bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Efficacy of SM-20302, a GP IIb/IIIa Receptor Antagonist, Correlates With Ex Vivo Platelet Inhibition in Heparinized Blood but Not in Citrated Blood
Authors: Rebello Sam S. , Huang Jinbao , Saito Kumi , Lucchesi Benedict R.
Published: June 2011

Abstract—We tested the hypothesis that the in vivo antithrombotic efficacy of SM-20302, a GP IIb/IIIa receptor antagonist, correlates with the ex vivo platelet inhibition in heparinized platelet rich plasma (hPRP) but not in citrated PRP (cPRP). The studies were performed in a canine model of carotid artery thrombosis in which thrombus formation was induced by electrolytic injury. Thrombosis of the right carotid artery was induced immediately after the administration of saline (n=12). Thirty minutes after persistent occlusive thrombosis was obtained, the vessel segment was ligated, and the time to occlusion and thrombus weight were noted. Subsequently, thrombosis of the left carotid artery was initiated in the presence of SM-20302 (100, 300, 600, or 1000 μg/kg IV; n=4 to 6). All the doses of SM-20302 inhibited (by ≥90%) the ex vivo platelet aggregation induced by ADP and arachidonic acid (AA) in cPRP. In hPRP, a dose-dependent inhibition of ex vivo platelet aggregation was observed. The maximal inhibition produced by 100 to 1000 μg/kg SM-20302 ranged from 18% to 80% for ADP and 44% to 88% for AA. Maximal prolongation of the template bleeding time induced by the 100-, 300-, 600-, and 1000-μg/kg doses were 2.5-, 9.5-, 10-, and >10-fold, respectively. All the injured carotid arteries (n=12) in the saline-treated group occluded. SM-20302 pretreatment produced a dose-dependent maintenance of the carotid artery patency, and the incidence of occlusion at 4 hours was 5/6, 3/6, 0/6, and 0/6 for the 100-, 300-, 600-, and 1000-μg/kg doses, respectively. The results indicate that SM-20302 prevents carotid artery thrombosis in response to electrolytic arterial wall injury and that its in vivo antithrombotic efficacy can be correlated accurately with the ex vivo platelet inhibition in PRP prepared from heparinized blood but not from citrated blood.
Hormone-Sensitive Lipase Overexpression Increases Cholesteryl Ester Hydrolysis in Macrophage Foam Cells
Authors: Escary Jean-Louis , Choy Henry A. , Reue Karen , Schotz Michael C.
Published: June 2011

Abstract
—Atherosclerosis is a complex physiopathologic process initiated by the formation of cholesterol-rich lesions in the arterial wall. Macrophages play a crucial role in this process because they accumulate large amounts of cholesterol esters (CEs) to form the foam cells that initiate the formation of the lesion and participate actively in the development of the lesion. Therefore, prevention or reversal of CE accumulation in macrophage foam cells could result in protection from multiple pathological effects. In this report, we show that the CE hydrolysis catalyzed by neutral cholesterol ester hydrolase (nCEH) can be modulated by overexpression of hormone-sensitive lipase (HSL) in macrophage foam cells. For these studies, RAW 264.7 cells, a murine macrophage cell line, were found to be a suitable model of foam cell formation. HSL expression and nCEH activity in these cells and in peritoneal macrophages were comparable. In addition, antibody titration showed that essentially all nCEH activity in murine macrophages was accounted for by HSL. To examine the effect of HSL overexpression on foam cell formation, RAW 264.7 cells were stably transfected with a rat HSL cDNA. The resulting HSL overexpression increased hydrolysis of cellular CEs 2- to 3-fold in lipid-laden cells in the presence of an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. Furthermore, addition of cAMP produced a 5-fold higher rate of CE hydrolysis in cholesterol-laden, HSL-overexpressing cells than in control cells and resulted in nearly complete hydrolysis of cellular CEs in only 9 hours, compared with <50% hydrolysis in control cells. Thus, HSL overexpression stimulated the net hydrolysis of CEs, leading to faster hydrolysis of lipid deposits in model foam cells. These data suggest that HSL overexpression in macrophages, alone or in combination with ACAT inhibitors, may constitute a useful therapeutic approach for impeding CE accumulation in macrophages in vivo.
Relation of a Postmortem Renal Index of Hypertension to Atherosclerosis and Coronary Artery Size in Young Men and Women
Authors: McGill Henry C. , McMahan C. Alex , Tracy Richard E. , Oalmann Margaret C. , Cornhill J. Frederick , Herderick Edward E. , Strong Jack P.
Published: June 2011

