Journals RSS : Gourt

Vascular Incorporation of Endothelial Colony-Forming Cells Is Essential for Functional Recovery of Murine Ischemic Tissue Following Cell Therapy [Integrative Biology/Experimental Medicine]

Objective— Cord blood–derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell–based therapy, including mesenchymal stem cells (MSCs). Methods and Results— ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor+ cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. Conclusion— Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell–based therapy represents a promising approach for vascular regeneration of ischemic tissue.

Targeting Angiogenesis as Treatment for Obesity [Editorial]

Humanizing the Problem of Transplant Vasculopathy [Editorials]

Endothelial-to-Mesenchymal Transition and MicroRNA-21: The Game Is On Again [Editorials]

Intraplaque Hemorrhage: An Imaging Marker for Atherosclerotic Plaque Destabilization? [Editorials]

Life After GWAS: Functional Genomics in Vascular Biology [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

Strategies Beyond Genome-Wide Association Studies for Atherosclerosis [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

Atherosclerotic diseases, including coronary artery disease (CAD) and myocardial infarction (MI), are the leading causes of death in the world. The genetic basis of CAD and MI, which are caused by multiple interacting endogenous and exogenous factors, has gained considerable interest in the last years as genome-wide association studies (GWASs) have identified many new susceptibility loci for CAD and MI, and the underlying genes provide new insights into the genetic architecture of these diseases. Here we summarize the recent findings from GWASs of atherosclerosis and discuss their functional and biological implications. We also discuss the different post-GWAS strategies that are currently used for refining the location of causal variants, understanding their role, and shedding light on molecular mechanisms explaining their association to CAD. We finally discuss potential clinical translations of GWAS findings for individual risk prediction, advanced clinical strategies, and personalized treatments.

The Use of High-Throughput Technologies to Investigate Vascular Inflammation and Atherosclerosis [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

The greatest challenge of scientific research is to understand the causes and consequences of disease. In recent years, great efforts have been devoted to unraveling the basic mechanisms of atherosclerosis (the underlying pathology of cardiovascular disease), which remains a major cause of morbidity and mortality worldwide. Because of the complex and multifactorial pathophysiology of cardiovascular disease, different research techniques have increasingly been combined to unravel genetic aspects, molecular pathways, and cellular functions involved in atherogenesis, vascular inflammation, and dyslipidemia to gain a multifaceted picture addressing this complexity. Thanks to the rapid evolution of high-throughput technologies, we are now able to generate large-scale data on the DNA, RNA, and protein levels. With the help of sophisticated computational tools, these data sets are integrated to enhance information extraction and are being increasingly used in a systems biology approach to model biological processes as interconnected and regulated networks. This review exemplifies the use of high-throughput technologies—such as genomics, transcriptomics, proteomics, and epigenomics—and systems biology to explore pathomechanisms of vascular inflammation and atherosclerosis.

Recent Studies of the Human Chromosome 9p21 Locus, Which Is Associated With Atherosclerosis in Human Populations [ATVB in Focus Life After GWAS: Functional Genomics in Vascular Biology]

The chromosome 9p21 (Chr9p21) locus was discovered in 2007 by independent genome-wide association studies for coronary artery disease. Since then, the locus has been replicated numerous times and can be considered the most robust genetic marker of coronary artery disease today. Subsequent work has shown associations of Chr9p21 with a number of additional cardiovascular disease traits, such as carotid artery plaque, stroke, aneurysms, peripheral artery disease, heart failure, and cardiovascular mortality, suggesting a more general role in vascular pathology. Importantly, Chr9p21 lacks associations with common cardiovascular risk factors, such as lipids and hypertension, indicating that the locus exerts its effect through a completely novel mechanism. One of the challenges is that the core haplotype block at Chr9p21 resides in a region of the genome devoid of protein-coding genes. Recent progress has been made by functional studies focusing on differential expression of antisense noncoding RNA in the INK4 locus (ANRIL), which is transcribed from the Chr9p21 locus, as well as neighboring protein-coding genes at the INK4/ARF locus. The emerging concept suggests that ANRIL might constitute a regulator of epigenetic modification and thus modulate cardiovascular risk. Here, we review the current clinical, mechanistic, and diagnostic implications of the Chr9p21 locus in cardiovascular disease.

Beyond Genome-Wide Association Studies: The Usefulness of Mouse Genetics in Understanding the Complex Etiology of Atherosclerosis [ATVB in Focus: Life After GWAS: Functional Genomics in Vascular Biology]

The development of population-based genome-wide association studies has led to the rapid identification of large numbers of genetic variants associated with coronary artery disease (CAD) and related traits. Together with large-scale gene-centric studies, at least 35 loci associated with CAD per se have been identified with replication. The majority of these associations are with common single-nucleotide polymorphisms exhibiting modest effects on relative risk. The modest nature of the effects, coupled with ethical/practical constraints associated with human sampling, makes it difficult to answer important questions beyond gene/locus localization and allele frequency via human genetic studies. Questions related to gene function, disease-causing mechanism(s), and effective interventions will likely require studies in model organisms. The use of the mouse model for further detailed studies of CAD-associated loci identified by genome-wide association studies is highlighted herein.

Use of Functional Genomics to Identify Candidate Genes Underlying Human Genetic Association Studies of Vascular Diseases [ATVB in Focus Life After GWAS: Functional Genomics in Vascular Biology]

Recent genome-wide association studies have identified hundreds of genetic loci as being associated with vascular diseases or traits and their risk factors. Many of the loci uncovered represent novel discoveries with no obvious candidate genes and molecular mechanisms, testifying to the complexity of vascular diseases. To understand the functional consequences of genetic variations and help pinpoint the underlying genes and mechanisms of common complex diseases, functional genomics that integrate genetic variations and intermediate molecular traits such as gene expression has been extensively studied in the past few years. This review summarizes the key concepts of functional genomics, the current state of the field, and its application in vascular diseases.

S100A8 and S100A9 in Cardiovascular Biology and Disease [Brief Review]

There is recent and widespread interest in the damage-associated molecular pattern molecules S100A8 and S100A9 in cardiovascular science. These proteins have a number of interesting features and functions. For example, S100A8 and S100A9 (S100A8/A9) have both intracellular and extracellular actions, they are abundantly expressed in inflammatory and autoimmune states, primarily by myeloid cells but also by other vascular cells, and they modulate inflammatory processes, in part through Toll-like receptor 4 and the receptor for advanced glycation end products. S100A8/A9 also have anti-inflammatory and immune regulatory actions. Furthermore, increased plasma levels of S100A8/A9 predict cardiovascular events in humans, and deletion of these proteins partly protects Apoe–/– mice from atherosclerosis. Understanding the roles of S100A8 and S100A9 in vascular cell types and the mechanisms whereby these proteins mediate their biological effects may offer new therapeutic strategies to prevent, treat, and predict cardiovascular diseases.

