK-1 transduction pathways and finally, activation of the transcription factors (c-fos, c-jun, Elk-1, etc) and modulation of gene expression. Altogether these reactions promote cell survival.
Fg:ICAM-1 engagement on the vascular cells promotes capture and localization of circulating platelets and inflammatory cells, transmigration of leukocytes, and deposition of Fg, and intriguingly is also involved in cellular survival. Functional importance of Fg:ICAM-1–mediated reactions have been largely demonstrated in in vitro studies. There is now emerging evidence from in vivo animal models (Massberg et al. 1999, Sriramarao et al. 1996) and to some extent in vessel contractility studies (Hicks et al. 1996). The increased expression of ICAM-1 and accumulation of Fg reported in a number of cardiovascular and inflammatory disorders could manifest some of the functional effects of Fg:ICAM-1 interaction. Figure 4 provides a framework for the vascular events mediated through this ligation. Another important aspect is mediated via the recently reported interaction of FgBb(15–42) with VE-cadherin, which results in upregulation of ICAM-1 expression. This pathway may be considered a self-sustaining positive feedback loop, promoting Fg:ICAM-1– mediated reactions. FgBb(15–42) is exposed as a result of Fg proteolysis by thrombin, uPA and/or tPA. FgBb(15– 42):VE-cadherin interaction may be activated whenever blood coagulation and/
Figure 4. Schematic summary of Fg:ICAM-1 mediated reactions. Increased expression of ICAM-1 and accumulation of Fg is known to occur in vascular diseases and inflammation. Fgg(117–133):ICAM-1(8–22) ligation promotes adhesion of circulating platelets and leukocytes to endothelial cells (EC) and transendothelial migration of leukocytes, deposition of Fg on endothelial cells, prevents endothelial cell apoptosis, and elicits direct vasomotor responses. Interaction of FgBb(15–42) with VE-cadherin enhances expression of ICAM-1, thus creating a feedback loop. These molecular events promote ongoing inflammation and vascular damage.
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or fibrinolysis pathways are induced and may have important clinical implications in cardiovascular diseases and in conditions such as disseminated intravascular coagulation; and may also be considered in therapeutic interventions, such as thrombolytic therapy. Similarly, ICAM-1 mediates Fg deposition on the endothelium, while Fg is capable of inducing ICAM-1 expression. This will result in the triggering of multiple pathophysiological effects mediated through ICAM-1, Fg, and/or ICAM-1:Fg interaction. Fibrin(ogen) or its derivatives stimulate release of uPA and tPA from vascular endothelium, thus creating a vicious circle. These reactions contribute to the complex interplay of mechanisms inducing transformation of endothelium into the proinflammatory and prothrombotic surface that promote ongoing inflammation, thrombogenesis, atherogenesis, stenosis and occlusion of the vessel.
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VEGF Gene Delivery for Treatment of Ischemic Cardiovascular Disease Mark L. Koransky, Robert C. Robbins, and Helen M. Blau*
There are increasing numbers of patients with ischemic myocardial disease not amenable to traditional methods of revascularization. These patients may benefit from new research into the use of naturally occurring angiogenic compounds, such as vascular endothelial growth factor (VEGF) for re-establishing blood flow into regions of hibernating myocardium. Animal studies and human clinical trials evaluating VEGF demonstrate increases in myocardial perfusion after treatment, with some patients reporting improvement in anginal symptoms. Further research into the ideal form of VEGF therapy (protein, plasmid, or adenoviral) and delivery method (intracoronary, intramyocardial, or epicardial) seems justified. (Trends Cardiovasc Med 2002;12:108–114) © 2002, Elsevier Science Inc.
Percutaneous transluminal angioplasty (PTCA) and stenting or operative coronary revascularization (coronary artery bypass grafting, CABG) are procedures that effectively re-establish blood flow to distal myocardial vascular beds and pro-
vide relief of angina for most patients with lesions from coronary artery disease. However, percutaneous interventions are plagued by high rates of restenosis and bypass grafts are subject to progressive atherosclerotic occlusion.
Mark L. Koransky is at the Department of Cardiothoracic Surgery and the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California. Robert C. Robbins is at the Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California. Helen M. Blau is at the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California. * Address correspondence to: Helen M. Blau, Department of Microbiology and Immunology, Stanford University Medical Center, 269 Campus Drive, CCSR 4215a, Stanford, CA 943055175, USA. Tel.: (+1) 650-723-6270; fax: (+1) 650-736-0080; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
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Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation 97: 1708–1715.
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Tyagi S, Campbell SE, Reddy HK, et al.: 1996a. Matrix metalloproteinase activity expression in infarcted, noninfarcted and dilated cardiomyopathic human hearts. Mol Cell Biochem 155:13–21.
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man matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J Clin Invest 107:1227–1234.