Abstract
—In a cooperative multicenter study, the Pathobiological Determinants of Atherosclerosis in Youth, we measured atherosclerosis of the aorta and right coronary artery (RCA) in 2403 black and white men and women 15 through 34 years of age who died of external causes and were autopsied in forensic laboratories. We measured the diameter of the opened, flattened, and fixed RCA and the diameter, intimal thickness, intimal cross-sectional area, medial thickness, and medial cross-sectional area of the pressure-perfused, fixed left anterior descending (LAD) coronary artery. Using the ratio of intimal thickness to outer diameter of the small renal arteries to predict mean arterial pressure during life, we classified the cases as normotensive (mean arterial pressure <110 mm Hg) or hypertensive (mean arterial pressure ≥110 mm Hg). The prevalence of hypertension by age, sex, and race corresponded closely with that measured in a survey of the living population. Hypertension had little or no effect on fatty streaks. Hypertension was associated with more extensive raised lesions in the abdominal aortas and RCAs of blacks >20 years of age and in the RCAs of whites >25 years of age. At all ages, women had less extensive raised lesions in the RCAs than did men, but the effect of hypertension on raised lesions was similar to that in men. Adjustment for serum lipoprotein cholesterol levels and smoking in a subset of cases yielded results similar to those obtained without adjustment. Hypertension was associated with larger diameters of the RCA and LAD coronary artery and with larger cross-sectional intimal and medial areas of the LAD coronary artery. Hypertension augments atherosclerosis in both men and women primarily by accelerating the conversion of fatty streaks to raised lesions beginning in the third decade of life, and the effect of hypertension increases with age.
Inhibition of LPL Expression in Human Monocyte–Derived Macrophages Is Dependent on LDL Oxidation State
Authors: Stengel Dominique , Antonucci Micheline , Gaoua Wassila , Dachet Christiane , Lesnik Philippe , Hourton Delphine , Ninio Ewa , Chapman M. John , Griglio Sabine
Published: June 2011

Abstract
—The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 μg protein per milliliter) led to marked reduction in the expression of LPL activity (−62%,
P
<0.01) and mRNA level (−47%,
P
<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (−31%,
P
<0.05). Among the biologically active sterols formed during LDL oxidation, only 7β-hydroxycholesterol (5 μg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 μmol/L (−34% to −53%,
P
<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7β-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte–derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.
Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions
Authors: Ramos Miguel A. , Kuzuya Masafumi , Esaki Teiji , Miura Satoru , Satake Shosuke , Asai Toshinobu , Kanda Shigeru , Hayashi Toshio , Iguchi Akihisa
Published: June 2011

Abstract—The interaction between macrophages and oxidatively modified low density lipoprotein (Ox-LDL) appears to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the induction of numerous cytokines and growth factors. The current study demonstrated that Ox-LDL upregulated vascular endothelial growth factor (VEGF) mRNA expression in RAW 264 cells, a monocytic cell line, in a time- and concentration-dependent manner and that Ox-LDL stimulated VEGF protein secretion from the cells. Lysophosphatidylcholine, a component of Ox-LDL, also enhanced VEGF mRNA expression in RAW 264 cells and VEGF secretion from RAW 264 cells, with a maximal effect at a concentration of 10 μmol/L lysophosphatidylcholine. Immunohistochemical studies showed that human early atherosclerotic lesions exhibited intense VEGF immunoreactivity in subendothelial macrophage-rich regions of the thickened intima. In atherosclerotic plaques, VEGF staining was also observed in foam cell–rich regions adjacent to the lipid core or the neovascularized basal regions of plaque consisting predominantly of smooth muscle cells. High-power-field observation revealed that VEGF was localized in the extracellular space as well as at the macrophage cell surface. These observations suggest the possible involvement of Ox-LDL in the development of human atherosclerosis through VEGF induction in macrophages.
Relation Between Plasma Tumor Necrosis Factor-α and Insulin Sensitivity in Elderly Men With Non–Insulin-Dependent Diabetes Mellitus
Authors: Nilsson Jan , Jovinge Stefan , Niemann Audrey , Reneland Richard , Lithell Hans
Published: June 2011

