bluuebunny/crossref_metadata_2025 huggingface dataset:
Colocalization of Thrombin, PAI-1, and Vitronectin in the Atherosclerotic Vessel Wall
Authors: Stoop A. Allart , Lupu Florea , Pannekoek Hans
Published: June 2011

Abstract
—The serine protease thrombin is a mitogen for vascular smooth muscle cells. To that end, thrombin cleaves the surface-exposed, protease-activated receptor type 1 (PAR-1), resulting in signal transduction and ultimately, proliferation of these cells. Regulation of thrombin activity in the human atherosclerotic vessel wall has not been studied in great detail, conceivably because the traditional plasma thrombin inhibitor, anti–thrombin III, is not encountered at this location. By using immunofluorescence confocal microscopy, we demonstrate that the antigens of thrombin, plasminogen activator inhibitor 1 (PAI-1), and vitronectin (Vn) colocalize in human neointimal atherosclerotic arterial tissue. Furthermore, it is shown by in situ reverse zymography that these specimens harbor the active form of PAI-1, which is the only configuration of PAI-1 capable of complexing with Vn and inhibiting serine proteases, eg, thrombin. Two different criteria were used to establish that neointimal atherosclerotic material contains active α-thrombin, namely, its ability to bind to the thrombin inhibitor hirudin and to convert the thrombin-specific chromogenic substrate S2238. The latter activity could be fully prevented by preincubation with the thrombin-specific inhibitor, phenyl-prolyl-arginyl-chloromethyl ketone. The thrombin concentration measured by conversion of the chromogenic substrate was 7 to 12 nmol/L in the vascular specimens studied. This concentration range suffices to activate the PAR-1 receptor on vascular smooth muscle cells and to cause neointimal proliferation. It is concluded that the human atherosclerotic arterial vessel wall provides conditions that favor a regulatory mechanism of thrombin activity by PAI-1/Vn complexes.
Differential Activation of Mitogen-Activated Protein Kinases in Smooth Muscle Cells by Angiotensin II
Authors: Viedt Christiane , Soto Ubaldo , Krieger-Brauer Heidemarie Ingrid , Fei Jianwei , Elsing Christoph , Kübler Wolfgang , Kreuzer Jörg
Published: June 2011

Abstract
—The atherogenic effect of the renin-angiotensin system can be explained, in part, by the influence of its effector, angiotensin II (Ang II), on vascular smooth muscle cell (VSMC) growth. There is evidence that reactive oxygen species (ROS) play a role in the atherogenesis and activation of mitogen-activating protein (MAP) kinases, which are involved in proliferation and differentiation. The study was performed to further characterize the role of ROS in Ang II–mediated MAP kinase activation and the regulation of the transcription factor activator protein-1 (AP-1). Rat VSMCs were stimulated with Ang II. The activities of MAP kinases were assessed by Western blot analysis or by immunocomplex kinase assay. AP-1 binding was determined by using an electrophoretic mobility shift assay. Rat VSMCs were treated with Ang II–activated MAP kinases, extracellular signal–regulated kinase (ERK), c-Jun amino terminal kinase (JNK), p38 MAP kinase (p38 MAPK), and their downstream effector, AP-1. Interestingly, only the activation of ERK1/2, but not JNK or p38 MAPK, was tyrosine kinase, protein kinase C, and MEK1/2 dependent. Ang II also induced the rapid formation of ROS, which could be inhibited by a specific antibody as well as by antisense against the p22phox subunit of the NAD(P)H oxidase. JNK and p38 MAPK, but not ERK, activation was inhibited by an inhibitor of NAD(P)H oxidase. Antisense against p22phox also solely inhibited p38 MAPK but did not affect ERK. The results indicate that in VSMCs, Ang II activates MAP kinases and AP-1 through different pathways; the results further suggest that ROS, generated by p22phox, mediate Ang II–induced JNK and p38 MAPK activation, which may contribute to the pathogenesis of atherosclerosis.
Endothelin-1 Inhibits Apoptosis of Vascular Smooth Muscle Cells Induced by Nitric Oxide and Serum Deprivation via MAP Kinase Pathway
Authors: Shichiri Masayoshi , Yokokura Masaaki , Marumo Fumiaki , Hirata Yukio
Published: June 2011