Matrisian LM: 1990. Metalloproteinases and their inhibitors in matrix remodeling. Trends in Genetics 6:121–125. Montfort I, Perez-Tamayo R: 1975. The distribution of collagenase in normal rat tissues. J Histochem Cytochem 23:910–920. Ohuchi E, Imai K, Fujii Y, et al.: 1997. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem 272:2446–2451. Peterson JT, Hallak H, Johnson L, et al.: 2001. Matrix metalloproteinase inhibition attenuates left ventriclar remodeling and dysfunction in a rat model of progressive heart function. Circulation 103:2303–2309. Peterson JT, Li H, Dillon L, Bryant JW: 2000. Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat. Cardiovascular Res 46:307–315.
Uitto J, Perejda AJ: 1987. Molecular pathology of the extracellular matrix. New York, Marcel Dekker. Vincenti MP, Coon CI, Mengshol JA, et al.: 1998. Cloning of the gene for interstitial collagenase-3 (matrix metalloproteinase-13) from rabbit synovial fibroblasts: differential expression with collagenase-1 (matrix metalloproteinase-1). Biochem J 331: 341–346. Weber KT: 1989. Cardiac interstitium in health and disease: The fibrillar collagen network. J Am Coll Cardiol 13:1637–1652.
Weber KT, Janicki JS, Shroff SG, et al.: 1988b. Collagen remodeling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res 62: 757–765. Weber K, Pick R, Janicki JS, et al.: 1988a. Inadequate collagen tethers in dilated cardiopathy. Am Heart J 116:1641–1646. Weber KT, Pick R, Silver MA, et al.: 1990. Fibrillar collagen and remodeling of dilated canine left ventricle. Circulation 82:1387–1401. Woodwiss AJ, Tsotetsi OJ, Sprott S, et al.: 2001. Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circ 103:155–160. PII S1050-1738(01)00160-8
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Interactions of Intercellular Adhesion Molecule-1 with Fibrinogen
Robert V, Besse S, Sabri A, et al.: 1997. Differential regulation of matrix metalloproteinases associated with aging and hypertension in the rat heart. Lab Invest 76:729–738.
Nina L. Tsakadze, Zhendong Zhao, and Stanley E. D’Souza*
Rohde LE, et al.: 1999. Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation 99:3063–3070.
The binding of plasma protein fibrinogen (Fg) to intercellular adhesion molecule-1 (ICAM-1) on endothelial cells mediates the attachment of leukocytes and platelets that may result in vascular occlusion. Fg:ICAM-1 interactions elicit an array of effects that could have implications in vascular pathology and inflammation. ICAM-1 expression is regulated during inflammation and upon Fg binding. The mechanistic model presented provides a framework to delineate the consequences of Fg binding to ICAM-1. (Trends Cardiovasc Med 2002;12:101–108) © 2002, Elsevier Science Inc.
Spinale F, Coker ML, Heung LJ, et al.: 1999. Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res 85:364–376. Spinale F, Coker ML, Heung LJ, et al.: 2000. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation 102:1944–1949. Spinale FG, Zellner JL, Johnson WS, et al.: 1996. Cellular and extracellular remodeling with the development and recovery from tachycardia-induced cardiomyopathy: changes in fibrillar collagen, myocyte adhesion capacity and proteoglycans. J Mol Cell Cardiol 28:1591–1608. Takahashi S, Barry AC, Factor SM: 1990. Collagen degradation in ischaemic rat hearts. Biochem J 265:233–241. Thomas CV, Coker ML, Zellner JL, et al.: 1998.
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Nina L. Tsakadze, Zhendong Zhao, and Stanley E. D’Souza are from the Department of Physiology & Biophysics, University of Louisville, Louisville, Kentucky, USA. * Address correspondence to: Stanley E. D’Souza, Department of Physiology & Biophysics, A-1101 Health Sciences Center, University of Louisville, Louisville, KY 40292, USA. Tel.: (11) 502-852-3194; fax: (11) 502-8526239; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
Several reviews deal with the structural and functional aspects of intercellular adhesion molecule-1 (ICAM-1) and their role in immune and adhesive interactions (Carlos and Harlan 1994, Springer 1994, Van de Stolpe and Van der Saag 1996). This article focuses on the binding of ICAM-1 to plasma protein fibrinogen (Fg) and its derivatives. The intracellular signals, generated through this interaction, and their functional effect