Abstract
—Human obesity is associated with an increased tumor necrosis factor-α (TNF-α) mRNA expression in adipose tissue. TNF-α decreases insulin-dependent glucose uptake by inhibiting autophosphorylation of the insulin receptor, suggesting that TNF-α may play a role in insulin resistance. In this study, we analyzed plasma levels of TNF-α in 40 70-year-old men with newly detected non–insulin-dependent diabetes mellitus and in 20 age-matched controls. Twenty of the patients had a moderate level of insulin resistance and 20 were severely insulin resistant. The plasma levels of TNF-α were higher in patients (4.00±1.53 pg/mL in moderately insulin resistant and 4.91±1.43 pg/mL in severely insulin resistant subjects) than in controls (3.27±0.79 pg/mL,
P
<0.001). TNF-α was significantly related to body mass index, fasting glucose levels, and serum triglyceride levels and inversely related to the high density lipoprotein cholesterol level. The finding of an association between high plasma levels of TNF-α and several metabolic abnormalities characteristic for the insulin resistance syndrome suggests that TNF-α may be involved in the pathogenesis of non–insulin-dependent diabetes mellitus.
Elevated LDL Triglyceride Concentrations in Subjects Heterozygous for the Hepatic Lipase S267F Variant
Authors: Hegele Robert A. , Breckenridge W. Carl , Cox Diane W. , Maguire Graham F. , Little J. Alick , Connelly Philip W.
Published: June 2011

Abstract
—Although naturally occurring loss-of-function mutations in human hepatic lipase (
HL
) have been described, the biochemical phenotype of heterozygous HL deficiency remains ill defined. This may be due to the relatively small numbers of heterozygous adult carriers of
HL
mutations in index kindreds. We have identified several new heterozygotes for the catalytically inactive, nonsecreted
HL
variant S267F in the kindred that was originally ascertained because of hypertriglyceridemia due to the mutant, secreted, circulating apolipoprotein (apo) CII variant apo CII-T. Pairwise comparisons with family controls showed that only the plasma low density lipoprotein triglycerides (LDL TGs) were higher in 11 simple heterozygotes for
HL
S267F (
P
=0.002). In contrast, both plasma total TGs and LDL TGs were significantly higher in 12 simple heterozygotes for apo CII-T than in family-matched control subjects (
P
=0.005 and 0.009, respectively). These findings suggest that the TG content of LDL is increased by heterozygosity for 2 different mutations that affect different proteins involved in lipolysis. However, the mechanisms underlying this compositional change in LDL appear to be different for the 2 mutations, because the total TGs are also elevated in subjects heterozygous for apo CII-T but not in subjects heterozygous for
HL
S267F.
Interleukin-1β Induces Tissue- and Cell Type–Specific Expression of Adhesion Molecules In Vivo
Authors: Tamaru Masahiro , Tomura Keiko , Sakamoto Shinji , Tezuka Katsunari , Tamatani Takuya , Narumi Shosaku
Published: June 2011

Abstract—We examined the tissue distribution of adhesion molecule gene expression in mice treated intravenously with interleukin (IL)-1β. E-selectin mRNA expression was selectively induced in the heart by IL-1β, but only slight or no induction was observed in other organs. On the other hand, intercellular adhesion molecule-1 mRNA expression was inducible in all organs examined, although it showed the strongest induction in the lung and the weakest responses in the brain and skin. Vascular cell adhesion molecule-1 mRNA was also inducible in all organs with the exception of the skin, but it was induced most markedly in the lung and the heart. The accessibility of IL-1β to the heart was less than that to other organs except the brain. Similar tissue-specific induction of these mRNAs was also seen when tumor necrosis factor (TNF)-α or lipopolysaccharide was substituted for IL-1β. Analysis of E-selectin mRNA expression in the heart by in situ hybridization indicated that expression was most prominent in microvascular endothelial cells and some other stromal cells, but this transcript was not seen in the lung. Although intercellular adhesion molecule-1 mRNA expression was restricted to the endothelium lining the capillaries and small arteries in the heart, its distribution in the lung covered not only the endothelium but also the cells composing the alveolar septa. In contrast, vascular cell adhesion molecule-1 mRNA expression was most prominent in endothelial cells of larger vessels in both the heart and the lung. Our results demonstrate that expression of adhesion molecules is tissue- and cell type–specific and that endothelial cells differentially express adhesion molecules depending on the size of the blood vessels.
Plasma Levels of Activated Protein C in Healthy Subjects and Patients With Previous Venous Thromboembolism
Authors: Cattaneo Marco , Franchi Franca , Zighetti Maddalena L. , Martinelli Ida , Asti Daniela , Mannucci P. Mannuccio
Published: June 2011