Abstract
—Endothelin (ET)-1, an endothelium-derived vasoconstrictor and mitogen, acts as an antiapoptotic factor against serum deprivation–induced apoptosis of endothelial cells and fibroblasts but enhances apoptosis of some cancer cells. In the present study, we examined whether nitric oxide (NO) and ET-1 modulate apoptosis of rat vascular smooth muscle cells (VSMCs) via the mitogen-activated protein (MAP) kinase pathway. Both serum deprivation and NO donors (FK409 and SNAP) caused apoptosis of VSMCs, as demonstrated by TdT-mediated dUTP-biotin nick end-labeling, appearance of fragmented DNA, and induction of caspase-3 activity. ET-1 dose-dependently antagonized apoptosis induced by serum deprivation and NO donors. A selective ET
A
receptor antagonist (BQ123) and a nonselective ET
A/B
receptor antagonist (TAK044), but not a selective ET
B
receptor antagonist (BQ788), inhibited the antiapoptotic effect of ET-1, indicating that the antiapoptotic effect of ET-1 is mediated via the ET
A
receptor. ET-1 activated MAP kinase, whose effect was inhibited by FK409. Transfection with an unphosphorylated wild-type MAP kinase kinase-1 (MAPKK-1) or its constitutively activated mutant protected VSMCs against apoptosis induced by serum deprivation and NO donors. Inhibition of MAP kinase activity with PD98059, a specific inhibitor of MAPKK-1, or by transfection of a dominant-negative MAPKK-1 mutant antagonized the antiapoptotic effect of ET-1, suggesting the involvement of MAP kinase in the antiapoptotic effect. The potent inhibitory effect of ET-1 on apoptosis of VSMCs induced by serum deprivation and NO suggests that the counterbalance between the 2 endothelium-derived factors contributes to the process of vascular remodeling by determining VSMC survival and death, respectively, via a common MAP kinase pathway.
Protein-Tyrosine Phosphatases in the Vessel Wall
Authors: Wright Matthew B. , Seifert Ronald A. , Bowen-Pope Daniel F.
Published: June 2011

Abstract
—Many protein-tyrosine phosphatases (PTPases) have now been identified, but little is known about PTPase expression and regulation in vascular tissue and in vascular disease. Polymerase chain reaction (PCR) amplification and cDNA fingerprinting of PTPase catalytic domains, combined with random sequencing of PCR product libraries, identified 18 (8 receptor-like and 10 cytosolic) PTPases in the rat carotid artery and revealed differential expression of 5 of these PTPases during neointima formation after balloon catheter injury. In situ hybridization was used to localize mRNA expression in vessel cross sections for the 5 differentially expressed PTPases. This revealed that for 3 PTPases (SHP1, CD45, and PTPβ), differential transcript abundance was due to appearance/loss of the cell types by which they were expressed (leukocytes for SHP1 and CD45, endothelial cells for PTPβ). However, mRNA expression of 2 PTPases (PTPL1 and PTP1B) was specifically upregulated by proliferating and migrating smooth muscle cells (SMCs) in characteristic temporal and regional patterns in response to vessel damage. Quantitative PCR analysis showed that PTP1B and PTPL1 were induced ≈30-fold and ≈60-fold, respectively, by 2 weeks after injury in the damaged vessels compared with the uninjured vessels. PTP1B was rapidly upregulated in the media after vessel injury and remained highly expressed in the developing neointima. By contrast, PTPL1 expression did not increase dramatically until the SMCs had migrated into the intima. The differential expression of PTP1B and PTPL1 by SMCs after injury suggests roles for these PTPases in the regulation of vessel wall remodeling.
Human ApoA-IV Overexpression in Transgenic Mice Induces cAMP-Stimulated Cholesterol Efflux From J774 Macrophages to Whole Serum
Authors: Fournier Natalie , Atger Véronique , Paul Jean-Louis , Sturm Marie , Duverger Nicolas , Rothblat George H. , Moatti Nicole
Published: June 2011

Abstract
—The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.
Common Polymorphism in Promoter of Microsomal Triglyceride Transfer Protein Gene Influences Cholesterol, ApoB, and Triglyceride Levels in Young African American Men
Authors: Juo Suh-Hang Hank , Han Zhihua , Smith Jonathan D. , Colangelo Laura , Liu Kiang
Published: June 2011

Abstract
—The microsomal triglyceride transfer protein (MTP) plays a key role in the assembly of apolipoprotein B (apoB)-containing lipoproteins. We investigated the relation between lipid profiles and a common functional polymorphism (−493G/T) of the MTP gene in a large sample of young black men in the Coronary Artery Risk Development in Young Adults (CARDIA) Study. We performed serial cross-sectional analyses on lipids of 586 black men in 5 exams over 10 years of follow-up. Total cholesterol, LDL cholesterol, and apoB levels were very similar between the GT and GG genotypes; therefore, the GT and GG genotypes were combined as 1 group when the 3 phenotypes were analyzed. The results from ANCOVA showed that the TT group (prevalence 7%) had higher levels of apoB-related lipids than did the GT+GG group: the difference in total cholesterol ranged from 2 (
P
=0.79) to 19 (
P
=0.002) mg/dL in exams 1 to 5; the difference in LDL cholesterol ranged from 10 (
P
=0.14) to 17 (
P
=0.003) mg/dL in exams 1 to 4, but in examination 5, the difference became negligible. The TT group had higher levels of apoB, measured in only 2 exams, by 6 (
P
=0.12) and 9 (
P
=0.03) mg/dL. The TT group had higher levels of triglycerides than did the TG or GG group by 3 to 34 (
P
=0.02 to ≈0.003) mg/dL in all 5 exams. HDL cholesterol and apolipoprotein A-I levels were similar among the 3 genotypes. Our serial cross-sectional analyses indicated that the TT genotype was associated with higher levels of total cholesterol, LDL cholesterol, triglycerides, and apoB in young black men. The broad effect of this polymorphism on several atherogenic traits suggests that the MTP gene could be influential in atherosclerosis.
Smooth Muscle Cell Matrix Metalloproteinase Production Is Stimulated via α
v
β
3
Integrin
Authors: Bendeck Michelle P. , Irvin Colleen , Reidy Michael , Smith Laura , Mulholland Diane , Horton Michael , Giachelli Cecilia M.
Published: June 2011

