Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are potent vasculogenic and angiogenic factors that hold promise as a means to produce therapeutic vascularization and angiogenesis. However, VEGF also acts as a proinflammatory cytokine by inducing adhesion molecules that bind leukocytes to endothelial cells, an initial and essential step toward inflammation. In the present study, we used human umbilical vascular endothelial cells (HUVECs) to examine the effect of Ang1 on VEGF-induced expression of three adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Interestingly, Ang1 suppressed VEGF-induced expression of these adhesion molecules. Furthermore, Ang1 reduced VEGF-induced leukocyte adhesion to HUVECs. These results demonstrate that Ang1 counteracts VEGF-induced inflammation by reducing VEGF-induced endothelial adhesiveness.     

Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

During inflammation, E-selectin expressed on cytokine-activated endothelial cells mediates leukocyte rolling under flow. E-selectin undergoes endocytosis and may associate with lipid rafts. We asked whether distribution of E-selectin in membrane domains affects its functions. E-selectin was internalized in transfected CHO cells or cytokine-activated human umbilical vein endothelial cells (HUVECs). Confocal microscopy demonstrated colocalization of E-selectin with alpha-adaptin, a clathrin-associated protein. Deleting the cytoplasmic domain of E-selectin or disrupting clathrin-coated pits with hypertonic medium blocked internalization of E-selectin, reduced colocalization of E-selectin with alpha-adaptin, and inhibited E-selectin-mediated neutrophil rolling under flow. Unlike CHO cells, HUVECs expressed a small percentage of E-selectin in lipid rafts. Even fewer neutrophils rolled on E-selectin in HUVECs treated with hypertonic medium and with methyl-beta-cyclodextrin, which disrupts lipid rafts. These data demonstrate that E-selectin clusters in both clathrin-coated pits and lipid rafts of endothelial cells but is internalized in clathrin-coated pits. Distribution in both domains markedly enhances E-selectin's ability to mediate leukocyte rolling under flow.     

T-cell P/E-selectin ligand alpha(1,3)fucosylation is not required for graft-vs-host disease induction

OBJECTIVE: Recognition of E- and P-selectins on vascular endothelium by their leukocyte glycoprotein counterreceptor P-selectin glycoprotein ligand-1 (PSGL-1) initiates and sustains leukocyte rolling, culminating in extravasation of lymphocytes from blood into organs. PSGL-1 is rendered functional by terminal glycosylation steps, which occur mainly in activated Th1 but not Th2 cells. alpha(1,3)Fucosyltransferases IV and VII control this glycosylation pathway. Mice lacking these transferases (Fuc-TIV(-/-)/Fuc-TVII(-/-)) lack functional E- and P-selectin ligands. We hypothesized that Fuc-TIV(-/-)/Fuc-TVII(-/-) donor T cells might have reduced capacity to roll on vessels of inflamed target tissues and mediate graft-vs-host disease (GVHD). MATERIALS AND METHODS: We compared the ability of Fuc-TIV(-/-)/Fuc-TVII(-/-) and wild-type (WT) C57BL/6 (B6) spleen cells (SPCs) to produce GVHD in lethally irradiated major histocompatibility complex (MHC) haplotype-mismatched B6D2F1 recipients. Clinical GVHD, GVHD pathology in target organs, memory phenotype conversion, proliferation of donor T cells, and tissue and serum cytokine expression were examined. RESULTS: Surprisingly, clinical GVHD was not reduced in lethally irradiated mice receiving full haplotype MHC mismatched or matched Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared to those receiving WT SPCs. GVHD pathology in target organs, memory phenotype conversion, and proliferation of donor T cells were similar in both groups. However, reduced interferon-gamma was detected in liver and lung, and serum levels of tumor necrosis factor-alpha were higher in mice receiving Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared with WT SPCs. CONCLUSIONS: These results suggest that donor T cells, including Th1, are capable of trafficking to GVHD target tissues independently of P- and E- selectin ligand in conditioned hosts.     

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.     