Abstract
—The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro; this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy). We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age- and sex-matched healthy controls and correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML). Forty-eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study. Because the plasma levels of APC and PC were correlated in healthy controls, the APC/PC ratios were also analyzed. Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (
P
=0.03 and 0.0004, respectively). Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy. Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls. There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls. The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy. This study indicates that APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and that Hcy does not interfere with the activation of PC in vivo.
The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-α and Protein Kinase C-ζ
Authors: Wellner Maren , Maasch Christian , Kupprion Christine , Lindschau Carsten , Luft Friedrich C. , Haller Hermann
Published: June 2011

Abstract
—The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca
2+
]
i
regulation and [Ca
2+
]
i
-dependent messenger systems; and (2) these mechanisms are important for VEGF’s proliferative effects. [Ca
2+
]
i
was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca
2+
]
i
followed by a sustained elevation. The sustained [Ca
2+
]
i
plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms α, δ, ε, and ζ were identified in endothelial cells. VEGF induced a translocation of PKC-α and PKC-ζ toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-δ and PKC-ε was not influenced. Cell exposure to TPA led to a down-regulation of PKC-α and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-α and PKC-ζ with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca
2+
influx. VEGF leads to the translocation of the [Ca
2+
]
i
-sensitive PKC isoform α and the atypical PKC isoform ζ. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms α and ζ are important for VEGF’s angiogenic effects.
Inflammation of the Atherosclerotic Cap and Shoulder of the Plaque Is a Common and Locally Observed Feature in Unruptured Plaques of Femoral and Coronary Arteries
Authors: Pasterkamp Gerard , Schoneveld Arjan H. , van der Wal Allard C. , Hijnen Dirk-Jan , van Wolveren Willem J. A. , Plomp Simon , Teepen Hans L. J. M. , Borst Cornelius
Published: June 2011

Abstract
—Retrospectively, plaque rupture is often colocalized with inflammation of the cap and shoulder of the atherosclerotic plaque. Local inflammation is therefore considered a potential marker for plaque vulnerability. However, high specificity of inflammation for plaque rupture is a requisite for application of inflammation markers to detect rupture-prone lesions. The objective of the present study was to investigate the prevalence and distribution (local versus general) of inflammatory cells in nonruptured atherosclerotic plaques. The cap and shoulder of the plaque were stained for the presence of macrophages and T lymphocytes in 282 and 262 cross sections obtained from 74 coronary and 50 femoral arteries, respectively. From most cases, 2 atherosclerotic arteries were studied to gain insight into the local and systemic distribution of the inflammatory process. In 45% and 41% of all cross sections, staining for macrophages was observed in the femoral and coronary arteries, respectively. Rupture of the fibrous cap was observed in 2 femoral and 3 coronary artery segments and was always colocalized with inflammatory cells. At least 1 cross section stained positively for CD68 or acid phosphatase in 84% and 71% of all femoral and coronary arteries, respectively. Only 1 femoral and 6 coronary arteries revealed a positive stain for CD68 in all investigated segments. Inflammation of the cap and shoulder of the plaque is a common feature, locally observed, in atherosclerotic femoral and coronary arteries. The high prevalence of local inflammatory responses should be considered if they are used as a diagnostic target to detect vulnerable, rupture-prone lesions.
Autocrine FGF-2 Is Responsible for the Cell Density–Dependent Susceptibility to Apoptosis of HUVEC
Authors: Kinoshita Mikio , Shimokado Kentaro
Published: June 2011