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on the vascular endothelium is discussed. On this subject, the review by Altieri (1999) is highly informative. • Structural Features and Expression of ICAM-1 ICAM-1 (CD 54)—a 90- to 110-kDa cell surface glycoprotein expressed on monocytes, T and B lymphocytes, macrophage and fibroblasts, epithelial cells, and endothelial cells, plays an important role in immune-mediated cell–cell adhesive interactions (Springer 1994). ICAM-1 is an integral membrane protein (Figure 1) with an extracellular region composed of five immunoglobulin (Ig)-like domains, a transmembrane region, and a short cytoplasmic tail (Staunton et al. 1988). The Ig domains create a rigid structural framework, while a flexible bend (“hinge”) appearing between the second and third domains provides molecular flexibility, maximizing the accessibility of domains 1 and 2 (Wang and Springer 1998). Although low under basal conditions, the expression of ICAM-1 can be induced to higher levels by inflammatory mediators, such as tumor necrosis factor a (TNFa), interleukin (IL)-1b, IL-4, and interferon g, as well as by shear stress. Glucocorticoids have an inhibitory effect on ICAM-1 expression. The induced cell surface expression is the result of de novo mRNA transcription and translation, but mobilizable intracellular pools of ICAM-1 have been identified in monocytes. ICAM-1 can be detected 4 hours after the induction, becomes maximal within 16–24 hours, and remains high for about 48 hours. • Functional Role of ICAM-1 ICAM-1 is a ligand for leukocyte integrins lymphocyte function-associated antigen-1 (LFA-1; aLb2) and Mac-1 (aMb2),
and provides a mechanism for leukocyte–endothelial interaction and for the migration of leukocytes to the sites of inflammation. LFA-1 binds to the first and second, and Mac-1 to the third, domain of ICAM-1. Human ICAM-1 also is a receptor for the rhinovirus, coxsackie A13 virus, and Plasmodium falciparum malaria-infected erythrocytes (Springer 1994, Van de Stolpe and Van der Saag 1996), and thus facilitates viral and parasite invasion. Ligand binding avidity toward ICAM-1 depends on the presence of the divalent cations and is probably mediated through the conformational changes of the molecule. ICAM-1 is heavily glycosylated and variability in carbohydrate content of ICAM-1 of different cell types suggests glycosylation as a possible mechanism for affinity regulation, presumably by shielding the ligand-binding epitope. The main function of ICAM-1 is to promote intercellular communication through cell adhesion. The ICAM-1– dependent adhesion plays an important role in both afferent and efferent immune responses, including antigen presentation, T-cell–dependent antibody responses, T-cell proliferation and cellmediated cytotoxicity, and leukocyte attachment and migration through endothelial cell monolayers. ICAM-1 plays a costimulatory role on antigen-presenting cells in major histocompatibility complex (MHC) class II restricted T-cell activation and in cell-mediated cytotoxicity by activation of non-MHC– restricted killer cells and MHC class I restricted T-lymphocytes. ICAM-1– deficient mice are viable, but display an impaired inflammatory reaction; however, ICAM-1 deficiency has been demonstrated to be protective against lethal septic shock and ischemia/reperfusion injury (Sligh et al. 1993).
Figure 1. The structural boundaries of the extracellular, transmembrane, and cytoplasmic domains of ICAM-1.
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• ICAM-1 Expression in Diseases Increased levels of ICAM-1 have been reported in a variety of conditions characterized by local or generalized inflammation—including atherosclerosis, ischemia and reperfusion, stroke, malignancies, and autoimmune disorders—and suggest a role for ICAM-1 in the clinical evolution of disease. Low expression of ICAM-1 on tumor cells is associated with a reduced in vitro sensitivity to lymphocyte cytotoxicity, correlating with more aggressive clinical course, and a higher risk of metastasis. Inflammatory reactions in animal models have been down-regulated by the monoclonal antibodies directed against ICAM-1.
• Soluble ICAM-1 The ICAM-1 ectodomain undergoes proteolytic cleavage releasing soluble ICAM-1 from the cell surface. Matrix metalloproteinases (MMP) are likely to be involved in the cleavage process, as synthetic inhibitors of MMP-reduced soluble ICAM-1 in cell culture medium. Elevated levels of soluble ICAM-1 occur in vascular disease and appear to parallel the severity of the disease process. Soluble ICAM-1 may compete with membrane-bound form for the binding to its ligands and thus may be capable of modulating ICAM-1–mediated inflammatory responses. In this respect, it is interesting to note that gene polymorphism at residue 469 in the fifth Ig-like domain has been linked to inflammatory bowel diseases (Yang 1997), probably interfering with the rate of ICAM-1 ectodomain cleavage.
• Fg: A ligand for ICAM-1 More recently, the plasma protein Fg has been demonstrated to serve as a ligand for ICAM-1. Fg:ICAM-1 interaction was initially shown to promote bridging of monocytic cells to endothelium (Languino et al. 1993). Fg (340 kDa), a plasma glycoprotein composed of three pairs of disulfide-bonded polypeptide chains (Aa, Bb, and g; Figure 2), plays a key role in blood coagulation, inflammation, and tissue repair (Doolittle 1984). Fg binds to integrin aIIbb3 on activated platelets, promoting platelet adhesion and aggregation, thus playing a central role in primary hemoTCM Vol. 12, No. 3, 2002
Figure 2. Structure of fibrinogen. FpA and FpB, fibrinopeptides A and B.