Abstract
—This study tests the hypothesis that α
v
β
3
integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that β
3
integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the β
3
integrin–blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8±30.8 cells/mm
2
in control rats to 10.29±7.03 cells/mm
2
in F11-treated rats (
P
=0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed α
v
β
3
integrin receptors, whereas adult SMCs did not. Stimulation of newborn (α
v
β
3
+) SMCs with osteopontin, a matrix ligand for α
v
β
3
, increased MMP-1 production from 114.4±35.8 ng/mL at 0 nmol/L osteopontin to 232.5±57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC α
v
β
3
integrin receptors play an important role in regulating migration by stimulating SMC MMP production.
Lipoteichoic Acid Induces Delayed Protection in the Rat Heart
Authors: Zacharowski Kai , Frank Stefan , Otto Mike , Chatterjee Prabal K. , Cuzzocrea Salvatore , Hafner Gerd , Pfeilschifter Josef , Thiemermann Christoph
Published: June 2011

Abstract
—Classic ischemic preconditioning transiently (30 to 120 minutes) protects the myocardium against subsequent lethal ischemia/reperfusion injury. After dissipation of this acute protection, a second window of protection (SWOP) appears 12 to 24 hours later; this SWOP lasts up to 3 days. Several triggers induce a SWOP, including brief repetitive cycles of coronary artery occlusion, rapid ventricular pacing, stimulation of adenosine A
1
receptors, and administration of wall fragments of Gram-negative bacteria, such as lipopolysaccharide (LPS). The aim of this study was to investigate whether lipoteichoic acid (LTA), a cell wall fragment of Gram-positive bacteria, can induce a SWOP in a rat model of left anterior descending coronary artery (LAD) occlusion (25 minutes) and reperfusion (2 hours). Thus, 166 male Wistar rats were pretreated (2 to 24 hours) with saline, LTA (1 mg/kg IP), or LPS (1 mg/kg IP) and subjected to LAD occlusion/reperfusion. Pretreatment with LTA or LPS for 16 hours led to a substantial, ≈65%, reduction in infarct size and a reduction in the release of cardiac troponin T into the plasma. The dose of LTA used had no toxic effect (on any of the parameters studied), whereas the same dose of LPS caused a time-dependent activation of the coagulation system and liver injury. By use of RNase protection assays, it was determined that LPS caused a time-dependent induction of tumor necrosis factor-α, interleukin-1β, and manganese superoxide dismutase mRNA content in the heart, whereas LTA failed to induce manganese superoxide dismutase. LPS also caused an upregulation of the expression of intercellular adhesion molecule-1 and P-selectin, whereas LTA downregulated these molecules and attenuated the accumulation of polymorphonuclear granulocytes caused by myocardial ischemia/reperfusion. This study demonstrates for the first time that pretreatment with LTA at 8 to 24 hours before myocardial ischemia significantly reduces (1) infarct size, (2) cardiac troponin T, and (3) the histological signs of tissue injury in rats subjected to LAD occlusion and reperfusion. The mechanism(s) underlying the observed cardioprotective effects of LTA warrants further investigation but is likely to be related to its ability to inhibit the interactions between the coronary vascular endothelium and polymorphonuclear granulocytes. Therefore, LTA represents a novel and promising agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury.
High Levels of Nonesterified Fatty Acids Are Associated With Increased Familial Risk of Cardiovascular Disease
Authors: Carlsson M. , Wessman Y. , Almgren P. , Groop L.
Published: June 2011

Abstract
—To address the question of whether elevated concentrations of nonesterified fatty acids (NEFAs) are associated with an increased risk of cardiovascular disease, we measured NEFA concentrations in 140 diabetic and 343 nondiabetic unrelated Swedish subjects with a family history of type 2 diabetes and related the findings to history of cardiovascular disease in their parents. Parents of nondiabetic offspring belonging to the quartile of highest NEFA concentrations had a higher risk of myocardial infarction (35% versus 16%,
P
<0.01) and stroke (45% versus 16%,
P
<0.0005) than did parents of offspring from the lowest NEFA quartile. In a multiple logistic regression analysis, a high NEFA concentration in offspring was significantly associated with myocardial infarction and stroke in their parents. No such relationship was observed between diabetic offspring and their parents. Assuming that the same relationship between NEFA concentrations and cardiovascular disease is seen in the offspring and their parents, the findings suggest that elevated NEFA concentration is a risk factor for cardiovascular disease and could be pathogenically involved in the atherosclerotic process.
Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin
Authors: Hop Caroline , Guilliatt Andrea , Daly Martina , de Leeuw Hubert P. , Brinkman Herm-Jan M. , Peake Ian R. , van Mourik Jan A. , Pannekoek Hans
Published: June 2011

