Blocking Very Late Antigen-4 Integrin Decreases Leukocyte Entry and Fatty Streak Formation in Mice Fed an Atherogenic Diet

Abstract

—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.

Oxidized Phospholipids Trigger Atherogenic Inflammation in Murine Arteries

“…33 However, binding of monocytes to OxPAPC-stimulated endothelial cells is mediated by CS-1 fibronectin 34 and blocking CS-1 reduced atherosclerotic lesion formation in mice. 35 In addition to expression of chemokines, we found that OxPAPC induced expression of several other atherosclerosis-related genes. We demonstrate that oxidized phospholipids induce IL-6 transcripts in a native murine artery in vivo.…”

VCAM-1 is critical in atherosclerosis

“…This was further supported by evidence that chronic application of a small peptide that inhibits ligand binding by α 4 β 1 delays the development of atherosclerosis in mice fed an atherogenic diet (7). However, interpretation of this study was complicated by the fact that activated endothelial cells can express a second ligand for α 4 β 1 integrin, alternatively spliced fibronectin containing the CS-1 peptide.…”

Endothelial cell regulation by phospholipid oxidation products

“…R-Ras was shown to activate PI3K, which then results in the activation of α5β1 integrin on the apical surface; this activated integrin binds the fibronectin 18 . In fact, it has been shown that inactivation of VLA-4 using a synthetic peptide mimetic of CS1 ( 4 ), decreased leukocyte entry and fatty streak formation in mice fed an atherogenic diet ( 19,20 ). Recent findings have also demonstrated a role for the prostaglandin receptor E2 (EP2), a G-protein coupled receptor (GPCR), in this pathway (Figure 1) 21 .…”