Reciprocal Metabolic Perturbations in the Adipose Tissue and Liver of GPIHBP1-Deficient Mice [Integrative Biology/Experimental Medicine]

Objective— Gpihbp1-deficient (Gpihbp1–/–) mice lack the ability to transport lipoprotein lipase to the capillary lumen, resulting in mislocalization of lipoprotein lipase within tissues, defective lipolysis of triglyceride-rich lipoproteins, and chylomicronemia. We asked whether GPIHBP1 deficiency and mislocalization of catalytically active lipoprotein lipase would alter the composition of triglycerides in adipose tissue or perturb the expression of lipid biosynthetic genes. We also asked whether perturbations in adipose tissue composition and gene expression, if they occur, would be accompanied by reciprocal metabolic changes in the liver. Methods and Results— The chylomicronemia in Gpihbp1–/– mice was associated with reduced levels of essential fatty acids in adipose tissue triglycerides and increased expression of lipid biosynthetic genes. The liver exhibited the opposite changes: increased levels of essential fatty acids in triglycerides and reduced expression of lipid biosynthetic genes. Conclusion— Defective lipolysis in Gpihbp1–/– mice causes reciprocal metabolic perturbations in adipose tissue and liver. In adipose tissue, the essential fatty acid content of triglycerides is reduced and lipid biosynthetic gene expression is increased, whereas the opposite changes occur in the liver.

Apolipoprotein B Secretion Is Regulated by Hepatic Triglyceride, and Not Insulin, in a Model of Increased Hepatic Insulin Signaling [Integrative Biology/Experimental Medicine]

Objective— States of insulin resistance, hyperinsulinemia, and hepatic steatosis are associated with increased secretion of triglycerides (TG) and apolipoprotein B (apoB), even though insulin targets apoB for degradation. We used hepatic-specific "phosphatase and tensin homologue deleted on chromosome 10" (Pten) knockout (hPten-ko) mice, with increased hepatic insulin signaling, to determine the relative roles of insulin signaling and hepatic TG in regulating apoB secretion. Methods and Results— TG and apoB secretion was elevated in hPten-ko mice. When hepatic TG was reduced by inhibition of diacylglycerol acyltransferase 1/diacylglycerol acyltransferase 2 or sterol regulatory element-binding protein-1c, both TG secretion and apoB secretion fell without changes in hepatic insulin signaling. Acute reconstitution of hPten reduced hepatic TG content, and both TG and apoB secretion fell within 4 days despite decreased hepatic insulin signaling. Acute depletion of hepatic Pten by adenoviral introduction of Cre into Pten floxed mice caused steatosis within 4 days, and secretion of both TG and apoB increased despite increased hepatic insulin signaling. Even when steatosis after acute Pten depletion was prevented by pretreatment with SREBP-1c antisense oligonucleotides, apoB secretion was not reduced after 4 days. Ex vivo results were in primary hepatocytes were similar. Conclusion— Either hepatic TG is the dominant regulator of apoB secretion or any inhibitory effects of hepatic insulin signaling on apoB secretion is very short-lived.

Accelerated Atherosclerosis in Apoe-/- Mice Heterozygous for the Insulin Receptor and the Insulin Receptor Substrate-1 [Integrative Biology/Experimental Medicine]

Objective— Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. Methods and Results— To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor (Insr)/insulin receptor substrate-1 (Insr1) double heterozygous apolipoprotein (Apoe)-knockout mice (Insr+/–Irs1+/–Apoe–/–) mice. Insr+/–Irs1+/–Apoe–/– mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr+/+Irs1+/+Apoe–/– mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr+/–Irs1+/–Apoe–/– and Insr+/+Irs1+/+Apoe–/– mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr+/–Irs1+/–Apoe–/– compared to Insr+/+Irs1+/+Apoe–/– mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr+/–Irs1+/– to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr+/–Irs1+/–Apoe–/– mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr+/–Irs1+/–Apoe–/– mice. Conclusion— These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr+/–Irs1+/–Apoe–/– mice by promoting vascular dysfunction and inflammation.

Aldosterone Induces Vascular Insulin Resistance by Increasing Insulin-Like Growth Factor-1 Receptor and Hybrid Receptor [Integrative Biology/Experimental Medicine]

Objective— We previously showed that aldosterone induces insulin resistance in rat vascular smooth muscle cells (VSMCs). Because insulin-like growth factor-1 receptor (IGF1R) affects insulin signaling, we hypothesized that aldosterone induces vascular insulin resistance and remodeling via upregulation of IGF1R and its hybrid insulin/insulin-like growth factor-1 receptor. Methods and Results— Hybrid receptor expression was measured by immunoprecipitation. Hypertrophy of VSMCs was evaluated by 3H-labeled leucine incorporation. Aldosterone (10 nmol/L) significantly increased protein and mRNA expression of IGF1R and hybrid receptor in VSMCs but did not affect insulin receptor expression. Mineralocorticoid receptor blockade with eplerenone inhibited aldosterone-induced increases in IGF1R and hybrid receptor. Aldosterone augmented insulin (100 nmol/L)-induced extracellular signal-regulated kinase 1/2 phosphorylation. Insulin-induced leucine incorporation and α-smooth muscle actin expression were also augmented by aldosterone in VSMCs. These aldosterone-induced changes were significantly attenuated by eplerenone or picropodophyllin, an IGF1R inhibitor. Chronic infusion of aldosterone (0.75 μg/hour) increased blood pressure and aggravated glucose metabolism in rats. Expression of hybrid receptor, azan-positive area, and oxidative stress in aorta was increased in aldosterone-infused rats. Spironolactone and tempol prevented these aldosterone-induced changes. Conclusion— Aldosterone induces vascular remodeling through IGF1R- and hybrid receptor–dependent vascular insulin resistance. Mineralocorticoid receptor blockade may attenuate angiopathy in hypertensive patients with hyperinsulinemia.