Abstract
—We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)–like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD’D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA–transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA–transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules.
G-250A Substitution in Promoter of Hepatic Lipase Gene Is Associated With Dyslipidemia and Insulin Resistance in Healthy Control Subjects and in Members of Families With Familial Combined Hyperlipidemia
Authors: Pihlajamäki Jussi , Karjalainen Leena , Karhapää Pauli , Vauhkonen Ilkka , Taskinen Marja-Riitta , Deeb Samir S. , Laakso Markku
Published: June 2011

Abstract
—Low activity of hepatic lipase (HL) has been associated with high levels of triglycerides and high density lipoproteins, but the association of the HL promoter variants with insulin sensitivity has not been investigated. Therefore, in this study, the relationship of the G-250A promoter variant of the HL gene to the rates of insulin-stimulated glucose uptake measured by the hyperinsulinemic euglycemic clamp was investigated in 110 control subjects (82 men and 28 women, aged 50.7±7.6 [mean±SD] years, body mass index 26.1±3.6 kg/m
2
) and in 105 first-degree relatives (65 men and 40 women, aged 47.8±16.0 years, body mass index 26.9±5.3 kg/m
2
) of 34 families with familial combined hyperlipidemia (FCHL). The A-250 allele of the HL promoter was associated with low rates of insulin-stimulated whole-body nonoxidative glucose disposal in control subjects (41.1±12.7 μmol · kg
−1
· min
−1
in subjects with the G-250G genotype, 36.9±13.1 μmol · kg
−1
· min
−1
in subjects with the G-250A genotype, and 29.9±13.5 μmol · kg
−1
· min
−1
in subjects with the A-250A genotype;
P
=0.012 adjusted for age and sex) and with low rates of insulin-stimulated whole-body glucose oxidation in FCHL family members (16.7±4.2 versus 15.0±4.4 versus 14.1±4.4 μmol · kg
−1
· min
−1
,
P
=0.024). In addition, the A-250 allele was associated with high levels of fasting insulin (
P
=0.047), very low density lipoprotein cholesterol (
P
=0.007), and total (
P
=0.009) and very low density lipoprotein (
P
=0.005) triglycerides in control subjects and with high levels of low density lipoprotein triglycerides (
P
=0.001) in FCHL family members (n=340). We conclude that the G-250A promoter variant of the HL gene is associated with dyslipidemia and insulin resistance. Mechanisms via which this polymorphism could affect insulin sensitivity remain to be elucidated.
Nitric Oxide Synthase in Atherosclerosis and Vascular Injury
Authors: Channon Keith M. , Qian HuSheng , George Samuel E.
Published: June 2011

Abstract
—Gene therapy aims to intervene in a disease process by transfer and expression of specific genes in a target tissue or organ. Cardiovascular gene therapy in humans remains in its infancy, but in the last decade, experimental gene transfer has emerged as a powerful biological tool to investigate the function of specific genes in vascular disease pathobiology. Nitric oxide synthases, the enzymes that produce nitric oxide, have received considerable attention as potential candidates for vascular gene therapy because nitric oxide has pleiotropic antiatherogenic actions in the vessel wall, and abnormalities in nitric oxide biology are apparent very early in the atherogenic process. In this article, we review the use of nitric oxide synthases in experimental vascular gene therapy and assess the utility of these approaches for investigating the role of nitric oxide in atherosclerosis and their potential for human gene therapy.
Early Effects of Arterial Hemodynamic Conditions on Human Saphenous Veins Perfused Ex Vivo
Authors: Mavromatis Kreton , Fukai Tohru , Tate Matthew , Chesler Naomi , Ku David N. , Galis Zorina S.
Published: June 2011