An important regulatory role for CD4+CD8 alpha alpha T cells in the intestinal epithelial layer in the prevention of inflammatory bowel disease

The normal immunoregulatory mechanisms that maintain homeostasis in the intestinal mucosa, despite continuous provocation by environmental antigens, are jeopardized in inflammatory bowel diseases. Although previous studies have suggested that intestinal intraepithelial lymphocytes prevent spontaneous intestinal inflammation, there is limited knowledge about the characteristics of regulatory cells in the intestinal intraepithelial lymphocytes population. Here we show that CD4(+)CD8 alpha alpha(+) double-positive cells present in the intestinal intraepithelial lymphocytes population can suppress T helper 1-induced intestinal inflammation in an IL-10-dependent fashion. CD4(+) T cells stimulated along the Th2 but not the Th1 lineage, when transferred to RAG-1-/- mice, acquire CD8 alpha alpha expression on reaching the intestinal epithelium, and on arrival there, augment their production of IL-10. We show that a precursor CD4(+) T cell after limited, but not repeated, stimulation by IL-4 is able to become a double-positive-regulatory cell on exposure to the intestinal microenvironment in mice. Both CD8 alpha alpha acquisition and IL-10 production depend critically on the NF-kappa B-GATA-3-axis that we have previously shown is essential for differentiation to the Th2 phenotype and for the induction of airway inflammation. Our studies identify a mechanism for the generation of regulatory T cells in the intestine that may play an important role in controlling inflammatory bowel disease.     

Relative contributions of alpha4 and alphaL integrins to IL-4-induced leukocyte rolling and adhesion

OBJECTIVE: To delineate the relative contributions of alpha4 and alphaL to mediate interleukin-4 (IL-4) induced leukocyte rolling, and the subsets of leukocytes that use these pathways to adhere. METHODS: Intravital microscopy was used to examine leukocytes in venules of cremaster muscles of mice receiving intrascrotal injections of IL-4. alpha4 and alphaL monoclonal antibodies (mAbs) were administrated either prior to (prophylactic) or 24 h following (therapeutic) treatment with IL-4. In addition, fluorescent microspheres coated with mAbs directed against CD4, CD8, or Gr-1 were injected into mice and the number of subset-specific adherent leukocytes was measured. RESULTS: Prophylactic inhibition of alpha4 and alphaL integrins prevented IL-4-induced leukocyte rolling flux (p< .05) and increased leukocyte rolling velocity twofold (p < .05), respectively, while blocking either integrin eliminated IL-4-induced leukocyte adhesion (p < .05). In contrast, therapeutic administration of both anti-alpha4 and anti-alphaL mAbs was necessary to completely inhibit IL-4-induced leukocyte adhesion (p < .05). Furthermore, CD8+ and Gr-1+ leukocytes utilized alpha4 and alphaL to adhere to postcapillary venules, whereas CD4+ leukocytes primarily utilized alpha4. CONCLUSIONS: Following tissue activation with IL-4, alpha4 and alphaL initiate the attachment and deceleration, respectively, of leukocytes during rolling, and are responsible for mediating the adhesion CD4+, CD8+, Gr-1+ leukocytes.     

The leukocyte cell adhesion cascade and its role in myocardial ischemia-reperfusion injury

Cell-cell and cell-matrix interactions are known to be mediated by specific cell adhesion receptors expressed on the cell surface. The characterization of these cell adhesion molecules has allowed researchers to examine their roles in a variety of physiologic and pathophysiologic conditions. Numerous studies have demonstrated that myocardial ischemia-reperfusion injury is an acute inflammatory process in which leukocytes are intimately involved. In this review, we summarize the current data on the leukocyte cell adhesion cascade, focus upon studies which have demonstrated specific cell adhesion molecule interactions which mediate the leukocyte involvement in myocardial ischemia-reperfusion injury, and suggest future avenues of exploration and possible clinical implications of the studies reviewed.R. J. Gumina is a fellow in the Medical Scientist Training Program at the Medical College of Wisconsin. This work was supported in part by a predoctoral fellowship to RJG from the American Heart Association, Wisconsin Affiliate and NIH Grants HL08311 (GJG), HL40926 (PJN), and HL54280 (DCW).     

