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Neuropeptide Y gene polymorphisms confer risk of early-onset atherosclerosis.

Shah SH, Freedman NJ, Zhang L, Crosslin DR, Stone DH, Haynes C, Johnson J, Nelson S, Wang L, Connelly JJ, Muehlbauer M, Ginsburg GS, Crossman DC, Jones CJ, Vance J, Sketch MH, Granger CB, Newgard CB, Gregory SG, Goldschmidt-Clermont PJ, Kraus WE, Hauser ER - PLoS Genet. (2009)

Bottom Line: In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families.A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04).We conclude that NPY contributes to atherosclerosis pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58-2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46-1.65, p = 0.01-0.05), showing stronger association in youngest cases (OR 1.84-2.20, p = 0.0004-0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79-2.06, p = 0.003-0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor-antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.

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Mouse carotid atherogenesis: cellular effects of a NPY Y1 receptor antagonist.Immediately after wire-mediated endothelial denudation, carotid arteries of apoe−/− mice were encased in Pluronic gel lacking (“None,” “control”) or containing the NPY1 receptor-selective antagonist BIBP 3226 (“BIBP”). Carotid arteries were harvested 6 wk (A–C) or 2 wk (D–G) postoperatively, and processed as described in Methods. A, B, Typical atherosclerosis in a representative control-treated carotid artery. Frozen sections were stained for DNA and (immunofluorescently) with IgG specific for SMC α-actin and either the macrophage marker Mac3 (A), or an isotype negative control rat IgG (B). Green autofluorescence of the elastic laminae can be seen in both A and B. Alternatively, frozen sections were stained for cholesteryl ester with Sudan IV and hematoxylin counterstain (C). Scale bar = 100 µm. D, E, Carotid sections were stained with IgG for cleaved caspase-3 (apoptosis marker) and counterstained for DNA; specimens stained with non-immune rabbit IgG yielded no red immunofluorescence (not shown). Scale bar = 50 µm. F, G, Carotid sections were stained with IgG for proliferating cell nuclear antigen (PCNA), counterstained for DNA, and oriented with the lumen side upward. The closed arrow designates a single PCNA-positive nucleus in the tunica media, representative of >20 such nuclei in the field shown; specimens stained with non-immune rabbit IgG (not shown) yielded only elastic lamina autofluorescence, like that observed in panels F and G. IEL, internal elastic lamina, indicated by the open arrows. Scale bar = 50 µm. All images are representative of ≥4 independent specimens stained with each modality.
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pgen-1000318-g004: Mouse carotid atherogenesis: cellular effects of a NPY Y1 receptor antagonist.Immediately after wire-mediated endothelial denudation, carotid arteries of apoe−/− mice were encased in Pluronic gel lacking (“None,” “control”) or containing the NPY1 receptor-selective antagonist BIBP 3226 (“BIBP”). Carotid arteries were harvested 6 wk (A–C) or 2 wk (D–G) postoperatively, and processed as described in Methods. A, B, Typical atherosclerosis in a representative control-treated carotid artery. Frozen sections were stained for DNA and (immunofluorescently) with IgG specific for SMC α-actin and either the macrophage marker Mac3 (A), or an isotype negative control rat IgG (B). Green autofluorescence of the elastic laminae can be seen in both A and B. Alternatively, frozen sections were stained for cholesteryl ester with Sudan IV and hematoxylin counterstain (C). Scale bar = 100 µm. D, E, Carotid sections were stained with IgG for cleaved caspase-3 (apoptosis marker) and counterstained for DNA; specimens stained with non-immune rabbit IgG yielded no red immunofluorescence (not shown). Scale bar = 50 µm. F, G, Carotid sections were stained with IgG for proliferating cell nuclear antigen (PCNA), counterstained for DNA, and oriented with the lumen side upward. The closed arrow designates a single PCNA-positive nucleus in the tunica media, representative of >20 such nuclei in the field shown; specimens stained with non-immune rabbit IgG (not shown) yielded only elastic lamina autofluorescence, like that observed in panels F and G. IEL, internal elastic lamina, indicated by the open arrows. Scale bar = 50 µm. All images are representative of ≥4 independent specimens stained with each modality.

Mentions: 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 apoe−/− 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 application 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 70±20% 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 1.7±0.3-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.