Abstract
—Exposure to the arterial hemodynamic environment is thought to be a potential trigger for the pathological remodeling of saphenous vein grafts. Using matched pairs of freshly isolated human saphenous vein, we analyzed the early effects of ex vivo hemodynamic conditions mimicking the venous (native) compared with arterial (graft) environment on the key components of vascular remodeling, ie, matrix metalloproteinase (MMP)-9 and MMP-2 and cell proliferation. Interestingly, we found that arterial conditions halved latent MMP-9 (50±11%,
P
=0.01) and MMP-2 (44±6%,
P
=0.005) levels relative to matched vein pairs maintained ex vivo under venous perfusion for up to 3 days. Immunostaining supported decreased MMP levels in the innermost area of arterially perfused veins. Either decreased synthesis or increased posttranslational processing may decrease MMP zymogen levels. Biosynthetic radiolabeling showed that arterial perfusion actually increased MMP-9 and MMP-2 production. When we then examined potential pathways for MMP zymogen processing, we found that arterial conditions did not affect the expression of MT-MMP-1, a cell-associated MMP activator, but that they significantly increased the levels of superoxide, another MMP activator, suggesting redox-dependent MMP processing. Additional experiments indicated that increased superoxide under arterial conditions was due to diminished scavenging by decreased extracellular superoxide dismutase. Arterial perfusion also stimulated cell proliferation (by 220% to 750%) in the majority of vein segments investigated. Our observations support the hypothesis that arterial hemodynamic conditions stimulate early vein graft remodeling. Furthermore, physiological arterial flow may work to prevent pathological remodeling, particularly the formation of intimal hyperplasia, through rapid inactivation of secreted MMPs and, possibly, through preferential stimulation of cell proliferation in the outer layers of the vein wall.
Modification of Protein Moiety of Human Low Density Lipoprotein by Hypochlorite Generates Strong Platelet Agonist
Authors: Volf Ivo , Bielek Edith , Moeslinger Thomas , Koller Franz , Koller Elisabeth
Published: June 2011

Abstract
—Conflicting reports exist about the effects of mildly or extensively oxidized low density lipoproteins (LDLs) on the reactivity of human platelets. This platelet response is mainly caused by modification of the protein and lipid moiety, giving rise to very differently modified species with hardly predictable properties. The aim of this study was to prepare oxidized LDL with modifications essentially restricted to the protein moiety and to determine the eventual platelet responses. We treated LDL at 0°C for 10 minutes with a 60- to 1000-fold molar excess of sodium hypochlorite in borate buffer in the presence of the radical scavenger butylated hydroxytoluene. Under these conditions, neither fragmentation of apolipoprotein B-100 nor formation of LDL aggregates was observed, and lipid oxidation products did not exceed the amount present in untreated LDLs. The degree of modification and the respective effects on platelet function were highly reproducible. Hypochlorite-modified LDLs act as strong platelet agonists, inducing morphological changes, dense granule release, and irreversible platelet aggregation. The evoked platelet effects are completely suppressed by inhibitors of the phosphoinositide cycle but not by EDTA or acetylsalicylic acid. Most likely, these effects are transmitted via high-affinity binding to a single class of sites, which does not recognize native or acetylated LDL. Obviously, modified lysines, and the secondary lipid modifications derived from them, are not essential for this interaction. We conclude that bioactive oxidized lipids are not directly involved in the stimulation of platelets by hypochlorite-modified LDLs.
Does ADMA Cause Endothelial Dysfunction?
Authors: Cooke John P.
Published: June 2011

Abstract
—Asymmetric dimethylarginine (ADMA) is an endogenous and competitive inhibitor of nitric oxide synthase. Plasma levels of this inhibitor are elevated in patients with atherosclerosis and in those with risk factors for atherosclerosis. In these patients, plasma ADMA levels are correlated with the severity of endothelial dysfunction and atherosclerosis. By inhibiting the production of nitric oxide, ADMA may impair blood flow, accelerate atherogenesis, and interfere with angiogenesis. ADMA may be a novel risk factor for vascular disease.
Stimulation of Receptor-Mediated Nitric Oxide Production by Vanadate
Authors: Hellermann Gary R. , Flam Brenda R. , Eichler Duane C. , Solomonson Larry P.
Published: June 2011

Abstract
—Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore– (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to ≈125 kDa and 2 minor bands corresponding to ≈105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.
Tobacco Smoking Modifies Association Between Gln-Arg192 Polymorphism of Human Paraoxonase Gene and Risk of Myocardial Infarction
Authors: Sen-Banerjee Sucharita , Siles Xinia , Campos Hannia
Published: June 2011

