Vascular cell adhesion molecule-1 expression and signaling during disease: regulation by reactive oxygen species and antioxidants

Abstract

The endothelium is immunoregulatory in that inhibiting the function of vascular adhesion molecules blocks leukocyte recruitment and thus tissue inflammation. The function of endothelial cells during leukocyte recruitment is regulated by reactive oxygen species (ROS) and antioxidants. In inflammatory sites and lymph nodes, the endothelium is stimulated to express adhesion molecules that mediate leukocyte binding. Upon leukocyte binding, these adhesion molecules activate endothelial cell signal transduction that then alters endothelial cell shape for the opening of passageways through which leukocytes can migrate. If the stimulation of this opening is blocked, inflammation is blocked. In this review, we focus on the endothelial cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1). Expression of VCAM-1 is induced on endothelial cells during inflammatory diseases by several mediators, including ROS. Then, VCAM-1 on the endothelium functions as both a scaffold for leukocyte migration and a trigger of endothelial signaling through NADPH oxidase-generated ROS. These ROS induce signals for the opening of intercellular passageways through which leukocytes migrate. In several inflammatory diseases, inflammation is blocked by inhibition of leukocyte binding to VCAM-1 or by inhibition of VCAM-1 signal transduction. VCAM-1 signal transduction and VCAM-1-dependent inflammation are blocked by antioxidants. Thus, VCAM-1 signaling is a target for intervention by pharmacological agents and by antioxidants during inflammatory diseases. This review discusses ROS and antioxidant functions during activation of VCAM-1 expression and VCAM-1 signaling in inflammatory diseases.

Figures

FIG. 1.

Leukocyte transendothelial migration. During inflammation, cytokines produced in the tissue induce endothelial cell adhesion molecule expression. In addition, chemoattractants released by both the tissue and endothelial cells increase leukocyte adhesion molecule affinity as well as provide direction for leukocyte migration. This vascular recruitment of leukocytes is a three-step process involving low affinity rolling of leukocytes on the endothelium followed by arrest of the leukocyte on the endothelium through high affinity adhesion, and then transmigration of the leukocyte through the endothelium. L, leukocyte.

FIG. 2.

VCAM-1 splice variants. Human VCAM-1 has two splice variants that contain either six or seven immunoglobulin-like domains with disulfide linkages. The six-domain form of human VCAM-1 lacks domain 4. Mouse VCAM-1 has the seven-domain form and unique three-domain form. The three-domain form is linked to glycophosphatidylinositol through a 36 amino acid glycophosphatidylinositol-linker. Within VCAM-1, domains 1 and 4 contain the binding sites for integrins. VCAM-1 is also N-glycosylated. VCAM-1, vascular cell adhesion molecule-1.

FIG. 3.

Ligand binding to VCAM-1. Integrin binding to VCAM-1 is regulated by the integrin activation state. α4β1-integrin binds readily to domain 1 but requires higher affinity activation for binding to domain 4. This integrin binding to domains 1 and 4 requires the amino acids D40 and L43, or D328 and L331, respectively. α4β7-integrin also binds to VCAM-1 (dashed arrow) but with a lower affinity than its binding to another adhesion molecule, mucosal addressin cell adhesion molecule-1 (not shown). Galectin 3 binds to N-glycosylation sites on VCAM-1. VCAM-1 has six N-glycosylation sites that may participate in galectin 3 binding. VCAM-1 also coimmunoprecipitates with ezrin and moesin. VCAM-1 cell surface expression requires associated tetraspanins CD151 or CD9. The tetraspanin long extracellular loop (LEL) is necessary for its binding to immunoglobulin superfamily members. This LEL contains a CCG and CC motif. VCAM-1 can also be clipped from the cell surface by ADAM17, ADAM8, and ADAM9. Solid arrow, major ligand binding site. Dashed arrow, ligand binding requires higher integrin activation. Large filled arrow, galectin3 binds to N-glycosylation sites. ADAM, a disintegrin and metalloprotease.

FIG. 4.

VCAM-1 is located in tetraspanin-enriched microdomains. VCAM-1 is found in a lipid-raft-like platform containing ICAM-1 and the tetraspanins CD9, CD81, and CD151, known as the tetraspanin-enriched microdomain. Upon ligand binding to VCAM-1 or ICAM-1, the membrane forms apical projections toward the leukocyte. ICAM-1, intercellular adhesion molecule-1.

FIG. 5.

