There is considerable evidence that oxidized lipoproteins play a role in the development of atherosclerosis. Early studies showed oxidized lipoproteins to be present in atherosclerotic lesions (1-3). Studies on hyperlipidemic animals demonstrated that oxidation products were increased in lesions (4). In addition, antioxidants such as probucol and butylated hydroxytoluene have been found to reduce the development and severity of lesions in a number of animal models (5, 6).Mononuclear cell adhesion to vascular endothelium was observed in the initial steps of fatty streak formation (7,8). Recent studies (9, 10) using mice null for monocyte chemotactic protein-1 (MCP-1) and the MCP-1 receptor have demonstrated the important role of monocytes in lesion development. Our group has used minimally modified low-density lipoprotein (MM-LDL)-stimulated aortic endothelial cells to model the development of the fatty streak (11). We have demonstrated that monocyte, but not neutrophil, adhesion and transmigration across the aortic endothelium is increased in response to MM-LDL (11). Active oxidized phospholipids isolated from MM-LDL were found to be increased in rabbit atherosclerotic lesions (12).Previous studies from our group have identified MM-LDL-induced molecules involved in several phases of monocyte/endothelial interactions. Previous work (13,14) suggests that in vivo entry of leukocytes into the vessel wall involves at least three steps; rolling, activation, and firm adhesion to the endothelium. The rolling step has been shown to involve the interaction of selectins on the endothelium, with their ligands on leukocytes. Studies from our group and others (15-18) suggest that Pselectin is an important rolling molecule for monocytes in atherosclerosis. Using in vitro studies, we have shown that levels of P-selectin in human aortic endothelial cells (HAEC) are increased by MM-LDL (18), whereas levels of E-selectin are decreased (19). We and others have also shown that highly oxidized low-density lipoprotein (LDL) leads to P-selectin release to the upper cell surface (18,20). Specific cytokines and chemokines that activate monocyte adhesion ligands have been found in lesions We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic α4β1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial α4β1 ligand in MM-LDLtreated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation ...

(J. Clin. Invest. 1994. 93:405416.)

—Atherosclerotic lesion development is characterized by the recruitment of leukocytes, principally monocytes, to the vessel wall. Considerable interest has been focused on the adhesion molecule(s) involved in leukocyte/endothelial interactions. The goal of the present study was to determine the role of the very late antigen-4 (VLA-4) integrin/ligand interaction in fatty streak development using murine models. Because α4 null mice are not viable, a peptidomimetic was used to block VLA-4–mediated leukocyte binding. The ability of a synthetic peptidomimetic of connecting segment-1 (CS-1 peptide) to block the recruitment of leukocytes and the accumulation of lipid in the aortic sinus of either wild-type mice (strain C57BL/6J) or mice with a low-density lipoprotein null mutation (LDLR−/−) maintained on an atherogenic diet was assessed. The active (Ac) CS-1 peptide or scrambled (Sc) CS-1 peptide was delivered subcutaneously into mice using a mini osmotic pump. Mice were exposed to the peptide for 24 to 36 hours before the onset of the atherogenic diet. In C57BL/6J mice, leukocyte entry into the aortic sinus, as assessed by en face preparations, was inhibited by the active peptide (Ac=28±4, Sc=54±6 monocytes/valve; P =0.004). Additionally, frozen sections stained with Oil Red O were analyzed to assess lipid accumulation in the aortic sinus. C57BL/6J mice that received the (Ac) compound demonstrated significantly reduced lesion areas as compared with mice that received the (Sc) peptide (Ac=4887±4438 μm 2 , Sc=15 009 ±5619 μm 2 ; P <0.0001). In a separate study, LDLR−/− mice were implanted with pumps containing either the (Ac) or (Sc) peptide before initiation of the atherogenic diet. Because LDLR−/− mice fed a chow diet displayed small lesions at 14 weeks, the effects of the peptide seen in these animals represented a change in early lipid accumulation rather than initiation. By using whole-mount preparations, the (Ac) but not the (Sc) peptide significantly reduced the area of lipid accumulation in the aortic sinus, resulting in an approximate 66% decrease. Plasma analysis from all studies revealed concentrations of peptide to be present at levels previously determined by in vitro analysis to block adhesion. (Ac) CS-1 peptide, which blocks VLA-4 on the leukocyte surface, is effective in reducing leukocyte recruitment and lipid accumulation in the aortic sinus. The present study provides in vivo evidence that the VLA-4 integrin plays an important role in the initiation of the atherosclerotic lesion and lipid accumulation, and it suggests a potential therapeutic strategy for this disease.