Abstract
—Paraoxonase, a high density lipoprotein–associated human serum enzyme, plays a role in atherosclerosis by protecting against lipid peroxidation. Its activity is modulated by 2 common amino acid polymorphisms at positions 192 (Gln→Arg) and 55 (Met→Leu) in the paraoxonase gene (
PON1
)
.
We studied the association of
PON1
polymorphisms and myocardial infarction (MI) in a population-based study consisting of 492 cases and 518 controls matched for age, sex, and area of residence, all living in Costa Rica. The allele frequency of
PON1
192Arg
was higher in cases (0.27) than in controls (0.24,
P
=0.008), whereas that of
PON1
55Leu
was identical (0.26). Compared with
PON1
192Gln-Gln
, the
PON1
192Arg
allele was associated with an increased risk of MI (odds ratio [OR] 1.36, CI 1.06 to 1.75), and this association was independent of the
PON1
55
polymorphism, which was not associated with MI (OR 1.10, CI 0.82 to 1.48). Adjustment for lipid and nonlipid risk factors strengthened the association between
PON1
192Arg
and the risk of MI (OR 1.51, CI 1.13 to 2.03). Interestingly, this association was evident only among nonsmokers (OR 1.90, CI 1.29 to 2.79): there was no evidence of an association in smokers (OR 0.95, CI 0.57 to 1.79). The interaction between
PON1
192
and smoking status was statistically significant (
P
=0.04). Thus, the
PON1
192
but not the
PON1
55
gene polymorphism is associated with an increased risk of MI. This association is not evident among smokers.
Clinical Expression of Familial Hypercholesterolemia in Clusters of Mutations of the LDL Receptor Gene That Cause a Receptor-Defective or Receptor-Negative Phenotype
Authors: Bertolini S. , Cantafora A. , Averna M. , Cortese C. , Motti C. , Martini S. , Pes G. , Postiglione A. , Stefanutti C. , Blotta I. , Pisciotta L. , Rolleri M. , Langheim S. , Ghisellini M. , Rabbone I. , Calandra S.
Published: June 2011

Abstract
—Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelated Italian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases, we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutations on the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative, according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients’ age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDL cholesterol was higher (+18%) and high density lipoprotein cholesterol lower (−5%) than the values found in receptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higher in receptor-negative subjects. In patients >30 years of age in both groups, the presence of CAD was related to age, arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in the receptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negative subjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact than in receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptor-defective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. Apolipoprotein E genotype analysis showed a lowering effect of the ε2 allele and a raising effect of the ε4 allele on the LDL cholesterol level in both groups; however, the apolipoprotein E genotype accounted for only 4% of the variation in LDL cholesterol. Haplotype analysis showed that all families of the major clusters shared the same intragenic haplotype cosegregating with the mutation, thus suggesting the presence of common ancestors.
Simvastatin Has Anti-Inflammatory and Antiatherosclerotic Activities Independent of Plasma Cholesterol Lowering
Authors: Sparrow Carl P. , Burton Charlotte A. , Hernandez Melba , Mundt Steven , Hassing Heide , Patel Sushma , Rosa Ray , Hermanowski-Vosatka Anne , Wang Pei-Ran , Zhang Donghui , Peterson Larry , Detmers Patricia A. , Chao Yu-Sheng , Wright Samuel D.
Published: June 2011

Abstract
—Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (n=20) contained 56±4 nmol total cholesterol/mg wet wt tissue, 38±2 nmol free cholesterol/mg, and 17±2 nmol cholesteryl ester/mg. Simvastatin (n=22) significantly (
P
<0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol–lowering activity.
Potassium Channel Function in Vascular Disease
Authors: Sobey Christopher G.
Published: June 2011

Abstract
—Potassium ion (K
+
) channel activity is a major regulator of vascular muscle cell membrane potential (
E
m
) and is therefore an important determinant of vascular tone. There is growing evidence that the function of several types of vascular K
+
channels is altered during major cardiovascular diseases, such as chronic hypertension, diabetes, and atherosclerosis. Vasoconstriction and the compromised ability of an artery to dilate are likely consequences of defective K
+
channel function in blood vessels during these disease states. In some instances, increased K
+
channel function may help to compensate for increased vascular tone. Endothelial cell dysfunction is commonly associated with cardiovascular disease, and altered activity of nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor could also contribute to changes in resting K
+
channel activity,
E
m
, and K
+
channel–mediated vasodilatation. Our current knowledge of the effects of disease on vascular K
+
channel function almost exclusively relies on interpretation of data obtained by using pharmacological modulators of K
+
channels. As further progress is made in the development of more selective drugs and through molecular approaches such as gene targeting technology in mice, specific K
+
channel abnormalities and their causes in particular diseases should be more readily identified, providing novel directions for vascular therapy.
Circulating Autoantibodies to Oxidized LDL Correlate With Arterial Accumulation and Depletion of Oxidized LDL in LDL Receptor–Deficient Mice
Authors: Tsimikas Sotirios , Palinski Wulf , Witztum Joseph L.
Published: June 2011