An important role for Akt3 in platelet activation and thrombosis

The Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3(-/-) mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A? (TXA?), but not collagen or VWF. In contrast, platelets from Akt1(-/-) or Akt2(-/-) mice are defective in platelet activation induced by thrombin, TXA?, and VWF, but only Akt1(-/-) platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3(-/-) platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3β (GSK-3β) at Ser9, which is known to inhibit GSK-3β function. Thus, Akt3 is important in inhibiting GSK-3β. Accordingly, treatment of Akt3(-/-) platelets with a GSK-3β inhibitor rescued the defect of Akt3(-/-) platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3β may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3(-/-) mice showed retardation in FeCl?-induced carotid artery thrombosis in vivo. Thus, Akt3 plays an important and distinct role in platelet activation and in thrombosis.     

Conversion of the severe to the moderate disease phenotype with donor leukocyte microchimerism in canine leukocyte adhesion deficiency

Leukocyte adhesion deficiency-1 (LAD-1), a genetic immunodeficiency disease characterized by life-threatening bacterial infections, results from the defective adherence and migration of leukocytes due to mutations in the leukocyte integrin CD18 molecule. Canine LAD (CLAD) represents the canine homologue of the severe phenotype of LAD-1 in children. In previous studies we demonstrated that non-myeloablative stem cell transplantation from matched littermates resulted in mixed donor-host chimerism and reversal of the disease phenotype in CLAD. In this study, we describe two CLAD dogs with less than 2% donor leukocyte chimerism following non-myeloablative transplant. Both dogs are alive more than 24 months after transplant with an attenuated CLAD phenotype resembling the moderate deficiency phenotype of LAD. The improvement in the CLAD phenotype with very low levels of donor CD18(+) leukocytes correlated with the preferential egress of the CD18(+) neutrophils into extravascular sites. The clinical response with very low levels of donor CD18(+) leukocytes in CLAD supports using this model for testing gene therapy strategies since the low levels of gene-corrected hematopoietic cells expected with hematopoietic gene therapy would likely have a therapeutic effect in CLAD.     

Intercellular adhesion molecule-1 is required for chemoattractant-induced leukocyte adhesion in resting, but not inflamed, venules in vivo

The leukocyte integrins LFA-1 and Mac-1 bind to endothelial intercellular adhesion molecule-1 (ICAM-1). Leukocyte adhesion induced by micropipette injection of formylmethionylleucylphenylalanine (fMLP) or macrophage inflammatory protein 2 (MIP-2) next to a venule in the exteriorized mouse cremaster muscle was almost completely blocked after intravenous injection of the ICAM-1 mAb YN-1. In contrast, after 2-h pretreatment with TNF-alpha, leukocyte adhesion induced in postcapillary venules by fMLP or MIP-2 was not blocked by the ICAM-1 mAb. Leukocyte adhesion was significantly reduced by mAb GAME-46 to CD18 even after TNF-alpha treatment. We conclude that ICAM-1 is necessary for neutrophil adhesion to unstimulated endothelium, but not for adhesion to cytokine-stimulated endothelium. Although ICAM-1 is expressed at high levels after TNF-alpha, ICAM-1 either is not functional or is redundant with other endothelial ligands for beta(2) integrins.     

Soluble intercelluar adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1 and von Willebrand factor in stroke.

We investigated the role of endothelial cell and leukocyte adhesion in the pathophysiology of acute stroke. The immunocytochemical expression of adhesion molecules in brain tissue from six patients who died following acute ischaemic stroke showed weak endothelial expression of intercellular adhesion molecule-1 (ICAM-1), but intense expression of vascular cell adhesion molecule-1 (VCAM-1) by astrocytes and endothelial cells from the infarcted, but not the non-infarcted areas. We also measured soluble adhesion molecules E-selectin, ICAM-1, VCAM-1 and von Willebrand factor (all by enzyme-linked immunosorbent assay) in 21 patients after an acute ischaemic stroke (ictus < 12 h), and again 3 months later. Blood levels in the stroke patients were compared with 82 healthy controls and 22 subjects with carotid atherosclerosis. Compared with healthy controls, both patient groups had raised levels of von Willebrand factor (P < 0.02) but the level of soluble VCAM-1 was raised only in patients with acute stroke (P < 0.02). Levels of von Willebrand factor and soluble VCAM-1 in the stroke patients were still high at 3 months follow-up. We suggest that there might be changes in adhesion molecule expression and release in the acute and chronic stages of ischaemic stroke, where blood levels are related to immunocytochemical findings on infarcted brain. These changes may perhaps be part of the complex pathophysiological responses to infarction and repair of brain tissue following stroke.     