stasis. Fg is spatially arranged into three globular domains: a central E domain and two peripheral D domains. Under the action of thrombin, fibrinopeptides A and B are cleaved from the amino-terminal of chains Aa and Bb of Fg, with a resultant polymerization into fibrin. Release of FpA and FpB exposes the A and B polymerization sites, respectively, that function cooperatively in the self-assembly process. The human polymerization site located at the aminoterminal end of the fibrin a chain (Aa 17–20) contains the sequence gly-proarg-val, while that located in the aminoterminal region of the fibrinogen Bb chain, in the close proximity to position 14, contains the sequence gly-his-argpro. The synthetic peptide gly-pro-argpro, which encompasses both these sequences, indeed binds to both the sites and prevents polymerization of fibrin monomer (Mosesson 1990). During fibrinolysis, fibrin(ogen) is proteolytically cleaved by plasmin, releasing fibrinogen degradation products (fragments D and E). Generation of fibrin and Fg-dependent formation of platelet plugs are essential components of both normal hemostasis and occlusive vascular diseases such as atherosclerosis and thrombosis. Accumulating evidence, however, suggests much broader physiological and pathophysiological roles for Fg than simply triggering blood coagulation and thrombosis. • Effect of Fg and Its Derivatives on the Vessel Wall Function Fg accumulation, at the site of tissue injury, occurs in a variety of pathologic processes, including inflammation, ischemia, trauma, and neoplasm. As an acute phase reactant, Fg readily responds to inflammatory stimuli. Plasma Fg level TCM Vol. 12, No. 3, 2002
is a strong and consistent predictor of cardiovascular events in individuals with or without clinically manifested vascular disease. Increased plasma Fg is associated with a significantly increased risk of myocardial infarction, stroke, peripheral arterial disease, atrial fibrillation, heart failure, and restenosis following angioplasty (Ernst and Resch 1993, Lowe 1997). There are several interactive, plausible, potential biological mechanisms by which Fg might be involved in cardiovascular disease pathogenesis, apart from its effects on blood coagulation and blood viscosity. Fibrin(ogen) and its degradation products can display mitogenic (Levesque et al. 1986) and angiogenic (Thompson et al. 1992) effects on endothelial cells, and increase endothelial cell permeability and detachment (Ge et al. 1992). It can stimulate the release of growth factors (Lorenzet et al. 1992), tissue plasminogen activator (t-PA), prostacycline (Kaplan et al. 1989), urokinase plasminogen activator (uPA) and von Willebrand factor (Ribes et al. 1989). • Structural Basis of the Fg:ICAM-1 Recognition The recognition sequences located on the (chain of Fg and within the first Ig-like domain of the ICAM-1 mediate Fg:ICAM-1 interaction. Altieri et al. (1995), utilizing synthetic peptides, identified amino-acid sequence 117–133 on the g chain of Fg as the region involved in binding to ICAM-1. Peptide g (117– 133) (117NQKIVNLKEKVAQLEA133), designated g3, blocked Fg-mediated adhesion of leukocytes to ICAM-1–expressing cells. Amino-acids encompassing sequence 8–21 within the first Ig domain of the ICAM-1 (8KVILPRGGSVLVTC21) interacts with intact Fg and with fragment D (fragment D contains the Fgg(117–133) sequence). ICAM-1(8–21) inhibited adhesion of Fg to ICAM-1– expressing lymphoid cells (D’Souza et al. 1996). Duperray et al. (1997) verified the location of the Fg binding epitope within the first domain of ICAM-1 using mutational analyses and a panel of inhibitory anti–ICAM-1 antibodies. In this analysis, however, aspartic acid (D26) and proline (P70) residues within ICAM-1 were determined to be critical for Fg binding. Therefore, there is a discrepancy between the sites identified by
D’Souza et al. (1996) and Duperray et al. (1997), even though they lie within the first domain of ICAM-1. It is quite likely that the critical residues in the sites reported are proximal to each other. Nevertheless, this discrepancy warrants further clarification. It is quite possible, that conformational changes of the Fg molecule may be required in order to expose g3 sequence for Fgg(117– 133):ICAM-1(8–21) interaction to occur. Altieri et al. (1995) proposed that the exposure of the g3 site may occur upon Fg binding to Mac-1 subsequent to Mac-1 activation by inflammatory stimuli. On the other hand, exposure of ICAM-1(8–21) may be regulated by initial low-affinity Fg binding sites on endothelial cells. These may be regulatory mechanisms limiting the Fg:ICAM-1 pathway activation under physiologic conditions. • Cellular Bridging through ICAM-1:Fg Increased levels of plasma Fg often coexist with an upregulated ICAM-1 expression in a number of cardiovascular disorders and are essential components of the inflammatory reaction. The mechanism for Fg:ICAM-1 interaction and their mutually potentiating interrelationship is now emerging. Fg functions as a coupling molecule between Mac-1 on leukocytes and ICAM-1 on endothelial cells, thus