VCAM-1 signal transduction. (A) Model for VCAM-1 signaling. Crosslinking of VCAM-1 activates calcium fluxes and Rac-1, which then activates endothelial cell NOX2. Nox2 catalyzes the production of superoxide that then dismutates to H2O2. VCAM-1 induces the production of only 1 μM H2O2. H2O2 activates endothelial cell-associated MMPs that degrade extracellular matrix and endothelial cell surface receptors in cell junctions. The endothelial cell-derived H2O2 also mediates a 2–5 h delayed activation of lymphocyte-associated MMPs by inducing the degradation of leukocyte TIMPs. H2O2 diffuses through membranes at 100 μm/s to activate p38MAPK. H2O2 also oxidizes and transiently activates endothelial cell PKCα. PKCα phosphorylates and activates PTP1B on the endoplasmic reticulum. PTP1B is not oxidized. These signals through ROS, MMPs, PKCα, and PTP1B are required for VCAM-1-dependent leukocyte transendothelial migration. The G protein Gαi is also involved in VCAM-1 signaling. (B) Mouse lung tissue section from antigen-challenged lungs was labeled with anti-VCAM-1 and a TRITC-conjugated secondary antibody. VCAM-1 labels the luminal and lateral, but not the basal surface of vascular endothelial cells in vivo. ER, endoplasmic reticulum; H2O2, hydrogen peroxide; MMP, matrix metalloproteinase; oxPKCα, oxidized protein kinase Cα; PTP1B, protein tyrosine phosphatase 1B; ROS, reactive oxygen species; TIMP, tissue inhibitor of metalloproteinase.

FIG. 6.

Activation of VCAM-1 signals. Cells: (A, D) Cytokine-activated primary cultures of endothelial cells express multiple receptors for leukocyte adhesion. (B–D) Immortalized endothelial cell lines (mHEV) constitutively express VCAM-1 but not other ligands for leukocytes. The mHEV cells also express MCP-1 that induces leukocyte transmigration. Stimulation: (A, C) Anti-VCAM-1-coated beads crosslink VCAM-1 and activate VCAM-1 signaling. In contrast, soluble anti-VCAM-1 antibodies do not activate VCAM-1 signaling. (B) Leukocyte binding to VCAM-1 on mHEV cells crosslinks VCAM-1 and activates VCAM-1 signaling. (D) Exogenous 1 μM H2O2 activates VCAM-1 signals downstream of NOX2 to determine whether H2O2 is sufficient for the signaling. MCP-1, monocyte chemoattractant protein; mHEV, lymph node-derived high endothelial venule-like cells.

FIG. 7.

The level of hydrogen peroxide produced during VCAM-1 signaling induces degradation of TIMP-2 on leukocytes. BALB/c mouse spleens were collected and red blood cells were lysed by hypotonic shock. The leukocytes were nontreated (NT) or treated with the proteosome inhibitor MG132, and then 1 μM H2O2 was added. This is the level of H2O2 generated during VCAM-1 signaling. After 5 h, the cells were lysed and TIMP-2 expression was examined by western blot. H2O2 induced the loss of TIMP-2 on leukocytes, and this loss was blocked by the proteosome inhibitor. The treatments had no affect on cell viability (data not shown). The western blots are representative from three independent experiments. The data are presented as mean ± SEM. *p < 0.05 as compared to the NT control.

FIG. 8.

VCAM-1-dependent eosinophil recruitment during allergic lung inflammation. After sensitization with the antigen OVA in the adjuvant alum, the lung is challenged with OVA. In this model, eosinophil recruitment from the blood is blocked with anti-VCAM-1 blocking antibodies. In contrast, lymphocytes, monocytes, and neutrophils migrate on ICAM-1 or PECAM-1. After the leukocytes undergo transendothelial migration, the leukocytes migrate through the tissue, across the epithelium and into the airway spaces. Chemokines in the tissue direct the leukocyte migration. OVA, chicken egg ovalbumin; PECAM-1, platelet-endothelial cell adhesion molecule-1.

FIG. 9.

VCAM-1-dependent eosinophil transendothelial migration in the lung is blocked in mice deficient in nonhematopoietic NOX2. Adapted from ref. (2). CYBB mice that lack NOX2 activity were irradiated and received a bone marrow transplant with wild-type bone marrow. Thus, the leukocytes expressed wild-type NOX2, but the nonhematopoietic cells, including endothelial cells, were NOX2 deficient. Control wild-type mice received wild-type bone marrow transplants. The mice were sensitized with OVA/alum intraperitoneally and challenged intranasally with OVA in saline. Lung tissue sections were collected and stained with hematoxylin and eosin. (A) Representative lung tissue section from OVA-challenged chimeric CYBB mice. Arrows indicate an accumulation of eosinophils bound to the luminal surface of the endothelium. (B) Representative lung tissue section from OVA-challenged chimeric wild-type control mice. Leukocytes are in the tissue and do not accumulate on the endothelium. L, vessel lumen.

FIG. 10.

Alpha and gamma-tocopherol. Tocopherols are lipids. α-Tocopherol differs from γ-tocopherol by one methyl group (arrows).

FIG. 11.

Tocopherol regulation of VCAM-1-induced ROS. Tocopherols are lipids in the plasma membrane. The tocopherol head group is external to the membrane and is thus poised for scavenging of extracellular ROS. Tocopherols are also found in membranes of organelles and can scavenge intracellular ROS. There is ∼10-fold more α-tocopherol than γ-tocopherol in membranes in vivo.

FIG. 12.

α-tocopherol and γ-tocopherol in dietary oils. Adapted from ref. (30). Tocopherols were extracted from dietary oils and measured by high pressure liquid chromatography with an electrochemical detector.