Vascular adhesion protein-1, intercellular adhesion molecule-1 and P-selectin mediate leukocyte binding to ischemic heart in humans

OBJECTIVES: The expression of endothelial adhesion molecules and their functional significance in leukocyte adhesion to human myocardial blood vessels in acute myocardial infarction (AMI) were studied. BACKGROUND: Leukocyte extravasation, mediated by specific adhesion molecules, exacerbates tissue injury after restoration of blood supply to an ischemic tissue. Experimental myocardial reperfusion injury can be alleviated with antibodies that block the function of adhesion molecules involved in leukocyte emigration, but the relevant molecules remain poorly characterized in human AMI. METHODS: Semiquantitative immunohistochemistry and in vitro adhesion assays were used to study the expression and granulocyte binding abilities of different endothelial adhesion molecules in human AMI. Changes in the molecular nature of vascular adhesion protein-1 (VAP-1) were evaluated using immunoblotting. RESULTS: Certain endothelial adhesion molecules (intercellular adhesion molecule [ICAM-2], CD31 and CD73) were expressed in myocardial blood vessels homogeneously in normal and ischemic hearts, whereas others (E-selectin and peripheral lymph node addressin) were completely absent from all specimens. The synthesis of ICAM-1 was locally, and that of P-selectin regionally, upregulated in the infarcted hearts when compared with nonischemic controls. Vascular adhesion protein-1 showed ventricular preponderance in expression and alterations in posttranslational modifications during ischemia-reperfusion. Importantly, P-selectin, ICAM-1 and VAP-1 mediated granulocyte binding to blood vessels in the ischemic human heart. CONCLUSIONS: Human P-selectin, ICAM-1 and VAP-1 appear to be the most promising targets when antiadhesive interventions preventing leukocyte-mediated tissue destruction after myocardial ischemia are planned.     

Circulating intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in patients with type 2 diabetes mellitus

Serum levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in patients with type 2 diabetes mellitus (n = 64) and control subjects (n = 40) were studied. Serum ICAM-1 concentrations in diabetic patients were significantly higher than those of control subjects (378.2 +/- 70.0 versus 220.4 +/- 31.8 ng/ml, P < 0.01). By multiple regression analysis, hemoglobin A1c was independently associated with serum ICAM-1 concentration in patients with diabetes. The serum VCAM-1 concentration of diabetic patients with macroangiopathy was higher than those of patients without macroangiopathy and of control subjects (806.9 + 276.5 versus 639.0 +/- 146.0 (P < 0.01), and 652.1 +/- 146.9 ng/ml (P < 0.01), respectively). There was no difference in serum E-selectin concentration between diabetic patients with or without macroangiopathy and normal control subjects. These results suggest that adhesion molecules may contribute to the development of atherosclerosis in the diabetic state.     

Fibronectin in artery subendothelium is important for platelet adhesion

The role of subendothelial fibronectin in platelet interaction with subendothelium was studied. Human umbilical artery subendothelium was exposed to flowing blood containing 111In-labeled platelets in an annular perfusion chamber. Platelet adhesion was determined from the 111In radioactivity on the vessel wall. When perfusions were performed for five minutes at a wall shear rate of 1,800 s-1, platelet adhesion was the same whether normal plasma or fibronectin-free plasma was used. Preincubation of subendothelium with rabbit anti-human fibronectin serum, however, resulted in a marked inhibition of platelet adhesion. Preincubation with normal rabbit serum had no effect. Platelet adhesion was also diminished when the vessel wall was preincubated with anti- fibronectin IgG fraction or F(ab')2 fragment. After the latter preincubations, frozen sections of 4 micron were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, F(ab')2 fragment specific. Fluorescence was seen throughout the subendothelium both before and after perfusion. No fluorescence was seen when subendothelium was preincubated with normal rabbit IgG or F(ab')2 or with anti-fibronectin IgG that had been absorbed with purified fibronectin. After absorption of anti-fibronectin IgG with purified fibronectin, the inhibiting effect on platelet adhesion was also no longer present. Preincubation of the vessel wall with anti-fibronectin IgG reduced platelet adhesion significantly at a wall shear rate of 800 s-1. This effect was even greater at 1,800 s-1. At low shear rate (400 s-1), there was no inhibition.     