Abstract
—Autoantibodies to oxidized low density lipoprotein (OxLDL) are elevated in some human populations with increased risk of atherosclerosis. To determine whether autoantibody levels to epitopes of OxLDL reflect the extent of aortic atherosclerosis and the content of OxLDL, we measured IgG and IgM autoantibody titers to malondialdehyde (MDA)-LDL and copper-oxidized LDL (Cu-OxLDL) in 43 LDL receptor–deficient mice consuming atherogenic and regression diets. Antibody titers were correlated to percent atherosclerotic surface area, aortic weight, and aortic OxLDL content, measured as the in vivo uptake of
125
I-MDA2, a monoclonal antibody to MDA-LDL. All mice were fed an atherogenic diet for 6 months, and 1 group was euthanized. The other 3 groups were fed an atherogenic diet (fat/CHOL group), normal mouse chow (chow group), or mouse chow supplemented with vitamins E and C (chow+VIT group) for an additional 6 months. After dietary intervention, compared with their own baseline, autoantibody titers to MDA-LDL and Cu-OxLDL increased significantly in the fat/CHOL group, whereas they did not change or decreased significantly in the chow and chow+VIT groups. Aortic weight and surface area showed significant progression in the fat/CHOL group, mild progression in the chow group, and no progression in the chow+VIT group (
P
<0.001), whereas OxLDL content actually decreased in the latter 2 groups (
P
<0.001). Significant correlations were seen with MDA-LDL autoantibody titers and OxLDL content (IgM,
R
=0.64 and
P
=0.0009; IgG,
R
=0.52 and
P
=0.009), as well as with percent surface area and aortic weight. These data support the hypothesis that autoantibody titers to OxLDL reflect changes in OxLDL content in atherosclerotic lesions of LDL receptor–deficient mice. Whether autoantibody titers to OxLDL will provide similar valuable insights into the extent of human atherosclerosis, particularly anatomic measurements of plaque burden and OxLDL content, remains to be determined.
Proatherogenic Role of Elevated CE Transfer From HDL to VLDL
1
and Dense LDL in Type 2 Diabetes
Authors: Guérin Maryse , Le Goff Wilfried , Lassel Taous S. , Van Tol Arie , Steiner George , Chapman M. John
Published: June 2011

Abstract
—Plasma cholesteryl ester transfer protein (CETP) facilitates intravascular lipoprotein remodeling by promoting the heteroexchange of neutral lipids. To determine whether the degree of triglyceridemia may influence the CETP-mediated redistribution of HDL CE between atherogenic plasma lipoprotein particles in type 2 diabetes, we evaluated CE mass transfer from HDL to apoB-containing lipoprotein acceptors in the plasma of type 2 diabetes subjects (n=38). In parallel, we investigated the potential relationship between CE transfer and the appearance of an atherogenic dense LDL profile. The diabetic population was divided into 3 subgroups according to fasting plasma triglyceride (TG) levels: group 1 (G1), TG<100 mg/dL; group 2 (G2), 100<TG<200 mg/dL; and group 3 (G3), TG>200 mg/dL. Type 2 diabetes patients displayed an asymmetrical LDL profile in which the dense LDL subfractions predominated. Plasma levels of dense LDL subfractions were strongly positively correlated with those of plasma triglyceride (TG) (
r
=0.471;
P
=0.0003). The rate of CE mass transfer from HDL to apoB-containing lipoproteins was significantly enhanced in G3 compared with G2 or G1 (46.2±8.1, 33.6±5.3, and 28.2±2.7 μg CE transferred · h
−1
· mL
−1
in G3, G2, and G1, respectively;
P
<0.0001 G3 versus G1,
P
=0.0001 G2 versus G1, and
P
=0.02 G2 versus G3). The relative capacities of VLDL and LDL to act as acceptors of CE from HDL were distinct between type 2 diabetes subgroups. LDL particles represented the preferential CE acceptor in G1 and accounted for 74% of total CE transferred from HDL. By contrast, in G2 and G3, TG-rich lipoprotein subfractions accounted for 47% and 72% of total CE transferred from HDL, respectively. Moreover, the relative proportion of CE transferred from HDL to VLDL
1
in type 2 diabetes patients increased progressively with increase in plasma TG levels. The VLDL
1
subfraction accounted for 34%, 43%, and 52% of total CE transferred from HDL to TG-rich lipoproteins in patients from G1, G2, and G3, respectively. Finally, dense LDL acquired an average of 45% of total CE transferred from HDL to LDL in type 2 diabetes patients. In conclusion, CETP contributes significantly to the formation of small dense LDL particles in type 2 diabetes by a preferential CE transfer from HDL to small dense LDL, as well as through an indirect mechanism involving an enhanced CE transfer from HDL to VLDL
1
, the specific precursors of small dense LDL particles in plasma.
Familial Clustering of High Factor VIII Levels in Patients With Venous Thromboembolism
Authors: Schambeck C. M. , Hinney K. , Haubitz I. , Taleghani B. Mansouri , Wahler D. , Keller F.
Published: June 2011