ApoE Suppresses Atherosclerosis by Reducing Lipid Accumulation in Circulating Monocytes and the Expression of Inflammatory Molecules on Monocytes and Vascular Endothelium [Integrative Biology/Experimental Medicine]

Objective— We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. Methods and Results— We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoeh/hLdlr–/–) were compared to Apoe–/–Ldlr–/– mice. Despite 4-fold more plasma apoE than WT mice, Apoeh/hLdlr–/– mice displayed similar plasma cholesterol as Apoe–/– Ldlr–/– mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoeh/hLdlr–/– mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoeh/hLdlr–/– mice had less circulating leukocytes and proinflammatory Ly6Chigh monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoeh/hLdlr–/– mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. Conclusions— Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

Interleukin-17A Deficiency Accelerates Unstable Atherosclerotic Plaque Formation in Apolipoprotein E-Deficient Mice [Integrative Biology/Experimental Medicine]

Objective— Interleukin(IL)-17A, an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results— To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE–/–IL-17A–/–) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6 to 8 weeks. We found that splenic CD4+ T-cells produced high amounts of IL-17A in ApoE–/– mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE–/–IL-17A–/– mice compared with ApoE–/– mice. Splenic CD4+ T-cells of ApoE–/–IL-17A–/– mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased interferon gamma and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE–/–IL-17A–/– mice after HFD feeding for 8 weeks. Conclusion— These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating interferon gamma and IL-5 production from CD4+ T-cells.

Transsignaling of Interleukin-6 Crucially Contributes to Atherosclerosis in Mice [Integrative Biology/Experimental Medicine]

Objective— Transsignaling of interleukin (IL)-6 is a central pathway in the pathogenesis of disorders associated with chronic inflammation, such as Crohn disease, rheumatoid arthritis, and inflammatory colon cancer. Notably, IL-6 also represents an independent risk factor for coronary artery disease (CAD) in humans and is crucially involved in vascular inflammatory processes. Methods and Results— In the present study, we showed that treatment with a fusion protein of the natural IL-6 transsignaling inhibitor soluble glycoprotein 130 (sgp130) and IgG1-Fc (sgp130Fc) dramatically reduced atherosclerosis in hypercholesterolemic Ldlr–/– mice without affecting weight gain and serum lipid levels. Moreover, sgp130Fc treatment even led to a significant regression of advanced atherosclerosis. Mechanistically, endothelial activation and intimal smooth muscle cell infiltration were decreased in sgp130Fc-treated mice, resulting in a marked reduction of monocyte recruitment and subsequent atherosclerotic plaque progression. Of note, patients with CAD exhibited significantly lower plasma levels of endogenous sgp130, suggesting that a compromised counterbalancing of IL-6 transsignaling may contribute to atherogenesis in humans. Conclusion— These data clarify, for the first time, the critical involvement of, in particular, the transsignaling of IL-6 in CAD and warrant further investigation of sgp130Fc as a novel therapeutic for the treatment of CAD and related diseases.

Xanthine Oxidoreductase Is Involved in Macrophage Foam Cell Formation and Atherosclerosis Development [Integrative Biology/Experimental Medicine]

Objective— Hyperuricemia is common in patients with metabolic syndrome. We investigated the role of xanthine oxidoreductase (XOR) in atherosclerosis development, and the effects of the XOR inhibitor allopurinol on this process. Methods and Results— Oral administration of allopurinol to ApoE knockout mice markedly ameliorated lipid accumulation and calcification in the aorta and aortic root. In addition, allopurinol treatment or siRNA-mediated gene knockdown of XOR suppressed transformation of J774.1 murine macrophage cells, treated with acetylated LDL or very low density lipoprotein (VLDL) into foam cells. This inhibitory effect of allopurinol was also observed in primary cultured human macrophages. In contrast, overexpression of XOR promoted transformation of J774.1 cells into foam cells. Interestingly, SR-A1, SR-B1, SR-B II, and VLDL receptors in J774.1 cells were reduced by XOR knockdown, and increased by XOR overexpression. Conversely, expressions of ABCA1 and ABCG1 were increased by XOR knockdown and suppressed by XOR overexpression. Finally, productions of inflammatory cytokines accompanied by foam cell formation were also reduced by allopurinol administration. Conclusion— These results strongly suggest XOR activity and/or its expression level to contribute to macrophage foam cell formation. Thus, XOR inhibitors may be useful for preventing atherosclerosis.

Pharmacological Suppression of Hepcidin Increases Macrophage Cholesterol Efflux and Reduces Foam Cell Formation and Atherosclerosis [Integrative Biology/Experimental Medicine]

Objective— We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. Methods and Results— To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E–/– mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O–positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. Conclusion— These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

G Protein-Coupled Receptor Kinase-5 Attenuates Atherosclerosis by Regulating Receptor Tyrosine Kinases and 7-Transmembrane Receptors [Integrative Biology/Experimental Medicine]

Objective— G protein–coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-B (NF-B). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. Methods and Results— Grk5–/–/Apoe–/– mice developed 50% greater aortic atherosclerosis than Apoe–/– mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe–/– mice, carotid interposition grafts from Grk5–/– mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5–/– with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-β in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-B-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-B activity in macrophages. Conclusion— GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-B activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-β.

Inhibitor of Differentiation-3 Mediates High Fat Diet-Induced Visceral Fat Expansion [Integrative Biology/Experimental Medicine]

Objective— Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. Methods and Results— Id3–/– mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3–/– mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. Conclusion— Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Resolution of Mitochondrial Oxidative Stress Rescues Coronary Collateral Growth in Zucker Obese Fatty Rats [Integrative Biology/Experimental Medicine]

Objective— We have previously found abrogated ischemia-induced coronary collateral growth in Zucker obese fatty (ZOF) rats compared with Zucker lean (ZLN) rats. Because ZOF rats have structural abnormalities in their mitochondria suggesting dysfunction and also show increased production of O2macr;, we hypothesized that mitochondrial dysfunction caused by oxidative stress impairs coronary collateral growth in ZOF. Methods and Results— Increased levels of reactive oxygen species were observed in aortic endothelium and smooth muscle cells in ZOF rats compared with ZLN rats. Reactive oxygen species levels were decreased by the mitochondria-targeted antioxidants MitoQuinone (MQ) and MitoTempol (MT) as assessed by MitoSox Red and dihydroethidine staining. Lipid peroxides (a marker of oxidized lipids) were increased in ZOF by 47% compared with ZLN rats. The elevation in oxidative stress was accompanied by increased antioxidant enzymes, except glutathione peroxidase-1, and by increased uncoupling protein-2 in ZOF versus ZLN rats. In addition, elevated respiration rates were also observed in the obese compared with lean rats. Administration of MQ significantly normalized the metabolic profiles and reduced lipid peroxides in ZOF rats to the same level observed in lean rats. The protective effect of MQ also suppressed the induction of uncoupling protein-2 in the obese rats. Resolution of mitochondrial oxidative stress by MQ or MT restored coronary collateral growth to the same magnitude observed in ZLN rats in response to repetitive ischemia. Conclusion— We conclude that mitochondrial oxidative stress and dysfunction play a key role in disrupting coronary collateral growth in obesity and the metabolic syndrome, and elimination of the mitochondrial oxidative stress with MQ or MT rescues collateral growth.