providing an alternative mechanism for their interaction and enhancing adhesion and migration of leukocytes (Languino et al. 1993 and 1995). Sriramarao et al. (1996), using intravital microscopy in rabbit mesentery, demonstrated that Fg induced a dose-dependent increase in the adhesion of monocytes to the vessel wall. The specificity of this cellular interaction was demonstrated by blockage in the presence of ICAM-1 monoclonal antibodies or Fgg(117–133) peptide. Interestingly, the adhesion was dependent on shear forces and occurred in venules, but not in arterioles or capillaries. These studies were the initial indication that cellular bridging mediated through Fg:ICAM-1 may be physiologically applicable. Sans et al. (2001), using Chinese hamster ovary cells expressing various constructs of ICAM-1, demonstrated that ICAM-1, through its interactions with Fg, is independently capable of mediating leukocyte transmigration in Transwell assays. Given these obser-
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vations, in experiments using intravital microscopy, one could speculate that ICAM-1–deficient mice may completely lack the capacity for Fg-dependent monocyte migration. Fg:ICAM-1 bridging is implicated in the adhesion of activated platelets to endothelial cells (Bombeli et al. 1998). In these studies, platelet adhesion to endothelial cells was markedly reduced in the presence of monoclonal antibodies to ICAM-1, aVb3, and adhesive proteins, including Fg. Fg deposition on endothelium has been demonstrated by intravital microscopy in a model of intestinal ischemia/reperfusion in mice (Massberg et al. 1999). Deposition of Fg induced platelet recruitment and adhesion to postischemic endothelium that was attenuated in the presence of antiFg antibodies and demonstrated to be integrin aIIb/b3 mediated. Fg deposition in postischemic reperfusion was markedly decreased in ICAM-1–deficient mice. • Other Functions Implicating Fg:ICAM-1 Interaction Fg ligation to ICAM-1 elicits vasomotor responses in the human saphenous veins (vasodilatation followed by vasoconstriction at higher concentrations), perhaps through the production of vasoactive mediators (Hicks et al. 1996). Fg-mediated relaxation was inhibited in the presence of anti–ICAM-1 antibodies and K1 channel blockers. Indomethacin and Nv-nitro-L-arginine methyl ester, inhibitors of endothelium-derived vasodilators (prostacycline and nitric oxide, respectively), did not significantly affect Fg-mediated relaxation. The vasomotor response was increased after exposure of the vessel to the arterial flow and correlated with the enhanced expression of ICAM-1 under conditions of high shear stress. Fg is known to induce mitogenesis (Levesque et al. 1986). Fg was found to induce proliferation of B-lymphoid Raji cells mediated through Fg:ICAM-1 interaction (Gardiner and D’Souza 1997). Similar mitogenic response was elicited by Fgg(117–133) and was blocked by ICAM-1(8–22) peptide. The phosphorylation of pp60Src and extracellular signalregulated kinase (ERK-1/2) appear to regulate mitogenic signals (Gardiner and D’Souza 1999). These signals also mediate endothelial cell survival and prevent
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TNFa-induced apoptosis (Pluskota and D’Souza 2000). We have observed, however, that proliferative response in endothelial cells is mediated mainly though Fg interaction with aVb3 integrin. Fg:ICAM-1 interaction plays only a minor role, even under conditions wherein ICAM-1 was upregulated in the presence of TNFa. This is likely due to the apoptotic effect of TNFa that may counteract the proliferative effect of Fg:ICAM-1 ligation. It has been suggested that release of vasoactive mediators coupled with the mitogenic response promoted through Fg may have a role in the causation of vascular diseases (Hicks et al. 1996). Interestingly, apart from the proinflammatory cytokine-induced ICAM-1 expression, fibrin and Fg itself have also been shown to upregulate ICAM-1 on the endothelial cells and human synovial fibroblasts (Harley et al. 2000, Liu and Piela-Smith 2000, Qi et al. 1997) perhaps due to FgBb15–42 ligation to the endothelial cadherin (VE-cadherin; Harley et al. 2000). VE-cadherin mediates homotypic cell adhesion and formation of the adherens junctions (Dejana et al. 1999). VE-cadherin has been identified as a receptor for the Bb15–42 sequence on the N-terminus of fibrin (Bach et al. 1998). For this interaction to occur, Bb15–42 needs to be exposed as a result of proteolytic degradation of fibrin(ogen) and cleavage of fibrinopeptide B (Bb1–14) by thrombin, uPA, or tPA. Interestingly, exposure of the FgBb15–42 sequence has a mitogenic effect on endothelial cells (Sporn et al. 1995). In synovial fibroblasts, fibrin(ogen)-induced ICAM-1 upregulation was regulated through the transcription factor nuclear factor-kB (NF-kB) that also caused an increase in IL-8 production. The increases in IL-8 and ICAM-1 may result in the recruitment of lymphocytes and in sustaining ongoing inflammation (Liu and Piela-Smith 2000). Interestingly, when incubated with endothelial cells, activated platelets (but not resting platelets) also promoted ICAM-1 expression, secretion of monocyte chemotactic protein-1, and activation of the transcription factor NF-kB (Gawaz et al. 1998). Other sequences within Fg or fibrin molecules exposed during fibrinogenesis and/or fibrin polymerization may be capable of increasing ICAM-1 expression (Liu and Piela-Smith 2000). This at-
