Intercellular adhesion molecule-1 (ICAM-1), 3 a cell adhesion molecule expressed in various cell types, plays a key role in mediating the inflammatory and immune responses (1). It functions as a co-stimulatory molecule during antigen presentation to T cells. ICAM-1 interaction with leukocyte integrins LFA-1 and Mac-1 promotes firm adhesion of leukocytes and their transmigration to the sites of inflammation (1-3). ICAM-1 consists of five Ig-like domains, a single transmembrane domain, and a short cytoplasmic tail. ICAM-1 is shed as soluble ICAM-1 (sICAM-1) in blood and other biological fluids. Elevated plasma levels of sICAM-1 have been reported in inflammatory, neoplastic, autoimmune, and vascular diseases (4), and sICAM-1 is utilized as a marker of inflammation and a predictor of prognosis (5, 6). Multiple signaling pathways regulate the shedding of ICAM-1 in 293 cells transfected with ICAM-1 (293 ICAM-1 ), including mitogen-activated protein kinase and phosphatidylinositol 3-kinases (7). How these signaling cascades mediate ICAM-1 release is currently unknown. Previous reports indicate that ICAM-1 is a substrate for matrix metalloprotease (MMP)-9 (8) as well as human leukocyte elastase and cathepsin G (9, 10).Ectodomain shedding is an important regulatory mechanism in the function of membrane-bound cell-surface molecules (reviewed in Refs. 11 and 12). Cytokines and their receptors, growth factors, ectoenzymes, adhesion molecules, and proteoglycans undergo shedding (13-16). The most widely studied inducer of shedding is phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (16, 18 -20). Calcium ionophores, cytokines, growth factors, and chemotactic peptides also induce shedding (22). The majority of the shedding events are mediated by the zinc-dependent metalloproteinases of the metzincin family, which includes MMPs and proteases containing a disintegrin and metalloproteinase (ADAM) domain. Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of MMPs and some ADAMs. There are four distinct TIMPs. TIMP-1-4 inhibit a wide range of MMPs, whereas TIMP-3 is also an inhibitor of ADAM-17. ADAM-10 is inhibited by TIMP-1 and -3, whereas ADAM-8 and -9 are very poorly inhibited by, and are unlikely to be physiologically regulated by, TIMPs. To date, no ADAM has been shown to be inhibited by TIMP-2. ADAMs are type I transmembrane glycoproteins; and in addition to their proteolytic activity, they also mediate cell adhesion. Tumor necrosis factor-␣ (TNF␣)-converting enzyme (TACE) plays a central role in ectodomain shedding (16). Cell lines derived from TACE-deficient mice (TACE Ϫ/Ϫ ) verify that TACE is involved in the PMA-induced shedding of a number of structurally and functionally diverse proteins, including pro-TNF␣, L-selectin, ␤-amyloid precursor protein, transforming growth factor-␣, heparin-binding epidermal growth factor (EGF), and vascular cell adhesion molecule-1 (VCAM-1) (14, 17-21), suggesting a common shedding mechanism regulated by a protein kinase C-TACE axis. Mice lacking T...