Calpains Contribute to Vascular Repair in Rapidly Progressive Form of Glomerulonephritis: Potential Role of Their Externalization [Integrative Biology/Experimental Medicine]

Objective— Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. Methods and Results— A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. Conclusion— This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.

Effect of Ionizing Radiation Induced Damage of Endothelial Progenitor Cells in Vascular Regeneration [Integrative Biology/Experimental Medicine]

Objective— A number of studies have revealed that stress signaling and subsequent stress responses in stem/progenitor cells are responsible for attenuated regeneration or degenerative disease. Because ionizing radiation (IR), which sensitizes diverse types of stem cells, reportedly induces cardio-circulatory diseases, we hypothesized that IR-induced vascular abnormalities are associated with defects in endothelial progenitor cells (EPCs) that are responsible for vascular homeostasis. Methods and Results— We used an irradiated mouse model to mimic the IR effect on vasculogenesis. Mouse EPCs isolated from irradiated mice and human EPCs exposed to IR were used for functional analysis and gene expression study. Under IR exposure, EPCs were depleted, and their function for vasculogenesis in vitro and in vivo was significantly reduced. In such IR-mediated stress responses, upregulating p21Cip1 and downregulating vascular endothelial growth factor (VEGF) were mediated by p53 transcriptional activity. Conclusion— The results of the present study suggest that suppression of p53 would be clinically applicable to (1) minimize the functional defects in EPCs in order to prevent the onset of vascular diseases caused by radiation therapy or radiation exposure and also to (2) provide novel insight into the mechanisms of IR-induced vascular damage and a possible strategy to minimize vascular damage by IR.

Reperfusion Injury Intensifies the Adaptive Human T Cell Alloresponse in a Human-Mouse Chimeric Artery Model [Integrative Biology/Experimental Medicine]

Objective— Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. Methods and Results— Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell–mediated injury to human artery segments. Conclusion— We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Transforming Growth Factor-{beta}-Induced Endothelial-to-Mesenchymal Transition Is Partly Mediated by MicroRNA-21 [Integrative Biology/Experimental Medicine]

Objective— MicroRNAs are a class of small ribonucleotides regulating gene/protein targets by transcript degradation or translational inhibition. Transforming growth factor-β (TGF-β) is involved in cardiac fibrosis partly by stimulation of endothelial-to-mesenchymal transition (EndMT). Here, we investigated whether microRNA (miR)-21, a microRNA enriched in fibroblasts and involved in general fibrosis, has a role in cardiac EndMT. Methods and Results— TGF-β treatment of endothelial cells significantly increased miR-21 expression and induced EndMT characterized by suppression of endothelial and increase of fibroblast markers. Overexpression of miR-21 alone also stimulated EndMT. Importantly, miR-21 blockade by transfection of specific microRNA inhibitors partly prevented TGF-β-induced EndMT. Mechanistically, miR-21 silenced phosphatase and tensin homolog in endothelial cells, resulting in activation of the Akt-pathway. Akt inhibition partly restored TGF-β-mediated loss of endothelial markers during EndMT. In vivo, pressure overload of the left ventricle led to increased expression of miR-21 in sorted cardiac endothelial cells, which displayed molecular and phenotypic signs of EndMT. This was attenuated by treatment of mice subjected to left ventricular pressure overload with an antagomir against miR-21. Conclusion— TGF-β-mediated EndMT is regulated at least in part by miR-21 via the phosphatase and tensin homolog/Akt pathway. In vivo, antifibrotic effects of miR-21 antagonism are partly mediated by blocking EndMT under stress conditions.

Dynamic Synchrotron Imaging of Diabetic Rat Coronary Microcirculation In Vivo [Integrative Biology/Experimental Medicine]

Objective— In diabetes, long-term micro- and macrovascular damage often underlies the functional decline in the cardiovascular system. However, it remains unclear whether early-stage diabetes is associated with in vivo functional impairment in the coronary microvasculature. Synchrotron imaging allows us to detect and quantify regional differences in resistance microvessel caliber in vivo, even under conditions of high heart rate. Methods and Results— Synchrotron cine-angiograms of the coronary vasculature were recorded using anesthetized Sprague-Dawley rats 3 weeks after treatment with vehicle or streptozotocin (diabetic). In the early diabetic state, in the presence of nitric oxide and prostacyclin, vessel diameters were smaller (P<0.01) and endothelium-dependent vessel recruitment was already depressed (P<0.05). Endothelium-dependent and -independent vasodilatory responses in individual coronary vessels were not different in vivo. Inhibition of NO and PGI2 production in diabetes uncovered early localized impairment in dilation. Diabetic animals displayed focal stenoses and segmental constrictions during nitric oxide synthase/cyclooxygenase blockade, which persisted during acetylcholine infusion (P<0.05), and a strong trend toward loss of visible microvessels. Conclusion— Synchrotron imaging provides a novel method to investigate coronary microvascular function in vivo at all levels of the arterial tree. Furthermore, we have shown that early-stage diabetes is associated with localized coronary microvascular endothelial dysfunction.

The Proteoglycan Syndecan 4 Regulates Transient Receptor Potential Canonical 6 Channels via RhoA/Rho-associated Protein Kinase Signaling [Integrative Biology/Experimental Medicine]

Objective— Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular permeability, in a RhoA/Rho-associated protein kinase-dependent manner. Methods and Results— Sdc4 knockout (Sdc4–/–) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P<0.05). Using reverse transcription–polymerase chain reaction and immunoblotting, Sdc4–/– mice showed reduced TRPC6 mRNA by 79% and TRPC6 protein by 82% (each P<0.05). Sdc4–/– mice showed an increased RhoA activity by 87% and increased phosphorylation of ezrin in glomeruli by 48% (each P<0.05). Sdc4 knockdown in cultured podocytes reduced TRPC6 gene expression and reduced the association of TRPC6 with plasma membrane and TRPC6-mediated calcium influx and currents. Sdc4 knockdown inactivated negative regulatory protein Rho GTPase activating protein by 33%, accompanied by a 41% increase in RhoA activity and increased phosphorylation of ezrin (P<0.05). Conversely, overexpression of Sdc4 reduced RhoA activity and increased TRPC6 protein and TRPC6-mediated calcium influx and currents. Conclusion— Our results establish a previously unknown function of Sdc4 for regulation of TRPC6 channels and support the role of Sdc4 for the regulation of glomerular permeability.

