Smoking is a leading cause of preventable death, causing approximately five million premature deaths world-wide each year 1, 2 . Evidence for genetic influence on smoking behaviour and nicotine dependence (ND) 3-8 has prompted a search for susceptibility genes. Furthermore, assessing the impact of sequence variants on smoking-related diseases is important for public health reasons 9, 10 . Smoking is the major risk factor for lung cancer (LC) [11][12][13][14] , and one of the main risk factors for peripheral arterial disease (PAD) [15][16][17] . We have identified a common variant in the nicotinic acetylcholine receptor gene cluster on chromosome 15q24 with an effect on smoking quantity, ND and the risk of two smoking-related diseases in populations of European descent. The variant has an effect on the number of cigarettes smoked per day in 15,771 smokers (P=6×10 −20 ). The same variant associated with ND in a previous genome-wide association study using low quantity smokers as controls (OR=1.3, P=1×10 −3 ) 18,19 , and with a similar approach we observe a highly significant association with ND (OR =1.40, P=7×10 −15 ). Comparison of LC (N=1,024) and PAD (N= 2,738) cases with about 30,000 population controls each showed that the variant confers risk of LC (OR=1.31, P=1.5×10 −8 ) and PAD (OR=1.19, P=1.4×10 −7 ). The findings highlight the role of nicotine addiction in the pathogenesis of other serious diseases and provide a case study of the role of active gene-environment correlation 20 in the pathogenesis of disease.To perform a genome-wide association (GWA) study of smoking quantity (SQ), we utilised questionnaire data limited to basic questions on smoking behaviour that were available for a large number of lifetime smokers. The GWA scan comprises 10,995 Icelandic smokers who Reprints and permissions information is available at www.nature.com/reprints.

Recently, two common sequence variants on 9p21, tagged by rs10757278-G and rs10811661-T, were reported to be associated with coronary artery disease (CAD) and type 2 diabetes (T2D), respectively. We proceeded to further investigate the contributions of these variants to arterial diseases and T2D. Here we report that rs10757278-G is associated with, in addition to CAD, abdominal aortic aneurysm (AAA; odds ratio (OR) = 1.31, P = 1.2 x 10(-12)) and intracranial aneurysm (OR = 1.29, P = 2.5 x 10(-6)), but not with T2D. This variant is the first to be described that affects the risk of AAA and intracranial aneurysm in many populations. The association of rs10811661-T to T2D replicates in our samples, but the variant does not associate with any of the five arterial diseases examined. These findings extend our insight into the role of the sequence variant tagged by rs10757278-G and show that it is not confined to atherosclerotic diseases.

Sonic hedgehog (Shh) is a morphogen regulating crucial epithelial-mesenchymal interactions during embryonic development, but its signaling pathway is considered generally silent in post-natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant upregulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signaling pathway has an important regulatory role on injury-induced angiogenesis, as inhibition of Shh function results in impaired upregulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1alpha, decreased muscle blood flow, and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf-5 and MyoD, decreases the upregulation of insulin-like growth factor (IGF)-1, and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction, and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post-natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.

OBJECTIVEHigh-mobility group box-1 (HMGB1) protein is a nuclear DNA-binding protein released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis. Diabetic human and mouse tissues contain lower levels of HMGB1 than their normoglycemic counterparts. Deficient angiogenesis after ischemia contributes to worse outcomes of peripheral arterial disease in patients with diabetes. To test the hypothesis that HMGB1 enhances ischemia-induced angiogenesis in diabetes, we administered HMGB1 protein in a mouse hind limb ischemia model using diabetic mice.RESEARCH DESIGN AND METHODSAfter the induction of diabetes by streptozotocin, we studied ischemia-induced neovascularization in the ischemic hind limb of normoglycemic, diabetic, and HMGB1-treated diabetic mice.RESULTSWe found that the perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic control mice. Interestingly, HMGB1 protein expression was lower in the ischemic tissue of diabetic mice than in normoglycemic mice. Furthermore, we observed that HMGB1 administration restored the blood flow recovery and capillary density in the ischemic muscle of diabetic mice, that this process was associated with the increased expression of vascular endothelial growth factor (VEGF), and that HMGB1-induced angiogenesis was significantly reduced by inhibiting VEGF activity.CONCLUSIONSThe results of this study show that endogenous HMGB1 is crucial for ischemia-induced angiogenesis in diabetic mice and that HMGB1 protein administration enhances collateral blood flow in the ischemic hind limbs of diabetic mice through a VEGF-dependent mechanism.