Abstract
—To elucidate the factors affecting endothelial susceptibility to apoptosis, we studied the effects of cell density on endothelial cell apoptosis induced by deprivation of serum and fibroblast growth factor-2 (FGF-2/basic FGF). On deprivation, more cells became apoptotic in a dense culture (5×10
2
cells/mm
2
) than in a sparse culture (1×10
2
cells/mm
2
) of human umbilical vein endothelial cells. FGF-2, hepatocyte growth factor, and vascular endothelial cell growth factor, but not insulin-like growth factor-I, decreased apoptosis in the dense culture to a level similar in the sparse culture. An anti–FGF-2 antibody significantly increased the apoptosis in the sparse culture, suggesting that the sparse culture was resistant to apoptosis because of the greater autocrine production of FGF-2. Western blot analysis and metabolic labeling revealed that the sparse culture has, in fact, more FGF-2 than the dense culture. The steady state level of mRNA for FGF-2 was not significantly different between the dense and sparse cultures. Among a panel of inhibitors for 2 major cytoplasmic proteolytic enzymes, calpain inhibitors increased FGF-2 in the dense culture, but proteasome inhibitors did not. Our findings demonstrate that cell density affects endothelial survival by regulating autocrine FGF-2 production through a calpain inhibitor–sensitive mechanism.
Complement and Atherogenesis
Authors: Bhakdi Sucharit , Torzewski Michael , Klouche Mariam , Hemmes Monika
Published: June 2011

Abstract—Complement activation occurs in temporal correlation with the subendothelial deposition of LDL during early atherogenesis, and complement also plays a pathogenetic role in promoting lesion progression. Two lesion components have been identified that may be responsible for complement activation. First, enzymatic degradation of LDL generates a derivative that can spontaneously activate complement, and enzymatically degraded LDL (E-LDL) has been detected in the lesions. Second, C-reactive protein (CRP) colocalizes with complement C5b-9, as evidenced by immunohistological studies of early atherosclerotic lesions, so the possibility exists that this acute phase protein also fulfills a complement-activating function. Here, we report that addition of LDL and CRP to human serum did not result in significant C3 turnover. Addition of E-LDL provoked complement activation, which was markedly enhanced by CRP. Binding of CRP to E-LDL was demonstrated by sucrose flotation experiments. Binding was Ca2+-dependent and inhibitable by phosphorylcholine, and the complement-activating property of E-LDL was destroyed by treatment with phospholipase C. These results indicated that CRP binds to phosphorylcholine groups that become exposed in enzymatically degraded LDL particles. Immunohistological studies complemented these findings in showing that CRP colocalizes with E-LDL in early human atherosclerotic lesions. Thus enzymatic, nonoxidative modification of tissue-deposited LDL can be expected to confer CRP-binding capacity onto the molecule. The ensuing enhancement of complement activation may be relevant to the development and progression of the atherosclerotic lesion.
Role of Plasmalogens in the Enhanced Resistance of LDL to Copper-Induced Oxidation After LDL Apheresis
Authors: Hahnel Daniela , Thiery Joachim , Brosche Thorolf , Engelmann Bernd
Published: June 2011

Abstract
—Extracorporeal reduction of plasma low density lipoproteins (LDLs) by LDL apheresis was shown to attenuate the proatherogenic influences of LDL, such as impairment of vasodilation and increased monocyte adhesion to the endothelium. In 16 patients with familial hypercholesterolemia, we analyzed whether LDL apheresis by the heparin precipitation procedure affected the oxidative resistance of LDL. Plasma LDL cholesterol concentrations were reduced by 65% after the apheresis. The lag time of copper-mediated LDL oxidation was increased from 103 to 117 minutes (
P
<0.0005). The LDL contents of α-tocopherol and β-carotene, as well as the ratio of monounsaturated to polyunsaturated fatty acids in LDL, were not altered. However, the LDL apheresis induced a 15% increase in the LDL contents of plasmalogen phospholipids (
P
<0.0005), a class of ether phospholipids that were recently shown to prevent lipid oxidation. The phosphatidylcholine (PC) to lysoPC ratio was elevated by 16% after the apheresis (
P
<0.0005). The percent increase in LDL plasmalogen phospholipids showed a close association with the increased lag time after apheresis (
P
<0.0005). The LDL plasmalogen contents of the blood samples from patients and from normolipidemic donors were also positively related to the lag time (
P
<0.005). In vitro loading of LDL with plasmalogen phospholipids resulted in a prolongation of the lag time and an increase in the PC/lysoPC ratio. In conclusion, the rapid rise in LDL contents of plasmalogen phospholipids most probably causes the increase in lag time after LDL apheresis. Plasmalogens appear to play an important role in the oxidation resistance of LDL in vivo.
Human Growth Hormone Increases Apo(a) Expression in Transgenic Mice
Authors: Tao Ruixian , Acquati Francesco , Marcovina Santica M. , Hobbs Helen H.
Published: June 2011