Syndecan-1 Displays a Protective Role in Aortic Aneurysm Formation by Modulating T Cell-Mediated Responses [Integrative Biology/Experimental Medicine]

Objective— Chronic inflammation drives progressive and pathological remodeling inherent to formation of abdominal aortic aneurysm (AAA). Syndecan-1 (Sdc-1) is a cell surface heparan sulfate proteoglycan that displays the capacity to modulate inflammatory processes within the vascular wall. In the current investigation, the role of Sdc-1 in AAA formation was examined using 2 models of experimental aneurysm induction, angiotensin II infusion and elastase perfusion. Methods and Results— Sdc-1 deficiency exacerbated AAA formation in both experimental models and was associated with increased degradation of elastin, greater protease activity, and enhanced inflammatory cell recruitment into the aortic wall. Bone marrow transplantation studies indicated that deficiency of Sdc-1 in marrow-derived cells significantly contributed to AAA severity. Immunostaining revealed augmented Sdc-1 expression in a subset of AAA localized macrophages. We specifically characterized a higher percentage of CD4+ T cells in Sdc-1-deficient AAA, and antibody depletion studies established the active role of T cells in aneurysmal dilatation. Finally, we confirmed the ability of Sdc-1 macrophage to modulate the inflammatory chemokine environment. Conclusion— These investigations identify cross-talk between Sdc-1-expressing macrophages and AAA-localized CD4+ T cells, with Sdc-1 providing an important counterbalance to T-cell–driven inflammation in the vascular wall.

Regulation of Peroxisome Proliferator-Activated Receptor-{gamma} by Angiotensin II Via Transforming Growth Factor-{beta}1-Activated p38 Mitogen-Activated Protein Kinase in Aortic Smooth Muscle Cells [Cell Biology/Signaling]

Objective— Peroxisome proliferator–activated receptor- (PPAR) ligands attenuate angiotensin II (Ang II)–induced atherosclerosis through interactions with vascular smooth muscle cell (VSMC)–specific PPAR in hypercholesterolemic mice. Therefore, the purpose of this study was to determine the mechanism of Ang II–mediated intracellular regulation of PPAR in VSMCs. Methods and Results— Incubation of cultured mouse aortic VSMCs with Ang II for 24 hours reduced abundance of PPAR protein, mRNA, and transcriptional activity (P<0.001). This effect was attenuated by an angiotensin type 1 receptor antagonist, losartan. Ang II–induced PPAR reduction was dependent on stimulation of transforming growth factor (TGF)-β1 as demonstrated using either a neutralizing antibody or small interfering RNA (siRNA). Ang II–induced TGF-β1 secretion was dependent on epidermal growth factor receptor kinase activation through reactive oxygen species production. Inhibition of p38 mitogen-activated protein kinase by SB203580 or siRNA inhibited both Ang II– and TGF-β1–induced PPAR reduction. Blockade of TGF-β1 decreased p38 phosphorylation induced by Ang II. siRNA–mediated inhibition of histone deacetylase 3 attenuated p38-mediated reductions in PPAR abundance. Conclusion— These findings suggest that Ang II decreases PPAR abundance in cultured VSMCs via an angiotensin type 1 receptor–dependent secretion of TGF-β1 via phosphorylation of p38 mitogen-activated protein kinase and histone deacetylase 3.

Fatty Acid-Induced Nuclear Translocation of Heparanase Uncouples Glucose Metabolism in Endothelial Cells [Cell Biology/Signaling]

Objective— Heparanase is an endoglycosidase that specifically cleaves carbohydrate chains of heparan sulfate. We have recently reported that high fatty acid increased the nuclear content of endothelial heparanase. Here, we examined the mechanism and the consequences behind this nuclear translocation of heparanase. Methods and Results— Bovine coronary artery endothelial cells were grown to confluence and incubated with palmitic acid. Palmitic acid induced rapid nuclear accumulation of heparanase that was dependent on Bax activation and lysosome permeabilization. Heat shock protein 90 was an important mediator of palmitic acid-induced shuttling of heparanase to the nucleus. Nuclear heparanase promoted cleavage of heparan sulfate, a potent inhibitor of histone acetyltransferase activity and gene transcription. A TaqMan gene expression assay revealed an increase in genes related to glucose metabolism and inflammation. In addition, glycolysis was uncoupled from glucose oxidation, resulting in accumulation of lactate. Conclusion— The results presented in this study demonstrate that fatty acid can provoke lysosomal release of heparanase, its nuclear translocation, activation of genes controlling glucose metabolism, and accumulation of lactate. Given that lactate and inflammation have been implicated in the progression of atherosclerosis, our data may serve to reduce the associated cardiovascular complications seen during diabetes.

NADPH Oxidase 4 Mediates Monocyte Priming and Accelerated Chemotaxis Induced by Metabolic Stress [Cell Biology/Signaling]

Objective— Metabolic disorders increase monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis in mice. The goal of this study was to determine the molecular mechanisms responsible for the enhanced responsiveness of monocytes to chemoattractants induced by metabolic stress. Methods and Results— Chronic exposure of monocytes to diabetic conditions induced by human LDL plus high D-glucose concentrations (LDL+HG) promoted NADPH Oxidase 4 (Nox4) expression, increased intracellular H2O2 formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, platelet-derived growth factor B, and RANTES. Both H2O2 added exogenously and overexpression of Nox4 mimicked LDL+HG-induced monocyte priming, whereas Nox4 knockdown protected monocytes against metabolic stress-induced priming and accelerated chemotaxis. Exposure of monocytes to LDL+HG promoted the S-glutathionylation of actin, decreased the F-actin/G-actin ratio, and increased actin remodeling in response to MCP-1. Preventing LDL+HG-induced protein S-glutathionylation by overexpressing glutaredoxin 1 prevented monocyte priming and normalized monocyte chemotaxis in response to MCP-1. Induction of hypercholesterolemia and hyperglycemia in C57BL/6 mice promoted Nox4 expression and protein S-glutathionylation in macrophages, and increased macrophage recruitment into MCP-1–loaded Matrigel plugs implanted subcutaneous in these mice. Conclusion— By increasing actin-S-glutathionylation and remodeling, metabolic stress primes monocytes for chemoattractant-induced transmigration and recruitment to sites of vascular injury. This Nox4-dependent process provides a novel mechanism through which metabolic disorders promote atherogenesis.

Factor VII-Activating Protease Promotes the Proteolysis and Inhibition of Tissue Factor Pathway Inhibitor [Cell Biology/Signaling]

Objective— Factor VII–activating protease (FSAP) activates both factor VII and pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating hemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk of carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Methods and Results— Using screening procedures to determine the influence of FSAP on various hemostasis-related processes on endothelial cells, we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anticoagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting, and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP that specifically inhibited FSAP binding to TFPI reversed the inhibitory effect of FSAP on TFPI. Conclusion— The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating hemostasis and proliferative remodeling events in the vasculature.

