VCAM-1 and ICAM-1: Roles in Inflammation and Atherosclerosis
My research program focuses on inducible endothelial cell adhesion molecules for leukocytes, which are members of the immunoglobulin gene superfamily (VCAM-1 and ICAM-1) and studies their functions in inflammation and atherosclerosis. This research focus began with the identification of upregulated VCAM-1 expression on arterial endothelium in the earliest stages of atherosclerotic lesion formation (Science 251:788-791, 1991), which provided evidence of endothelial cell activation during early atherogenesis. Subsequent studies focused on VCAM-1 expression patterns, identified alternate splice forms of VCAM-1, defined VCAM-1 genomic structure, chromosomal localization and investigated mechanisms of expression.
In the last 5 years, the goal of the laboratory was to use transgenic approaches and in vivo models to investigate functions of VCAM-1 and ICAM-1 in the recruitment of monocytes and lymphocytes to sites of chronic inflammation and to atherosclerotic lesions. VCAM-1 knockout mice were produced by embryonic stem cell technology (1). These mice revealed a novel role for VCAM-1 during embryonic development - mediation of allantoic fusion to the chorion, resulting in the formation of the umbilical cord and placenta (1).
Subsequently, we produced VCAM-1 domain 4-deficient (D4D) mice (2), which circumvented the embryonic lethality of VCAM-1 knockout mice. These mice expressed a mutant form of VCAM-1 with only 1 ligand-binding site (compared to 2 in wild type) and had markedly reduced expression levels of D4D VCAM-1 (3-8% of wild type). VCAM-1 D4D mice had a number of inflammatory defects. Among these was diminished mononuclear leukocyte recruitment into the peritoneal cavity of mice immunized and subsequently challenged with ovalbumin. The chronic inflammatory reaction to an intraperitoneal thioglycollate injection was normal in VCAM-1 D4D mice, but was reduced in mice with combined VCAM-1 D4D and ICAM-1 deficiency (2).
These data indicate that in response to different types of inflammatory stimuli VCAM-1 alone or together with ICAM-1 participates in the recruitment of inflammatory cells. We have collaborated with Dr. Gutierrez-Ramos and demonstrated that eosinophil recruitment to the lung in response to allergic inflammation was abrogated in a VCAM-1 D4D mice (3).
In the last 2 years we have extended our research to evaluate leukocyte integrins that bind VCAM-1 and ICAM-1. Integrins on circulating leukocytes do not bind ligand and must be 'activated' to do so. We developed and characterized an in vitro assay to measure the strength of leukocyte adhesion to purified recombinant VCAM-1 and ICAM-1. This assay was used to make a novel observation that binding of a4 integrins to VCAM-1 induces a signal transduction pathway in leukocytes that activates b2 integrins and increases the strength of adhesion to ICAM-1 (4).
In addition to inflammation, we continue to study the role of endothelium and adhesion molecules in atherogenesis. One aspect of this endeavor is to evaluate atherosclerotic lesion formation in mice with different adhesion molecule deficiencies. Over the last 2 years, VCAM-1 D4D and ICAM-1-/- mice were bred onto the LDL receptor knockout background and lesion formation is being quantified in the aorta after feeding a cholesterol-enriched diet. We have developed a defined semi-purified diet without cholate for these studies, which avoids numerous problems associated with standard cholesterol-rich diets containing cholate (5). Preliminary data indicate that VCAM-1, but not ICAM-1, influences early atherosclerotic lesion formation and we expect to complete these experiments within 6 months.
Current and future research directions will continue to focus on basic mechanisms of inflammation and atherosclerosis. We will determine by what mechanism and at what stages of leukocyte emigration (i.e., tethering, rolling, stabilization of adhesion and transendothelial migration) do VCAM-1 and ICAM-1 contribute. We will label monocytes or lymphocytes with a fluorochrome and perform intravital microscopy in VCAM-1 D4D or ICAM-1-/- mice and visually assess each step of leukocyte emigration in real time. The production of transgenic mice is and will remain an ongoing activity in the laboratory. Recently, VCAM-1 domain 6-deficient mice were produced, as well as mice with the neomycin resistance gene inserted in the intron between domains 5 and 6. Both express a PI-linked form of VCAM-1 at reduced levels, which are comparable to VCAM-1 D4D mice. Intravital microscopy will be performed on the above mice and data will be compared to VCAM-1 D4D and wild-type mice.
With respect to leukocyte integrins, we will pursue signal transduction mechanisms by which a4 integrin/VCAM-1 interactions activate b2 integrins and define its pathophysiological significance. Studies on atherogenesis will be continued. We will compare chemoattractant- and adhesion molecule-dependent mechanisms of leukocyte recruitment to the arterial intima and will study the role of chronic hemodynamic perturbations on endothelial cell NF-kB signal transduction pathway, which is relevant to the induction of adhesion molecule and chemokine expression.