Abstract
—Levels of Lp(a), an atherogenic lipoprotein that circulates in human plasma, are increased by the administration of growth hormone (GH). Many of the physiological effects of GH are mediated through insulin-like growth factor-1 (IGF-1), but ironically, IGF-1 treatment of humans is associated with a fall in plasma Lp(a) levels. To glean insight into the mechanism responsible for the GH-associated increase in plasma levels of Lp(a), we administered recombinant human GH (rhGH) to mice expressing a 370-kb human genomic fragment containing the apo(a) gene, 40 kb of 5′-, and 200 kb of 3′-flanking sequence [YAC-apo(a) transgenic mice]. The plasma levels of apo(a) and hepatic levels of apo(a) mRNA rose dramatically in the post-pubertal male mice in response to rhGH treatment. To determine whether the increase in plasma apo(a) was mediated by IGF-1, we treated castrated and noncastrated YAC-apo(a) transgenic mice with a continuous infusion of IGF-1 (100 μg/d) for 2 weeks, and plasma levels of apo(a) fell by ≈50%. Thus the effects of rhGH and IGF-1 administration on plasma levels of apo(a) in the YAC-apo(a) transgenic mice simulate those seen in humans. The coordinate changes in apo(a) mRNA and plasma levels of apo(a) in response to rhGH and IGF-1 strongly suggest that these 2 hormones have independent effects on the transcription of the apo(a) gene.
Increased Systemic Oxidative Stress After Elective Endarterectomy
Authors: Mezzetti Andrea , Guglielmi Maria D. , Pierdomenico Sante D. , Costantini Fabrizio , Cipollone Francesco , De Cesare Domenico , Bucciarelli Tonino , Ucchino Sante , Chiarelli Francesco , Cuccurullo Franco , Romano Ferdinando
Published: June 2011

Abstract
—It has been reported that systemic and local redox state may have an important role in the functional and organic changes characterizing the process of vascular response to injury. Carotid endarterectomy to remove atherosclerotic plaque is followed by a long lasting healing and remodeling process that can be carefully followed over time with noninvasive ultrasonography. Plasma vitamin C concentration and native LDL (n-LDL) content in lipid peroxides, vitamin E, β-carotene, and lycopene as well as LDL susceptibility to peroxidation were assessed in 45 patients undergoing elective endarterectomy for internal carotid stenosis, at baseline, 24 hours, 3 and 15 days, and 1 month after surgery. Serial duplex scans were performed in all patients postoperatively and 3, 6, and 12 months. The changes in far wall thickness (FW) and % renarrowing from postoperatively to 12 months were used as remodeling indices. Plasma antioxidant vitamins and lag-phase showed a sharp and significant decrease during the first 24-hours after surgery remaining unchanged until the third day, whereas, an opposite trend was evidenced for n-LDL content in lipid peroxides and serum ceruloplasmin. After the third day all the parameters returned progressively to baseline within one month from endarterectomy. Interestingly, the n-LDL lipid peroxide content, the serum ceruloplasmin and the plasma vitamin C concentration, measured at 24 and 3 days from surgery, were significantly associated to the change in % renarrowing from postoperatively to 12 months. The higher the LDL content in lipid peroxides, the higher the serum level of ceruloplasmin, the lower the plasma content in vitamin C and the higher the % of vessel renarrowing. In conclusion, carotid endarterectomy with atherosclerotic plaque removal is associated with an acute and prolonged increase in systemic oxidative stress that influences vascular healing and late luminal loss.
Comparison of Plasminogen Activator Inhibitor-1 Concentration in Insulin-Resistant Versus Insulin-Sensitive Healthy Women
Authors: Abbasi Fahim , McLaughlin Tracey , Lamendola Cindy , Lipinska Izabella , Tofler Geoffrey , Reaven Gerald M.
Published: June 2011