Rap1-Rac1 Circuits Potentiate Platelet Activation [Cell Biology/Signaling]

Objective— The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. Methods and Results— We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI– and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. Conclusion— We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI– and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

Deleted in Malignant Brain Tumors 1 is Present in the Vascular Extracellular Matrix and Promotes Angiogenesis [Cell Biology/Signaling]

Objective— Deleted in malignant brain tumors 1 (DMBT1) belongs to the scavenger receptor cysteine-rich superfamily of proteins and is implicated in innate immunity, cell polarity, and differentiation. Here we studied the role of DMBT1 in endothelial cells. Methods and Results— DMBT1 was secreted into the extracellular matrix (ECM) by endothelial cells in vitro and in situ and the presence of DMBT1 in the ECM increased endothelial cell adherence. Endothelial cell-derived DMBT1 associated with galectin-3 (coprecipitation), and human recombinant DMBT1 bound EGF, vascular endothelial growth factor and Delta-like (Dll) 4 (specific ELISAs). Compared to cells from wild-type mice, endothelial cells from DMBT1–/– mice demonstrated reduced migration, proliferation, and tube formation. In vivo recovery from hindlimb ischemia was attenuated in DMBT1–/– animals as was vascular endothelial growth factor -induced endothelial sprouting from isolated aortic rings; the latter response could be rescued by the addition of recombinant DMBT1. The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation and we found that endothelial cells from DMBT1–/– mice expressed more EphrinB2 than cells from wild-type mice. Levels of Dll1, Dll4, Hes1, Hey1, and EphB4, on the other hand, were decreased. Conclusion— Taken together, the results of this study indicate that DMBT1 functions as an important endothelium-derived ECM protein that is able to bind angiogenic factors and promote adhesion, migration, proliferation, and angiogenesis as well as vascular repair. Mechanistically, DMBT1 interacts with galectin-3 and modulates the Notch signaling pathway as well as the differential expression of ephrin-B2 and EphB4.

Urokinase-Type Plasminogen Activator Downregulates Paraoxonase 1 Expression in Hepatocytes by Stimulating Peroxisome Proliferator-Activated Receptor-{gamma} Nuclear Export [Cell Biology/Signaling]

Objective— The atherosclerotic lesion is characterized by lipid peroxide accumulation. Paraoxonase 1 (PON1) reduces atherosclerotic lesion oxidative stress, whereas urokinase-type plasminogen activator (uPA) increases oxidative stress in atherosclerotic lesions and contributes to the progression and complications of atherosclerosis. We hypothesized that uPA may promote oxidative stress in the arterial wall via modulation of PON1 activity. Because the liver is the main site for PON1 production, in the present study, we tested whether uPA influences PON1 expression in hepatocytes. Methods and Results— HuH7 hepatocytes were incubated in culture with increasing concentrations of uPA. uPA decreased PON1 gene expression and activity in a dose-dependent manner and accordingly suppressed PON1 secretion from hepatocytes. This effect required uPA/uPA receptor interaction. uPA downregulated PON1 gene expression via inactivation of peroxisome proliferator–activated receptor- (PPAR) activity, and this effect was dependent on uPA-mediated mitogen-activated protein kinase kinase activation. Mechanistic studies showed that uPA enhanced mitogen-activated protein kinase kinase–PPAR interaction, resulting in PPAR nuclear export to the cytosol. Conclusion— This study provides the first evidence that uPA interferes with PPAR transcriptional activity in hepatocytes, resulting in downregulation of PON1 expression and its secretion to the medium. This may explain, at least in part, the prooxidative effect of uPA in the vascular wall.

Apolipoprotein B-100-Containing Lipoprotein Metabolism in Subjects With Lipoprotein Lipase Gene Mutations [Clinical and Population Studies]

Objective— We investigated the impact of lipoprotein lipase (LPL) gene mutations on apolipoprotein B (apoB)-100 metabolism. Methods and Results— We studied 3 subjects with familial LPL deficiency; 14 subjects heterozygous for the LPL gene mutations Gly188Glu, Trp64Stop, and Ile194Thr; and 10 control subjects. Very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL)-apoB-100 kinetics were determined in the fed state using stable isotope methods and compartmental modeling. Compared with controls, familial LPL deficiency had markedly elevated plasma triglycerides and lower VLDL-apoB-100 fractional catabolic rate (FCR), IDL-apoB-100 FCR, VLDL-to-IDL conversion, and VLDL-apoB-100 production rate (P<0.01). Compared with controls, Gly188Glu had higher plasma triglyceride and VLDL- and IDL-apoB-100 concentrations and lower VLDL- and IDL-apoB-100 FCR (P<0.05). Plasma triglycerides were not different, but IDL-apoB-100 concentration and production rate and VLDL-to-IDL conversion were lower in Trp64Stop compared with controls (P<0.05). No differences between controls and Ile194Thr were observed. Conclusion— Our results confirm that hypertriglyceridemia is a key feature of familial LPL deficiency. This is due to impaired VLDL- and IDL-apoB-100 catabolism and VLDL-to-IDL conversion. Single-allele mutations of the LPL gene result in modest to elevated plasma triglycerides. The changes in plasma triglycerides and apoB-100 kinetics are attributable to the effects of the LPL genotype.

Arteriolar Function in Visceral Adipose Tissue Is Impaired in Human Obesity [Clinical and Population Studies]

Objective— The purpose of this study was to characterize the relationship between adipose tissue phenotype and depot-specific microvascular function in fat. Methods and Results— In 30 obese subjects (age 42±11 years, body mass index 46±11 kg/m2) undergoing bariatric surgery, we intraoperatively collected visceral and subcutaneous adipose tissue and characterized depot-specific adipose phenotypes. We assessed vasomotor function of the adipose microvasculature using videomicroscopy of small arterioles (75–250 μm) isolated from different fat compartments. Endothelium-dependent, acetylcholine-mediated vasodilation was severely impaired in visceral arterioles, compared to the subcutaneous depot (P<0.001 by ANOVA). Nonendothelium dependent responses to papaverine and nitroprusside were similar. Endothelial nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester reduced subcutaneous vasodilation but had no effect on severely blunted visceral arteriolar responses. Visceral fat exhibited greater expression of proinflammatory, oxidative stress-related, hypoxia-induced, and proangiogenic genes; increased activated macrophage populations; and had a higher capacity for cytokine production ex vivo. Conclusion— Our findings provide clinical evidence that the visceral microenvironment may be intrinsically toxic to arterial health providing a potential mechanism by which visceral adiposity burden is linked to atherosclerotic vascular disease. Our findings also support the evolving concept that both adipose tissue quality and quantity may play significant roles in shaping cardiovascular phenotypes in human obesity.