Background and Purpose-Proinflammatory genetic profiles, resulting from the combination of single nucleotide polymorphisms in genes encoding inflammatory molecules, may contribute to the development and progression of cardiovascular diseases. We evaluated the association between history of ischemic stroke and genetic profiles determined by the synergistic effects of polymorphisms in genes encoding prototypical inflammatory proteins. Methods-The study included 237 individuals with history of ischemic stroke and 223 age-matched and gender-matched controls. The polymorphisms of the C-reactive protein (CRP), interleukin-6 (IL-6), macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin (E-sel), and matrix metalloproteinase-3 (MMP-3) genes were studied. Results-IL-6 GG, IL-6 GC, MCP-1 GG, ICAM-1 EE, E-sel AA, and MMP-3 5A5A genotypes were significantly and independently associated with stroke history. The odds of stroke increased with the number of high-risk genotypes: carrying 1 proinflammatory gene variant conferred a risk of 3.3 (1.6 to 6.9), whereas individuals concomitantly carrying 2 and 3 proinflammatory gene variants had adjusted odds ratios of 21.0 (7.6 to 57.5) and 50.3 (10.2 to 248.1), respectively. Conclusions-Proinflammatory genetic profiles are significantly more common in subjects with stroke history. Synergistic effects between proinflammatory genotypes might be potential markers for cerebrovascular diseases.

OBJECTIVE-Peroxisome proliferator-activated receptors (PPARs) are therapeutic targets for fibrates and thiazolidinediones, which are commonly used to ameliorate hyperlipidemia and hyperglycemia in type 2 diabetes. In this study, we evaluated whether activation of PPAR␣ and PPAR␥ stimulates neoangiogenesis.RESEARCH DESIGN AND METHODS-We used selective synthetic PPAR␣ and PPAR␥ agonists and investigated their angiogenic potentials in vitro and in vivo.RESULTS-Activation of PPAR␣ and PPAR␥ leads to endothelial tube formation in an endothelial/interstitial cell co-culture assay. This effect is associated with increased production of the angiogenic cytokine vascular endothelial growth factor (VEGF). Neovascularization also occurs in vivo, when PPAR␣ and PPAR␥ agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPAR␣ and PPAR␥ agonists are respectively used in PPAR␣ knockout mice and mice treated with a specific PPAR␥ inhibitor, demonstrating that this angiogenic response is PPAR mediated. PPAR␣-and PPAR␥-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPAR␣-and PPAR␥-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPAR␣ and PPAR␥ agonists, there is increased phosphorylation of endothelial nitric oxide synthase and Akt.CONCLUSIONS-These findings demonstrate that PPAR␣ and PPAR␥ activation stimulates neoangiogenesis through a VEGFdependent mechanism. Neoangiogenesis is a crucial pathological event in type 2 diabetes. The ability of PPAR␣ and PPAR␥ agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of type 2 diabetes. Diabetes 57:1394-1404, 2008 P eroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors that belong to the nuclear hormone receptor superfamily (1). The clinical importance of PPARs originates with fibrates and thiazolidinediones (TZDs), which respectively act on PPAR␣ and PPAR␥ and are used to ameliorate hyperlipidemia and hyperglycemia in subjects with type 2 diabetes. Fibrates (gemfibrozil, clofibrate, fenofibrate, and bezofibrate) are drugs that effectively reduce triglycerides (TGs) and free fatty acids (FFAs) and increase HDL cholesterol (2-5). Fibrates also improve glucose tolerance in type 2 diabetic patients, although this activity might be attributable to the fact that some of these compounds also have potential PPAR␥ activity (6). TZDs (such as rosiglitazone, troglitazone, pioglitazone, and ciglitazone) are insulin-sensitizing drugs and have constituted a major advance in the recent therapeutic management of type 2 diabetes (7-9). In addition to improving insulin sensitivity, TZDs have also effects on TG, FFA, and ketone body level in several animal models of type 2 diabetes. Recently, PPAR␣/␥ dual agonists have also been produced, hypothesizing that the simultaneous activa...

Peripheral artery disease (PAD) is one of the most relevant complications of diabetes. Although several pharmacological and revascularization approaches are available for treating patients with diabetes and PAD, an endovascular approach is often associated with postprocedural complications that can increase the risk for acute limb ischemia or amputation. However, no definitive molecular associations have been described that could explain the difference in outcomes after endovascular treatment in patients with diabetes, PAD, and chronic limb-threatening ischemia (CLTI). RESEARCH DESIGN AND METHODSWe evaluated the relationship between the levels of the main cytokines associated with diabetic atherosclerosis and the outcomes after endovascular procedures in patients with diabetes, PAD, and CLTI. RESULTSA total of 299 patients with below-the-knee occlusive disease who were undergoing an angioplasty procedure were enrolled. The levels of key cytokinesdosteoprotegerin (OPG), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and C-reactive protein (CRP)dwere measured, and major adverse limb events (MALE) and major adverse cardiovascular events (MACE) were assessed 1, 3, 6, and 12 months after the procedure. There was a linear trend from the lowest to the highest quartile for each cytokine at baseline and incident MALE. A linear association was also observed between increasing levels of each cytokine and incident MACE. Receiver operating characteristics models were constructed using clinical and laboratory risk factors, and the inclusion of cytokines significantly improved the prediction of incident events.

There is a synergistic effect of IL-6 G/C and ICAM-1 E/K gene polymorphisms in patients with stroke history. Reciprocal interactions between genotypes may contribute in determining the risk profile for cardiovascular diseases and may merit further investigation as potential therapeutic targets.