Abstract
—The primary goal of this investigation was to see whether plasminogen activator inhibitor-1 (PAI-1) concentrations varied as a function of differences in insulin-mediated glucose disposal in 2 groups of healthy women matched for every other variable that might play a role in regulation of PAI-1. For this purpose, we recruited 32 healthy women, divided on the basis of their steady-state plasma glucose (SSPG) concentrations during the insulin suppression test into an insulin-resistant (SSPG=216±12 mg/dL, n=16) and an insulin-sensitive (94±6 mg/dL, n=16) group. PAI-1 antigen concentrations were significantly higher (26±4 versus 14±3 ng/mL,
P
<0.02) in the insulin-resistant group. In addition, fasting plasma insulin (18±3 versus 11±2 μU/mL,
P
<0.02) and triglyceride (160±19 versus 93±10 mg/dL,
P
<0.001) concentrations were higher in the insulin-resistant individuals, whereas HDL concentrations were lower (44±3 versus 58±3 mg/dL,
P
<0.005). However, the 2 groups were essentially identical in terms of age, menopausal status, hormone replacement therapy, body mass index (BMI), ratio of waist-to-hip girth, and blood pressure. When the experimental population was considered as 1 group, there were statistically significant correlations between PAI-1 antigen and the following variables: adjusting for differences in age and BMI, SSPG (
r
=0.56,
P
<0.001); triglyceride (
r
=0.39,
P
<0.05); and HDL cholesterol (
r
=−0.65,
P
<0.001) concentrations. Finally, multiple regression analysis revealed the major determinants of PAI-1 to be insulin resistance, or insulin concentration, and HDL cholesterol. These results: 1) demonstrate that PAI-1 concentrations are higher in healthy, insulin-resistant women as compared with insulin-sensitive individuals, independent of differences in BMI or ratio of waist-to-hip girth; and 2) provide another mechanism by which insulin-resistant individuals are at increased thrombotic cardiovascular risk.
Procoagulant Expression in Platelets and Defects Leading to Clinical Disorders
Authors: Solum Nils Olav
Published: June 2011

Abstract—Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the “tenase” complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the “prothrombinase” complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid “translocase” and a “floppase” counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific “scramblase,” believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the α-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant microvesicles have been demonstrated in several clinical conditions, such as thrombotic and idiopathic thrombocytopenia, disseminated intravascular coagulation, and HIV-1 infection, and have been found to be associated with fibrin in thrombosis. Procoagulant microvesicles may also be formed from other cells as a result of apoptosis.
eNOS Gene Transfer Inhibits Smooth Muscle Cell Migration and MMP-2 and MMP-9 Activity
Authors: Gurjar Milind V. , Sharma Ram V. , Bhalla Ramesh C.
Published: June 2011

Abstract
—Vascular smooth muscle cell (SMC) migration is a critical step in the development of neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and the extracellular matrix, facilitating SMC migration. Transfer of the endothelial nitric oxide synthase (eNOS) gene to the injury site inhibits neointima formation. Neither the signaling pathways leading to NO-mediated inhibition of SMC migration and proliferation nor the alterations in these pathways have been characterized. We hypothesize that NO inhibits SMC migration in part by regulating MMP activity. To test this hypothesis, we transfected cultured rat aortic SMCs with replication-deficient adenovirus containing bovine eNOS gene and analyzed the conditioned medium for MMP activity. We observed that eNOS gene transfer significantly (
P
<0.05) inhibited SMC migration and significantly (
P
<0.05) decreased MMP-2 and MMP-9 activities in the conditioned medium. Similarly, addition of the NO donor DETA NONOate and 8-bromo-cGMP to the culture medium significantly decreased MMP-2 and MMP-9 activities in the conditioned medium collected 24 hours after treatment. Furthermore, Western blot analysis of the conditioned medium collected from eNOS gene–transfected SMCs showed a significant increase in tissue inhibitor of metalloproteinases-2 (TIMP-2) levels. Our data suggest that NO decreases MMP-2 and MMP-9 activities and increases TIMP-2 secretion, and this shifts the balance of MMP activity, which may favor the inhibition of cell migration because of inhibition of extracellular matrix degradation.
A Common Mutation of the Insulin Receptor Substrate-1 Gene Is A Risk Factor for Coronary Artery Disease
Authors: Baroni Marco Giorgio , D’Andrea Maria Pina , Montali Anna , Pannitteri Gaetano , Barillà Francesco , Campagna Filomena , Mazzei Ercole , Lovari Sarah , Seccareccia Fulvia , Campa Pietro Paolo , Ricci Giorgio , Pozzilli Paolo , Urbinati Giancarlo , Arca Marcello
Published: June 2011