Preventive Effects of Exenatide on Endothelial Dysfunction Induced by Ischemia-Reperfusion Injury via KATP Channels [Clinical and Population Studies]

Objective— The purpose of this study was to evaluate whether exenatide administration can prevent impairment in endothelium-dependent vasodilatation induced by ischemia-reperfusion (IR) injury and whether this effect is mediated by KATP channel opening. Methods and Results— In a double-blind, placebo-controlled, crossover design, 20 volunteers were randomly assigned to 2 groups: subcutaneous exenatide (10 μg) or placebo administration. At 30 minutes after the study drug administration, endothelium-dependent flow-mediated dilatation (FMD) of the radial artery was measured before and after IR (15 minutes of ischemia at the level of the brachial artery followed by 15 minutes of reperfusion) injury. Seven days later, both groups were crossed over and received the other treatment (ie, placebo or exenatide) and underwent the same protocol. Pre-IR radial artery diameter, FMD, and baseline radial artery diameter after IR injury were similar between 2 groups (P=no significant difference). After placebo administration, IR significantly blunted FMD (before IR: 12.0±6.23%; after IR: 4.6±3.57%, P=0.02). Exenatide prevented this impairment (FMD before IR: 15.0±7.14%; FMD after IR: 15.0±5.96%, P=no significant difference; P<0.001 compared with placebo). In a separate protocol, this protective effect was completely abolished by pretreatment with glibenclamide (glyburide, 5 mg), a blocker of KATP channels (n=7; FMD before IR: 12.0±2.2%; after IR: 3.2±2.1%, P<0.001). Conclusion— The present study demonstrates that subcutaneous exenatide protects IR-induced endothelial dysfunction through opening of KATP channels in human IR injury model.

Association of Testosterone Levels With Endothelial Function in Men: Results From a Population-Based Study [Clinical and Population Studies]

Objective— Because population-based data are lacking, we assessed the cross-sectional association between serum testosterone levels and endothelial function, as measured by flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NMD) of the brachial artery, in men from the population-based Study of Health in Pomerania. Methods and Results— Personal characteristics, including major cardiovascular confounders, were collected in 722 men, aged 25 to 85 years. Serum total testosterone and sexual hormone-binding globulin (SHBG) levels were determined by chemiluminescence immunoassays. Free testosterone levels were calculated according to the law of mass action. FMD and NMD measurements were performed using standardized ultrasound techniques. FMD and NMD values below the 20th percentile were considered decreased. Multivariable logistic regression analyses revealed an association for each decrement of total testosterone standard deviation (6.0 nmol/L) with decreased FMD after adjustment for potential confounders (odds ratio 1.30, 95% confidence interval 1.04–1.63; P=0.023). Multiple adjusted findings for free testosterone were similar (odds ratio 1.37, 95% confidence interval 1.06–1.76; P=0.016). There was no such association of SHBG levels with decreased FMD. Neither testosterone nor SHBG levels were significantly associated with decreased NMD. Conclusion— Lower serum total and free testosterone levels are associated with impaired endothelial function in this population-based sample of men.

Competing Risk of Atherosclerotic Risk Factors for Arterial and Venous Thrombosis in a General Population: The Tromso Study [Clinical and Population Studies]

Objective— To investigate and compare the impact of traditional atherosclerotic risk factors for the risk of arterial and venous thrombosis, taking into account competing risks. Methods and Results— In 1994–1995, 26,185 subjects were screened in the Tromsø study. Information on traditional atherosclerotic risk factors was obtained by physical examination, blood samples, and questionnaires. Subjects were followed to the first incident event of myocardial infarction (MI) or venous thromboembolism (VTE), or December 31, 2005. During a median of 10.8 years of follow-up, there were 1279 cases of incident MI and 341 VTE events. Advancing age and high body mass index were both associated with MI and VTE. Hazard ratio per decade of age was 2.34 (95% CI: 2.25–2.43) for MI and 1.87 (1.74–2.01) for VTE, and 3 kg/m2 increase in body mass index was associated with 1.16 (1.11–1.21) and 1.20 (1.12–1.29) increased risk of MI and VTE, respectively. Blood pressure, high levels of triglycerides and total cholesterol, low HDL cholesterol, self-reported diabetes, and smoking were all associated with increased risk of MI but not associated with VTE. Conclusion— Our findings imply that traditional atherosclerotic risk factors, such as smoking, hypertension, dyslipidemia, and diabetes mellitus are not shared by arterial and venous thrombosis.

Relationships Between Recent Intraplaque Hemorrhage and Stroke Risk Factors in Patients With Carotid Stenosis: The HIRISC Study [Clinical and Population Studies]

Objective— Intraplaque hemorrhage (IPH) is an emerging marker of plaque instability. However, little is known about the relationships between IPH and traditional risk factors and whether these relationships differ between symptomatic and asymptomatic disease. Methods and Results— Two hundred thirty-four patients with symptomatic (n=114) or asymptomatic (n=120) carotid stenosis underwent high-resolution plaque magnetic resonance imaging. Seventy-five patients had recent IPH (symptomatic, 33%; asymptomatic, 31%). In symptomatic stenosis, recent IPH was independently associated with degree of stenosis (odds ratio [OR]=4.21, 1.61–10.98 for North American Symptomatic Carotid Endarterectomy Trial >35%; OR=2.92, 1.18–7.24 for European Carotid Surgery Trial >60%), qualifying event (OR=4.13; 1.11–15.32 for stroke or hemispheric transient ischemic attack ≥1 hour versus transient ischemic attack <1 hour or ocular symptoms), time from ischemic event (OR=6.65, 1.56–28.35 for ≤2 weeks; OR=2.24, 0.87–5.81 for 2–12 weeks versus >12 weeks; P for trend=0.03). In asymptomatic stenosis, IPH was only associated with stenosis severity >70% by ECST (OR=6.65; 1.95–22.73) but not by the NASCET method. Conclusion— Our findings support the potential link between recent IPH and risk of ipsilateral stroke in symptomatic disease but also imply that prognostic studies should adjust for known stroke risk factors in multivariate analyses. In asymptomatic stenosis, the potential predictive value of recent IPH is less likely to be confounded by stroke risk factors.

