SNP RS7800861 RS16148 RS16147 RS16143 RS16478 RS16142 RS16141 RS16140 RS16139 RS5572 RS9785023 RS16138 RS1468271 RS5574 RS16132 RS16131 RS16475 RS16126 RS16474 RS16473 RS16120 RS16119 RS17374047 Dom 0.25 0.38 1.88 0.20 0.16 0.24 1.62 0.01 0.03 0.89 1.87 0.19 0.09 2.13 0.40 1.42 0.10 0 2.72 1.91 1.63 1.58 0.07 Rec 0.09 0.25 1.39 0.05 0.17 0.08 1.15 0.01 0.01 0.46 1.17 0.06 0.26 1.26 0.39 0.86 0.29 0 2.26 1.62 1.33 1.14 0.28 Dom 0.43 0.45 2.10 0.47 0.27 0.19 1.92 0.27 0.03 0.24 2.13 0.41 0.16 2.73 1.07 1.09 0.70 0 1.72 1.56 1.32 1.62 0.18 Rec 0.35 0.31 1.16 0.18 0.21 0.11 1.19 0.19 0.02 0.31 1.03 0.20 0.68 1.37 0.92 0.68 0.76 0 1.13 0.94 0.73 0.85 0.16 0.49 0.10 0.05 0.69 0.41 0.41 0.12 0.62 1.00 1.00 0.04 1.00 0.78 0.02 0.17 0.08 0.16 1.00 0.04 0.08 0.03 0.04 0.74 0.50 0.25 0.03 0.50 0.29 0.29 0.17 0.67 1.00 1.00 0.05 0.39 0.78 0.05 0.17 0.08 0.16 1.00 0.02 0.18 0.04 0.08 0.57 Table 2. Linkage and family-based association of NPY SNPs with CAD (GENECARD). Neuropeptide Y and Early-Onset Atherosclerosis Overall Two-Point LOD All Families (N = 420) LOD OSA Very Young AOO Families (N = 97) PDT P-value Geno-PDT P-value PDT: Pedigree-Disequilibrium-Test. SNPs with PDT p-value#0.05 in bold. SNPs listed in order of genomic location (39R59). Dom: dominant; Rec: recessive. doi:10.1371/journal.pgen.1000318.t002 Allele-Specific Effects on NPY Levels To examine allele-specific effects, we assessed the relationship of NPY variants with peripheral NPY levels in 220 subjects randomly selected from the CATHGEN case/control dataset. We found that rs16147 was associated with higher NPY levels for the minor (risk, G) allele compared with the major (A) allele (46.3 vs. 41.0 pmol/L, p = 0.04, Figure 3). Concordantly, NPY levels were higher in CAD cases compared with controls (46.8 vs. 41.1 pmol/L, p = 0.02). Antagonism of NPY Reduces Atherosclerosis To determine whether NPY-evoked signaling contributes to atherosclerosis, we attenuated the vascular effects of NPY [26] with an NPY1 receptor antagonist that does not cross the blood- brain barrier: BIBP 3226 (BIBP) [8,26,27], which has been shown to reduce non-atherosclerotic neointimal hyperplasia in rats [8,26]. In order to accelerate typical atherosclerosis in a focal manner, we employed carotid endothelial denudation in apoe2/2 mice, as reported by other groups [28,29]. With this approach, we could apply BIBP just focally to the carotid artery in a peri-arterial Pluronic gel. Atherosclerosis developed typically in this carotid model, with abundant evidence of macrophage foam cells, SMC- like cells constituting fibrous caps of complex intimal lesions, and extracellular cholesteryl ester (Figure 4A–4C). Peri-carotid appli- cation of Pluronic gel by itself had no effect on extent of atherosclerosis (data not shown), and BIBP in the gel did not engender cell toxicity, as judged by apoptosis: cleaved caspase-3 levels were 70620% higher in control than in BIBP-treated carotids (p,0.02, Figure 4D–4E). While control specimens demonstrated greater apoptosis than BIBP-treated specimens, they also demonstrated 1.760.3-fold greater cell proliferation, as judged by immunofluorescence for proliferating cell nuclear antigen (PCNA, Figure 4F, 4G). However, while BIBP did not affect the prevalence of macrophages, SMCs or collagen in atherosclerotic plaques, it reduced total plaque cell number, collagen content and plaque area by 56% (p,0.03, Figure 5). Thus, it appears that NPY, through the NPY1 receptor and perhaps other NPY receptors, contributes to atherogenesis. Discussion Our data provide the first evidence that NPY gene variants associate with CAD in humans, particularly those with early-onset CAD, and that NPY contributes to atherosclerosis in mice through its NPY1 receptor. Our results supporting the role of NPY in CAD pathogenesis emerge from multiple lines of evidence, including linkage, family-based and case-control association studies in multiple cohorts, as well as allele-specific differential NPY levels. PLoS Genetics | www.plosgenetics.org 4 January 2009 | Volume 5 | Issue 1 | e1000318