Molecular cloning of the alpha subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: chromosomal localization and homology to the alpha subunits of integrins.

The cell-surface glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct alpha subunit noncovalently associated with a common beta subunit. Leu-CAMs play crucial roles in cell-cell and cell-matrix interactions. We describe the isolation and analysis of two partial cDNA clones encoding the alpha subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig alpha chain was used for immunoscreening a lambda gt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig alpha subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1 alpha chain. RNA gel blots revealed that mature Mo1 alpha chain mRNA is approximately 5 kilobases in guinea pigs and humans. Southern analysis of DNA from hamster-human hybrids localized the human Mo1 alpha chain to chromosome 16, which has been shown to contain the gene for the alpha chain of lymphocyte function-associated antigen 1. A comparison of the Mo1 alpha chain coding region revealed significant homologies with carboxyl-terminal portions of the alpha subunits of fibronectin, vitronectin, and platelet IIb/IIIa receptors. These data suggest that the alpha subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the alpha subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related.     

Prolonged E-selectin induction by monocytes potentiates the adhesion of flowing neutrophils to cultured endothelial cells

We investigated the hypothesis that the infiltration of monocytes into inflamed tissue or damaged vessels would induce a secondary accumulation of neutrophils. Confluent human umbilical vein endothelial cells (HUVEC) and blood monocytes (0.5 or 0.05 monocytes/endothelial cell) were co-incubated for 4 or 24 h. The adhesion of neutrophils flowing over HUVEC was then analysed by video microscopy. Co-incubation caused up to a 40-fold increase in neutrophil adhesion, dependent upon monocyte/HUVEC ratio and duration of incubation. At the lower monocyte/HUVEC ratio, rolling adhesion alone was induced after 4 h co-incubation; however, the full repertoire of rolling, immobilization and migration of neutrophils was observed at all other combinations of co-culture ratio and exposure time. After maximal stimulation by monocytes, antibody blockade of the neutrophil integrin CD18 inhibited neutrophil arrest and migration and revealed underlying rolling adhesion. Rolling was supported by endothelial E-selectin as demonstrated by the almost total abolition of adhesion by a blocking antibody. In a direct comparison, monocytes, tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were assessed for their ability to induce endothelial expression of E-selectin. E selectin was significantly increased by all agents at 4 h, but monocytes alone were able to maintain high levels of E-selectin expression for 24 h. We conclude that monocytes can induce prolonged neutrophil adhesion and migration by activating endothelial cells and causing expression of E-selectin.     

Serum levels of thrombomodulin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in the acute phase of Plasmodium vivax malaria

Elevated plasma or serum levels of thrombomodulin (TM), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin have been reported in several diseases. However, plasma or serum levels of TM, ICAM-1, VCAM-1, and E-selectin have not been investigated in the acute phase of Plasmodium vivax malaria. Serum TM, ICAM-1, VCAM-1, E-selectin, and creatinine levels were determined in six Japanese patients in the acute phase of vivax malaria and in seven healthy Japanese controls. Parasitemias of the peripheral blood were < 0.1% in five patients and 0.8% in one patient. The patients' mean +/- SD serum levels of TM, ICAM-1, VCAM-1, and E-selectin were 5.7 +/- 1.3 Fujirebio units/ml, 709 +/- 397 ng/ml, 2,112 +/- 782 ng/ml, and 99 +/- 28 ng/ml, respectively, and all were significantly greater than those in the controls (TM; P < 0.005, ICAM-1; P < 0.025, VCAM-1; P < 0.005, E-selectin; P < 0.025). However, no significant difference was identified between patients and controls for serum creatinine values. The serum levels of TM and VCAM-1 were not related to parasitemia. The elevation of serum TM levels suggests that endothelial cell damage occurs in the acute phase of vivax malaria.