Objectively Assessed Physical Activity, Sedentary Time, and Coronary Artery Calcification in Healthy Older Adults [Clinical and Population Studies]

Objective— Physical activity is related to lower risk of cardiovascular disease, but data relating to coronary lesions have been conflicting. These inconsistencies may in part be due to unreliable assessment of physical activity and limitations imposed by self-reported data. The purpose of this study was to determine the relationship between objectively measured physical activity and coronary artery calcium (CAC). Methods and Results— Participants were 443 healthy men and women (mean age=66±6 years), without history or objective signs of coronary heart disease, drawn from the Whitehall II epidemiological cohort. Physical activity was objectively measured using accelerometers worn during waking hours for 7 consecutive days (average daily wear time=889±68 minutes/day). CAC was measured in each participant using electron beam computed tomography and was quantified according to the Agatston scoring system. On average, 54.4% of the sample recorded at least 30 minutes/day of moderate to vigorous physical activity (MVPA). There was no association between MVPA and presence of detectable CAC. For the participants with detectable CAC (n=283) a weak inverse relationship between MVPA (minutes/day) and log Agatston score was observed (B=–0.008, 95% CI: –0.16 to 0.00, P=0.05), although the association was no longer present after adjustments for age, sex, and conventional risk factors. No associations were seen for light activity or sedentary time. Conclusion— Our results confirm no association between objectively assessed physical activity and CAC. Because CAC measures cannot identify more vulnerable lesions, additional studies are required to examine whether physical activity can promote plaque stability.

Genetic Variation in ABCG1 and Risk of Myocardial Infarction and Ischemic Heart Disease [Clinical and Population Studies]

Objective— ATP binding cassette transporter G1 (ABCG1) facilitates cholesterol efflux from macrophages to mature high-density lipoprotein particles. Whether genetic variation in ABCG1 affects risk of atherosclerosis in humans remains to be determined. Methods and Results— We resequenced the core promoter and coding regions of ABCG1 in 380 individuals from the general population. Next, we genotyped 10 237 individuals from the Copenhagen City Heart Study for the identified variants and determined the effect on lipid and lipoprotein levels and on risk of myocardial infarction (MI) and ischemic heart disease (IHD). g.–376C>T, g.-311T>A, and Ser630Leu predicted risk of MI in the Copenhagen City Heart Study, with hazard ratios of 2.2 (95% confidence interval: 1.2–4.3), 1.7 (1.0–2.9), and 7.5 (1.9–30), respectively. These results were confirmed for g.–376C>T in a case-control study comprising 4983 independently ascertained IHD cases and 7489 controls. Expression levels of ABCG1 mRNA were decreased by approximately 40% in g.–376C>T heterozygotes versus noncarriers (probability values: 0.005–0.009). Finally, in vitro specificity protein 1 (Sp1) bound specifically to a putative Sp1 binding site at position –382 to –373 in the ABCG1 promoter, and the presence of the –376 T allele reduced binding and transactivation of the promoter by Sp1. Conclusion— This is the first report of a functional variant in ABCG1 that associates with increased risk of MI and IHD in the general population.

Clinical and Genetic Association of Serum Ceruloplasmin With Cardiovascular Risk [Clinical and Population Studies]

Objective— Ceruloplasmin (Cp) is an acute-phase reactant that is increased in inflammatory diseases and in acute coronary syndromes. Cp has recently been shown to possess nitric oxide (NO) oxidase catalytic activity, but its impact on long-term cardiovascular outcomes in stable cardiac patients has not been explored. Methods and Results— We examined serum Cp levels and their relationship with incident major adverse cardiovascular events (MACE; death, myocardial infarction [MI], stroke) over 3-year follow-up in 4177 patients undergoing elective coronary angiography. We also carried out a genome-wide association study to identify the genetic determinants of serum Cp levels and evaluate their relationship to prevalent and incident cardiovascular risk. In our cohort (age 63±11 years, 66% male, 32% history of MI, 31% diabetes mellitus), mean Cp level was 24±6 mg/dL. Serum Cp level was associated with greater risk of MI at 3 years (hazard ratio [quartile 4 versus 1] 2.35, 95% confidence interval [CI] 1.79–3.09, P<0.001). After adjustment for traditional risk factors, high-sensitivity C-reactive protein, and creatinine clearance, Cp remained independently predictive of MACE (hazard ratio 1.55, 95% CI 1.10–2.17, P=0.012). A 2-stage genome-wide association study identified a locus on chromosome 3 over the CP gene that was significantly associated with Cp levels (lead single-nucleotide polymorphism rs13072552; P=1.90x10–11). However, this variant, which leads to modestly increased serum Cp levels (1.5–2 mg/dL per minor allele copy), was not associated with coronary artery disease or future risk of MACE. Conclusion— In stable cardiac patients, serum Cp provides independent risk prediction of long-term adverse cardiac events. Genetic variants at the CP locus that modestly affect serum Cp levels are not associated with prevalent or incident risk of coronary artery disease in this study population.

Soluble Vascular Adhesion Protein-1 Correlates With Cardiovascular Risk Factors and Early Atherosclerotic Manifestations [Clinical and Population Studies]

Objective— Vascular adhesion protein-1 is an endothelial enzyme that regulates leukocyte traffic and contributes to vascular damage in animal models. The relations of soluble vascular adhesion protein-1 (sVAP-1) with cardiovascular risk factors and markers of subclinical atherosclerosis at a population level have not been studied. Methods and Results— We developed a new high-throughput method and measured sVAP-1 activities in serum of 2183 persons (The Cardiovascular Risk in Young Finns Study). In women, sVAP-1 activity correlated indirectly with body mass index (r=–0.15, P<0.0001), triglycerides (r=–0.13, P<0.0001), C-reactive protein (r=–0.23; P<0.0001), and brachial artery flow-mediated vasodilatation (r=–0.076, P=0.0089) and directly with carotid plaques (r=0.066, P=0.023). None of these correlations was significant in men. In women, all these univariate correlations remained significant after adjustment for body mass index, and direct correlations with LDL-cholesterol (r=0.094, P=0.0014) and carotid intima-media thickness (r=0.075, P=0.010) became evident. In men, sVAP-1 activity associated directly with glucose (r=0.074, P=0.020), intima-media thickness (r=0.072, P=0.025), metabolic syndrome (P=0.016), and type 1 (P=0.0002) and type 2 (P<0.0001) diabetes. In multivariable analyses, sVAP-1 activity was an independent determinant of carotid intima-media thickness (P=0.0072) and plaques [odds ratio 1.71 (95% confidence interval 1.07–2.72, P=0.025] in women, but not in men. Conclusion— sVAP-1 activity correlates directly with intima-media thickness and carotid plaques in general population and may play a role in the pathophysiology of preclinical atherosclerosis.