tributes a regulatory role to fibrin(ogen), which may expose neo-epitopes and trigger different molecular events depending on the environmental stimuli. Taken together, these studies suggest that Fg plays a myriad role—directly or through cellular bridging—in generation and maintenance of inflammatory processes, and provides a biochemical basis for the long-recognized link between activation of coagulation and inflammation. • Signaling through ICAM-1 Several reports indicate the participation of ICAM-1 in signal transduction. ICAM-1 crosslinking with anti–ICAM-1 antibodies results in an oxidative burst in mononuclear leukocytes in the presence of the bacterial proinflammatory peptide N-formyl-met-leu-phe (Rothlein et al. 1994), plays a co-stimulatory role in the expression of IL-2 receptors in B-cells (Poudrier and Owens 1994), induces an increase in intracellular calcium in Burkitt lymphoma (van Horssen et al. 1995), and activates transcription factor AP-1 and the transcription of the IL-1b gene in a rheumatoid synovial cell line (Koyama et al. 1996). Interestingly, ICAM-1 ligation also results in upregulation of adhesion molecules. ICAM-1 crosslinking, on human umbilical vein endothelial cells, caused expression of vascular cell adhesion molecule-1 (VCAM1) with activation of ERK-1 and transcription factor complex, but without any increase in NF-kB activity (Lawson et al. 1999). Clayton et al. (1998) reported de novo synthesis of ICAM-1 and VCAM-1 mRNA upon ICAM-1 and VCAM-1 crosslinking with a concomitant elevation of the cytosolic free Ca21, with a resultant increase of leukocyte adhesion to fibroblasts and endothelial cells. It is quite plausible that ICAM-1 ligation on one cell may result in transactivating adjacent cells, thereby contributing to the maintenance of the inflammatory response. The transactivation of endothelial cells through stimulated platelets has been likewise reported to cause an increase in levels of ICAM-1 and monocyte chemotactic protein-1 (Gawaz et al. 1998). • Tyrosine Phosphorylation Mediated through ICAM-1 Tyrosine phosphorylation of intracellular proteins is a key event for signal TCM Vol. 12, No. 3, 2002
transduction (Neel and Tonks 1997, Aplin et al. 1999). Fg:ICAM-1 ligation causes phosphorylation of two of the three tyrosines (tyrosines 474 and 485) within the cytoplasmic sequence of ICAM-1 (Pluskota et al. 2000, Pluskota and D’Souza 2002). ICAM-1 ligation in brain microvessel endothelial cells, mediated through antibody crosslinking, induced phosphorylation of the cytoskeletonassociated proteins cortactin (a substrate of Src kinase), focal adhesion kinase, paxillin, and the adaptor protein p130Cas (Durieu-Trautman et al. 1994, Etienne et al. 1998). The activation of the small GTP-binding protein, Rho, resulted in the facilitation of lymphocyte migration through Transwells (Adamson et al. 1999). The Rho-dependent leukocyte migration was abrogated when ICAM-1 cytoplasmic sequence was deleted (Sans et al. 2001). A direct in vivo evidence for this premise, however, is yet to be obtained. Tyrosine phosphorylation of cortactin, an actin-binding protein, occurs in endothelial cells upon Fg:ICAM-1 ligation (Harley and Powell 2000, Pluskota et al. 2000). For the transmigration of leukocytes through the endothelium, Rho activation and cortactin phosphorylation may act in concert to regulate cytoskeletal reorganization— that occurs upon engagement of the extracellular domain of ICAM-1—that is very likely dependent on the phosphorylation status of the cytoplasmic sequence of ICAM-1. The mitogen-activated protein kinase (MAPK)/ERK pathway plays a pivotal role in cell proliferation, migration, differentiation, and gene induction, and ERK1/2 phosphorylation is involved in a variety of cellular activities. Fg:ICAM-1 ligation, in Raji B-cells and in endothelial cells, resulted in ERK1/2 and pp60src phosphorylation, indicating activation of the classical MAPK cascade (Gardiner and D’Souza 1999, Pluskota and D’Souza 2000). Studies by Holland and Owens (1997) have reported the activation of the Src-related kinase Lyn in B-cells. ERK1/2 phosphorylation occurred within the first 5 min of ligation and persisted up to 60 min. By 120 min, ERK1/2 was completely dephosphorylated. This activation was mimicked upon Fg-g(117– 133) ligation to ICAM-1 and ICAM-1(8– 21) peptide blocked ERK1/2 activation. Fg:ICAM-1 ligation promotes endothelial cell survival and ERK1/2 activation TCM Vol. 12, No. 3, 2002