Neuropeptide Y gene polymorphisms confer risk of early-onset atherosclerosis.

Shah SH, Freedman NJ, Zhang L, Crosslin DR, Stone DH, Haynes C, Johnson J, Nelson S, Wang L, Connelly JJ, Muehlbauer M, Ginsburg GS, Crossman DC, Jones CJ, Vance J, Sketch MH, Granger CB, Newgard CB, Gregory SG, Goldschmidt-Clermont PJ, Kraus WE, Hauser ER - PLoS Genet. (2009)

Mouse carotid atherogenesis: cellular effects of a NPY Y1 receptor antagonist.Immediately after wire-mediated endothelial denudation, carotid arteries of apoe−/− mice were encased in Pluronic gel lacking (“None,” “control”) or containing the NPY1 receptor-selective antagonist BIBP 3226 (“BIBP”). Carotid arteries were harvested 6 wk (A–C) or 2 wk (D–G) postoperatively, and processed as described in Methods. A, B, Typical atherosclerosis in a representative control-treated carotid artery. Frozen sections were stained for DNA and (immunofluorescently) with IgG specific for SMC α-actin and either the macrophage marker Mac3 (A), or an isotype negative control rat IgG (B). Green autofluorescence of the elastic laminae can be seen in both A and B. Alternatively, frozen sections were stained for cholesteryl ester with Sudan IV and hematoxylin counterstain (C). Scale bar = 100 µm. D, E, Carotid sections were stained with IgG for cleaved caspase-3 (apoptosis marker) and counterstained for DNA; specimens stained with non-immune rabbit IgG yielded no red immunofluorescence (not shown). Scale bar = 50 µm. F, G, Carotid sections were stained with IgG for proliferating cell nuclear antigen (PCNA), counterstained for DNA, and oriented with the lumen side upward. The closed arrow designates a single PCNA-positive nucleus in the tunica media, representative of >20 such nuclei in the field shown; specimens stained with non-immune rabbit IgG (not shown) yielded only elastic lamina autofluorescence, like that observed in panels F and G. IEL, internal elastic lamina, indicated by the open arrows. Scale bar = 50 µm. All images are representative of ≥4 independent specimens stained with each modality.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2602734&req=5

pgen-1000318-g004: Mouse carotid atherogenesis: cellular effects of a NPY Y1 receptor antagonist.Immediately after wire-mediated endothelial denudation, carotid arteries of apoe−/− mice were encased in Pluronic gel lacking (“None,” “control”) or containing the NPY1 receptor-selective antagonist BIBP 3226 (“BIBP”). Carotid arteries were harvested 6 wk (A–C) or 2 wk (D–G) postoperatively, and processed as described in Methods. A, B, Typical atherosclerosis in a representative control-treated carotid artery. Frozen sections were stained for DNA and (immunofluorescently) with IgG specific for SMC α-actin and either the macrophage marker Mac3 (A), or an isotype negative control rat IgG (B). Green autofluorescence of the elastic laminae can be seen in both A and B. Alternatively, frozen sections were stained for cholesteryl ester with Sudan IV and hematoxylin counterstain (C). Scale bar = 100 µm. D, E, Carotid sections were stained with IgG for cleaved caspase-3 (apoptosis marker) and counterstained for DNA; specimens stained with non-immune rabbit IgG yielded no red immunofluorescence (not shown). Scale bar = 50 µm. F, G, Carotid sections were stained with IgG for proliferating cell nuclear antigen (PCNA), counterstained for DNA, and oriented with the lumen side upward. The closed arrow designates a single PCNA-positive nucleus in the tunica media, representative of >20 such nuclei in the field shown; specimens stained with non-immune rabbit IgG (not shown) yielded only elastic lamina autofluorescence, like that observed in panels F and G. IEL, internal elastic lamina, indicated by the open arrows. Scale bar = 50 µm. All images are representative of ≥4 independent specimens stained with each modality.
Mentions: 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 apoe−/− 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 application 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 70±20% 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 1.7±0.3-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.

Bottom Line: In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families.A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04).We conclude that NPY contributes to atherosclerosis pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58-2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46-1.65, p = 0.01-0.05), showing stronger association in youngest cases (OR 1.84-2.20, p = 0.0004-0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79-2.06, p = 0.003-0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor-antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.

Show MeSH
Related in: MedlinePlus