A major role for VCAM-1, but not ICAM-1, in early atherosclerosis

Abstract

VCAM-1 and ICAM-1 are endothelial adhesion molecules of the Ig gene superfamily that may participate in atherogenesis by promoting monocyte accumulation in the arterial intima. Both are expressed in regions predisposed to atherosclerosis and at the periphery of established lesions, while ICAM-1 is also expressed more broadly. To evaluate functions of VCAM-1 in chronic disease, we disrupted its fourth Ig domain, producing the murine Vcam1(D4D) allele. VCAM-1(D4D) mRNA and protein were reduced to 2-8% of wild-type allele (Vcam1(+)) levels but were sufficient to partially rescue the lethal phenotype of VCAM-1-null embryos. After crossing into the LDL receptor-null background, Vcam1(+/+) and Vcam1(D4D/D4D) paired littermates were generated from heterozygous intercrosses and fed a cholesterol-enriched diet for 8 weeks. The area of early atherosclerotic lesions in the aorta, quantified by en face oil red O staining, was reduced significantly in Vcam1(D4D/D4D) mice, although cholesterol levels, lipoprotein profiles, and numbers of circulating leukocytes were comparable to wild-type. In contrast, deficiency of ICAM-1 either alone or in combination with VCAM-1 deficiency did not alter nascent lesion formation. Therefore, although expression of both VCAM-1 and ICAM-1 is upregulated in atherosclerotic lesions, our data indicate that VCAM-1 plays a dominant role in the initiation of atherosclerosis.

Figure 1

Homologous recombination at the Vcam1 gene locus. (a) Alternative RNA splicing produces VCAM-1 isoforms with one or two α4 integrin binding sites (solid circles). Loops, disulfide-linked Ig domains 1–7. Arrowheads, exon splice junctions. (b) The wild-type murine Vcam1 gene, D4D targeting construct, and homologous recombination at the Vcam1 locus. D4 (exon 6) was disrupted by gene targeting. Black boxes, exons 1–10. D1–D7, Ig domains. PI, domain providing phosphatidylinositol linkage of 3 Ig VCAM-1. Restriction enzyme sites and the Southern blot probe are indicated. (c) Southern blot analysis of BamHI-digested genomic DNA from progeny of heterozygous mating. A solid line above the lanes indicates each litter. Vcam1⁺ and Vcam1^D4D alleles are 12 and 8 kb, respectively.

Figure 2

VCAM-1 expression in Vcam1^D4D/D4D mice. (a) Northern blot analysis of VCAM-1, ICAM-1, and β-actin expression using total or oligo dT–enriched RNA (mRNA) from hearts or lungs of control (C) or LPS-treated Vcam1^+/+ (+/+), Vcam1^+/D4D (+/D4D), and Vcam1^D4D/D4D (D4D/D4D) mice. Bars on the right indicate 18S ribosomal RNA migration. Transcripts corresponding to the 7 (asterisks) and 3 (filled diamonds) Ig VCAM-1 were found in Vcam1^+/+ and Vcam1^+/D4D mice, whereas expression of 6 (arrowheads) and possibly 4 (open circle) Ig domain forms was found in VCAM-1^D4D/D4D mice. (b) RT-PCR analysis of 7, 6, and 3 Ig VCAM-1 (7D, 6D, 3D) mRNA in lungs of LPS-treated mice. In Vcam1^D4D/D4D mice, domain-specific primers did not detect D4 found in 7 Ig VCAM-1. (c) Immunoprecipitation of cell surface VCAM-1 and ICAM-1 from Vcam1^+/+ and Vcam1^D4D/D4D biotin-labeled cultured lung endothelial cells. TNF induced expression of 90- to 95-kDa (asterisk) and 36- to 40-kDa (filled diamond) proteins, corresponding to 7 and 3 Ig VCAM-1, in Vcam1^+/+ cells, and 80- to 85-kDa (arrowhead) and possibly 50- to 60-kDa (open circle) proteins, corresponding to 6 and 4 Ig VCAM-1 in VCAM-1^D4D/D4D cells. No 7 Ig VCAM-1 was detected in VCAM-1^D4D/D4D cells. TNF-induced ICAM-1 expression was comparable.

Figure 3

VCAM-1 deficiency reduces the extent but does not alter the topography of atherosclerotic lesions. Representative oil red O–stained aortae from paired littermates demonstrate high lesion prevalence in the lesser curvature of the ascending aorta and arch. Scale bars are at 1-mm intervals.

Figure 4

VCAM-1 but not ICAM-1 deficiency reduces atherosclerotic lesion area in Ldlr^–/– mice. Data from the whole aorta and from different regions are shown. Each data point (left) represents a value from a single mouse, and the mean ± SEM of the group is on the right.