appears to be essential because survival was dramatically reduced in cells transfected with dominant-negative mutations within ERK-1/2. MAPK kinase (MEK) inhibitor PD98059 and pp60src inhibitors geldanamycin and herbimycin abrogated the survival and proliferative response, indicating involvement of these kinases in the Fg:ICAM-1 functions (Gardiner and D’Souza 1999). Moreover, studies indicate Fg:ICAM-1 ligation promotes endothelial cell survival and prevents TNFa-induced apoptosis through activation of the Bcl family of the survival factors, A1 (Pluskota and D’Souza 2000). In contrast to the positive survival signals through ICAM-1 in endothelial cells, crosslinking of ICAM-1 induced transient tyrosine phosphorylation and inactivation of cdc2 kinase on Tcells (Chirathaworn et al. 1995) that may be related to apoptosis through arrest of cell cycle. • Proteins Directly Associating with ICAM-1 Cytoplasmic Sequence The emerging paradigm is that ICAM-1 interacts with the actin cytoskeleton to form structural framework “scaffolds,” allowing efficient signal transduction to occur. In agreement with this concept, inhibitors of microtubule assembly and actin polymerization (nocodazole and cytochalasin, respectively) inhibited ERK1/2 phosphorylation, indicating that intact cytoskeleton architecture is required for ERK1/2 signaling (Pluskota and D’Souza 2000). Physical association of the ICAM-1 cytoplasmic tail with the actin cytoskeleton has been documented (Carpen et al. 1992). In studies by Pluskota et al. (2000), Fg:ICAM-1 ligation on endothelial cells resulted in tyrosine phosphorylation of ICAM-1 and of Src homology domain 2 (SH2) containing phosphatase-2 (SHP-2). In immunoprecipitation experiments, phosphorylated ICAM-1 coprecipitated with phosphorylated SHP-2 and vice versa. SHP-2, a widely expressed cytosolic protein tyrosine phosphatase involved in promoting mitogenic signals, is a mammalian homologue of the gene product of Drosophila corkscrew (Feng 1999, Stein-Gerlach et al. 1998). SHP-2 associates with phosphorylated cytoplasmic regions of several membranebound receptors, through the sequence termed immunoreceptor tyrosine-based inhibition motif (ITIM). SHP-2 is largely
a positive regulator involved also in promoting mitogenic signals. SHP-2 also serves as a scaffolding protein mediating the assembly of Grb2, which is bound to SOS and promotes activation of Ras, initiating the classical Raf-1/ MEK-1/ERK pathway. Activation of these transduction pathways finally results in the activation of the transcription factors and modulation of gene expression. In endothelial cells, SHP-2 phosphorylation follows a time course similar to that seen with ERK-1/2. SHP-2 is phosphorylated at early stages of ICAM-1 ligation (5 to 30 min) and becomes completely dephosphorylated by 2 h. The cytoplasmic tail of ICAM-1 (PKIKK485YRLQ) conforms poorly to the consensus ITIM sequence (I/V/L)XYXX(L/ V). This sequence in ICAM-1, however, binds directly to SHP-2 (Pluskota et al. 2000). In fact, the NH2-terminal SH2 domain of SHP-2 associates with the sequence surrounding phosphotyrosine at 485. Substitution of tyrosine 485 to alanine (Y485A) results in loss of ICAM-1 binding to SHP-2. Moreover, 293 cells expressing ICAM-1 (Y485A) have a reduced survival capacity compared to wildtype ICAM-1 upon adhesion of the cells to Fg. More recently, our results indicate that tyrosine 474 at the membrane proximal region of ICAM-1 also becomes phosphorylated. The sequence surrounding Y474 poorly resembles the consensus ITIM, yet this phosphopeptide is capable of directly associating with SHP-2. Interestingly, Y474 seems to specifically bind to carboxyl SH2 domain in SHP-2. Mutational studies suggest that 293 cells expressing ICAM-1 (Y474A) are also defective in maintaining cellular survival upon Fg ligation (Pluskota and D’Souza 2002; Figure 3). Moreover, mutational studies indicate that tyrosine 476 is either not phosphorylated or weakly phosphorylated upon Fg:ICAM1 ligation, and therefore shows no association with SHP-2 and is not involved in ICAM-1–mediated cellular survival. Several questions remain unanswered in our understanding of intracellular signals from ICAM-1, including the identity of the kinase involved in phosphorylating ICAM-1, the mechanism by which ICAM-1 is dephosphorylated, and if, in fact, the phosphatase SHP-2 is involved in ICAM-1 dephosphorylation. The key question is to relate the kinetics of ICAM-1 phosphorylation and its sig-
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nals in specific function of Fg:ICAM-1 interactions. • Conclusion
Figure 3. Signaling mediated through ICAM-1:Fg interaction. ICAM-1 is tyrosine phosphorylated upon ligation with Fg. Phosphorylated ICAM-1 associates with phosphatase SHP-2, which also becomes phosphorylated. The C-terminal SH2 domain of SHP-2 interacts with ICAM-1 through the ITIM (shown in bold circles) that encompasses phosphotyrosine 474. The N-terminal SH2 domain interacts with ITIM within phosphotyrosine 485, thus initiating signaling cascade, that promotes cell survival. SHP2 phosphorylation is followed by the activation of Ras, Raf-1/Mek/ERK-1 transduction pathways and finally, activation of the transcription factors (c-fos, c-jun, Elk-1, etc) and modulation of gene expression. Altogether these reactions promote cell survival.