Elevated fibrinogen (Fg) concentration in blood is a high risk factor for many cardiovascular diseases. We hypothesize that Fg and its early degradation product, fragment D, may result in arterial constriction by binding endothelial intercellular adhesion molecule-1 (ICAM-1). The vasoconstriction induced by Fg and fragment D was studied in third-and second-order arterioles (3As and 2As, respectively) of Sprague-Dawley rat cremaster muscle in vivo, in aortic and femoral artery rings, and in the segments of first-order arterioles (1As) isolated from rat cremaster muscle. Intravascular infusion of Fg induced significant constriction of 3As and 2As (by 33.4 Ϯ 3.4 and 23.7 Ϯ 4.3%, respectively) in vivo and was abolished in the presence of the specific endothelin type A receptor blocker BQ-610. Fg and fragment D produced significant constriction of both aortic and femoral artery rings. (25), and stroke (9). Increased blood Fg concentration results in an increase in blood viscosity (20,23) and, therefore, an increase in blood flow shear stress (7). These in turn contribute to increased peripheral vascular resistance and lead to a reduction of blood flow in muscle, which exacerbates complications during hypertension (40). In addition, higher blood viscosity-induced increases in blood flow shear stress can activate endothelial cells (8, 38) and platelets (36). Endothelial cell activation results in expression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) (39). Fg is a ligand for ICAM-1 (18, 41), and as such, Fg binding to shear stressactivated platelets through platelet GPIIb/IIIa (34) may serve as a bridging mechanism for platelet adhesion to the endothelium (5). Therefore, elevated blood Fg content may lead to a higher predisposition for platelet thrombogenesis by triggering both platelet and endothelial activation mechanisms.Fg deposition onto the microvascular wall of mice after ischemia-reperfusion has been demonstrated elsewhere (26). This Fg deposition was partially reduced in an ICAM-1-mutant mouse, which suggests that Fg deposition at the vascular endothelium mainly occurs through endothelial ICAM-1. Hicks et al. (16) showed that Fg has a vasodilatory effect on the human saphenous vein. The authors suggested that this vasorelaxation could be a result of Fg binding to endothelial ICAM-1 and was induced by the expression of vasoactive mediators other than nitric oxide and prostacyclin.Various reports show the vasoactive effects of isolated peptides derived from plasmin digestion of fibrin and Fg in several vascular beds, including the lung (17), heart (27, 33), femoral artery (37), and mesenteric arteries (3). However, the mechanism of these effects still remains unclear. An attempt to study vasoconstriction of rat pulmonary artery rings induced by early digestion products of Fg failed to show any effect of these Fg fragments including fragment D (6). The effects of vascular constriction by Fg degradation products on the arterial rings were tested in the presence of 4 ϫ 10 Ϫ8 M phen...

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ICAM-1, a membrane-bound receptor, is released as soluble ICAM-1 in inflammatory diseases. To delineate mechanisms regulating ICAM-1 cleavage, studies were performed in endothelial cells (EC), human embryonic kidney (HEK)-293 cells transfected with wild-type (WT) ICAM-1, and ICAM-1 containing single tyrosine-to-alanine substitutions (Y474A, Y476A, and Y485A) in the cytoplasmic region. Tyrosine residues at 474 and 485 become phosphorylated upon ICAM-1 ligation and associate with signaling modules. Cleavage was assessed by using an antibody against the cytoplasmic tail of ICAM-1, which recognizes intact ICAM-1 and the 7-kDa membrane-bound fragment remaining after cleavage. Cleavage in HEK-293 WT cells was accelerated by phorbol ester PMA, whereas in EC it was induced by tumor necrosis factor-alpha. In both cell types, a 7-kDa ICAM-1 remnant was detected. Tyrosine phosphatase inhibitors dephostatin and sodium orthovanadate augmented cleavage. PD-98059 (MEK kinase inhibitor), geldanamycin and PP2 (Src kinase inhibitors), and wortmannin (phosphatidylinositol 3-kinase inhibitor) dose-dependently inhibited cleavage in both cell types. SB-203580 (p38 inhibitor) was more effective in EC, and D609 (PLC inhibitor) mostly affected cleavage in HEK-293 cells. Cleavage was drastically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. Surprisingly, phosphorylation was not detectable on the 7-kDa fragment of ICAM-1. These results implicate distinct pathways in the cleavage process and suggest a preferred signal transmission route for ICAM-1 shedding in the two cell systems tested. Tyrosine residues Y474 and Y485 within the cytoplasmic sequence of ICAM-1 regulate the cleavage process.

Background-Conventional resuscitation (CR) from hemorrhagic shock (HS) often restores and maintains hemodynamics but fails to restore intestinal perfusion. Post-CR intestinal ischemia has been implicated in the initiation of a gut-derived exaggerated systemic inflammatory response and in the progressive organ failure following HS. We propose that intestinal ischemia can be prevented with hypertonic saline resuscitation (HTSR).