Abstract
—High levels of factor VIII (FVIII) but not von Willebrand factor (vWF) are known to increase the risk for venous thromboembolism. Whether high FVIII levels originate from hereditary defects or from acquired conditions remains unanswered. The objective of our study was to investigate whether there is evidence for familial clustering of elevated FVIII levels in families in which ≥1 member has been affected by a thromboembolic event and had reproducibly high FVIII levels. We investigated FVIII levels in 361 patients with previous venous thromboembolism. FVIII levels were measured by a chromogenic assay; the cutoff value was defined as the 98th percentile of FVIII plasma levels of 266 blood donors. vWF levels were determined by an enzyme immunoassay. After exclusion of known causes of FVIII elevation, such as the acute thrombotic event itself; inflammation; malignancy; liver, renal, or vascular disease; surgery; or pregnancy, we included 17 patients with unexplained, reproducibly high FVIII levels. The investigation was also extended to these patients’ relatives. Multiple regressive analysis of blood donors and asymptomatic family members showed that the affiliation with a family in which 1 member suffered from venous thromboembolism and had reproducibly high FVIII levels is the second most important predictor for FVIII levels. Familial clustering was analyzed by the Houwing-Duistermaat familial aggregation test. After adjustment for the influence of age, sex, blood group, and vWF, FVIII levels were significantly (
P
=0.038) clustered within families. In conclusion, FVIII levels seem to be familially determined in families in which a member showed high FVIII levels after previous venous thromboembolism.
Cell Lineages and Tissue Boundaries in Cardiac Arterial and Venous Poles
Authors: Ausoni Simonetta , Sartore Saverio
Published: June 2011

Abstract
—Multiple cell populations with different embryological histories are involved in the morphogenesis of the cardiac arterial and venous poles as well as in the correct alignment and connection of the developing vessels with the cardiac chambers. Formation of the aorta and the pulmonary trunk is a complicated process orchestrated via a specific sequence of highly integrated spatiotemporal events of cell proliferation, migration, differentiation, and apoptosis. The peculiar susceptibility of this intricate cell network to be altered explains the frequency of congenital cardiovascular diseases of the arterial and venous poles. We review this topic from the “vascular point of view,” putting major emphasis on (1) the existence of different cell lineages from which smooth muscle cells of the aorticopulmonary trunk can be derived, (2) the establishment of cell/tissue boundaries in the cardiovascular connecting regions, and (3) the animal models that can mimic human congenital defects of the arterial and venous poles of the heart.
Adenovirus-Mediated Transfer of Dominant-Negative Rho-Kinase Induces a Regression of Coronary Arteriosclerosis in Pigs In Vivo
Authors: Morishige Kunio , Shimokawa Hiroaki , Eto Yasuhiro , Kandabashi Tadashi , Miyata Kenji , Matsumoto Yasuharu , Hoshijima Masahiko , Kaibuchi Kozo , Takeshita Akira
Published: June 2011

Abstract
—Small GTPase Rho and its target Rho-kinase/ROK/ROCK play an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and cell adhesion and migration, all of which may be involved in the pathogenesis of arteriosclerosis. Here, we show that adenovirus-mediated transfer of dominant-negative Rho-kinase (DNRhoK) induces a marked regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1β, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin, as previously reported. Adenovirus-mediated transfer of DNRhoK, but not that of β-galactosidase, into the interleukin-1β–treated coronary segment caused a marked regression of the constrictive remodeling and abolished the vasospastic activity in 3 weeks. Western blot analysis showed that the phosphorylation of adducin and the ezrin/radixin/moesin family, the target proteins of Rho-kinase, were upregulated at the coronary lesions and were significantly suppressed by the transfer of DNRhoK. These results indicate that Rho-kinase is substantially involved in coronary constrictive remodeling and vasospastic responses, both of which can be reversed by the selective inhibition of the molecule in our porcine model in vivo.
3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition Prevents Endothelial NO Synthase Downregulation by Atherogenic Levels of Native LDLs
Authors: Martínez-González José , Raposo Berta , Rodríguez Cristina , Badimon Lina
Published: June 2011

Abstract
—Atherogenic levels of native low density lipoproteins (nLDLs) decrease the bioavailability of endothelium-derived NO and downregulate endothelial NO synthase (eNOS) expression in cultured human endothelial cells. Here, we show that simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, within the therapeutic range (0.01 to 1 μmol/L) prevented the downregulation of eNOS mRNA and protein promoted by nLDL (180 mg cholesterol/dL, 48 hours) in human umbilical vein endothelial cells. This effect of simvastatin was completely reversed by mevalonate, the product of the reaction, and to a lesser extent by farnesol and geranyl geraniol. Simvastatin significantly stabilized eNOS mRNA in cells treated with nLDL during 48 hours (eNOS mRNA half-life ≈11 hours in controls versus >24 hours in nLDL per 0.1 μmol/L simvastatin–treated cells). The downregulation of eNOS by nLDL was abrogated by cycloheximide, an inhibitor of protein synthesis, and by
N
-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor that reduces the catabolism of sterol regulatory element binding proteins. Sterol deprivation increased the downregulation produced by nLDL on eNOS and sterol regulatory element binding protein-2 expression levels. However, no differential modulation of the retardation bands corresponding to the putative sterol-responsive element present in the eNOS promoter was detected by electrophoretic mobility shift assay. Our results suggest that nLDL promote eNOS downregulation operating at a transcriptional level, whereas simvastatin prevents such an effect through a posttranscriptional mechanism.