Fg:ICAM-1 engagement on the vascular cells promotes capture and localization of circulating platelets and inflammatory cells, transmigration of leukocytes, and deposition of Fg, and intriguingly is also involved in cellular survival. Functional importance of Fg:ICAM-1–mediated reactions have been largely demonstrated in in vitro studies. There is now emerging evidence from in vivo animal models (Massberg et al. 1999, Sriramarao et al. 1996) and to some extent in vessel contractility studies (Hicks et al. 1996). The increased expression of ICAM-1 and accumulation of Fg reported in a number of cardiovascular and inflammatory disorders could manifest some of the functional effects of Fg:ICAM-1 interaction. Figure 4 provides a framework for the vascular events mediated through this ligation. Another important aspect is mediated via the recently reported interaction of FgBb(15–42) with VE-cadherin, which results in upregulation of ICAM-1 expression. This pathway may be considered a self-sustaining positive feedback loop, promoting Fg:ICAM-1– mediated reactions. FgBb(15–42) is exposed as a result of Fg proteolysis by thrombin, uPA and/or tPA. FgBb(15– 42):VE-cadherin interaction may be activated whenever blood coagulation and/
Figure 4. Schematic summary of Fg:ICAM-1 mediated reactions. Increased expression of ICAM-1 and accumulation of Fg is known to occur in vascular diseases and inflammation. Fgg(117–133):ICAM-1(8–22) ligation promotes adhesion of circulating platelets and leukocytes to endothelial cells (EC) and transendothelial migration of leukocytes, deposition of Fg on endothelial cells, prevents endothelial cell apoptosis, and elicits direct vasomotor responses. Interaction of FgBb(15–42) with VE-cadherin enhances expression of ICAM-1, thus creating a feedback loop. These molecular events promote ongoing inflammation and vascular damage.
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or fibrinolysis pathways are induced and may have important clinical implications in cardiovascular diseases and in conditions such as disseminated intravascular coagulation; and may also be considered in therapeutic interventions, such as thrombolytic therapy. Similarly, ICAM-1 mediates Fg deposition on the endothelium, while Fg is capable of inducing ICAM-1 expression. This will result in the triggering of multiple pathophysiological effects mediated through ICAM-1, Fg, and/or ICAM-1:Fg interaction. Fibrin(ogen) or its derivatives stimulate release of uPA and tPA from vascular endothelium, thus creating a vicious circle. These reactions contribute to the complex interplay of mechanisms inducing transformation of endothelium into the proinflammatory and prothrombotic surface that promote ongoing inflammation, thrombogenesis, atherogenesis, stenosis and occlusion of the vessel.
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VEGF Gene Delivery for Treatment of Ischemic Cardiovascular Disease Mark L. Koransky, Robert C. Robbins, and Helen M. Blau*
There are increasing numbers of patients with ischemic myocardial disease not amenable to traditional methods of revascularization. These patients may benefit from new research into the use of naturally occurring angiogenic compounds, such as vascular endothelial growth factor (VEGF) for re-establishing blood flow into regions of hibernating myocardium. Animal studies and human clinical trials evaluating VEGF demonstrate increases in myocardial perfusion after treatment, with some patients reporting improvement in anginal symptoms. Further research into the ideal form of VEGF therapy (protein, plasmid, or adenoviral) and delivery method (intracoronary, intramyocardial, or epicardial) seems justified. (Trends Cardiovasc Med 2002;12:108–114) © 2002, Elsevier Science Inc.
Percutaneous transluminal angioplasty (PTCA) and stenting or operative coronary revascularization (coronary artery bypass grafting, CABG) are procedures that effectively re-establish blood flow to distal myocardial vascular beds and pro-
vide relief of angina for most patients with lesions from coronary artery disease. However, percutaneous interventions are plagued by high rates of restenosis and bypass grafts are subject to progressive atherosclerotic occlusion.
Mark L. Koransky is at the Department of Cardiothoracic Surgery and the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California. Robert C. Robbins is at the Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California. Helen M. Blau is at the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California. * Address correspondence to: Helen M. Blau, Department of Microbiology and Immunology, Stanford University Medical Center, 269 Campus Drive, CCSR 4215a, Stanford, CA 943055175, USA. Tel.: (+1) 650-723-6270; fax: (+1) 650-736-0080; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
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