Abstract
—Insulin resistance is associated with increased risk of atherosclerosis. Insulin receptor substrate-1 (IRS-1) plays a key role in tissue insulin sensitivity. A common mutation (G972R) of the IRS-1 gene has been shown to impair IRS-1 function, and it has been associated with reduced insulin sensitivity and lipid abnormalities. This led us to investigate the role of the G972R mutation in predisposing individuals to coronary artery disease (CAD). The DNA of 318 subjects with angiographically documented coronary atherosclerosis (>50% stenosis) and 208 population control subjects was analyzed for the presence of the G972R mutation. This mutation was detected by nested polymerase chain reaction and
Bst
NI restriction enzyme digestion. The frequency of the G972R mutation was significantly higher among patients with CAD than controls (18.9% versus 6.8%, respectively;
P
<0.001). After controlling for other coronary risk factors, the relative risk of CAD associated with the G972R mutation was 2.93 (95% CI 1.30 to 6.60;
P
<0.02) in the entire cohort. This risk was found to be even higher in the subgroups of obese subjects (odds ratio [OR] 6.97, 95% CI 2.24 to 21.4;
P
<0.001) and subjects with clinical features of insulin resistance syndrome (OR 27.3, 95% CI 7.19 to 104.0;
P
<0.001). The IRS-1 gene variant was associated with a higher frequency of diabetes mellitus (14.9% among carriers versus 6.5% among noncarriers;
P
<0.01) and with a 60% increase of plasma total triglycerides (
P
<0.001). Also, plasma concentrations of total cholesterol and the ratio of total cholesterol to HDL cholesterol were significantly (
P
<0.001) higher among carriers than noncarriers, although to lesser a extent. These effects were independent of CAD status. The G972R mutation in the IRS-1 gene was found to be a significant independent predictor of CAD. Moreover, this mutation greatly increased the risk of CAD in obese subjects and in patients with the cluster of abnormalities of insulin resistance syndrome. Besides the increased frequency of diabetes, carriers showed a more atherogenic lipid profile, suggesting a potential role of the IRS-1 gene in the pathogenesis of lipid abnormalities associated with CAD.
Fluid Shear Stress Induction of the Tissue Factor Promoter In Vitro and In Vivo Is Mediated by Egr-1
Authors: Houston Parul , Dickson Marion C. , Ludbrook Valerie , White Brian , Schwachtgen Jean-Luc , McVey John H. , Mackman Nigel , Reese Jason M. , Gorman Daniel G. , Campbell Callum , Braddock Martin
Published: June 2011

Abstract
—Hemodynamic forces such as fluid shear stress have been shown to modulate the activity of an expanding family of genes involved in vessel wall homeostasis and the pathogenesis of vascular disease. We have investigated the effect of shear stress on tissue factor (TF) gene expression in human endothelial cells (ECs) and in a rat arterial model of occlusion. As measured by reverse transcriptase polymerase chain reaction, exposure of ECs to 1.5 N/m
2
shear stress resulted in a time-dependent induction of endogenous TF transcripts of over 5-fold. Transient transfection of TF promoter mutants into cultured ECs suggests the involvement of the transcription factor Egr-1 in mediating the response of the TF promoter to shear stress. To address the importance of flow induction of Egr-1 in vivo, we have established a flow-restricted rat arterial model and determined the level of expressed Egr-1 and TF at the site of restricted flow using immunohistochemistry. We report an increase in the level of Egr-1 and TF protein in ECs expressed at the site of restricted flow. Elevated expression of Egr-1 and TF is restricted to a highly localized area, as evidenced by the fact that no significant increase in level can be detected at arterial sites distal to the site of occlusion. These findings suggest a direct role for Egr-1 in flow-mediated induction of TF and further substantiate the importance of shear stress as a modulator of vascular endothelial gene function in vivo.
Single and Combined Prothrombotic Factors in Patients With Idiopathic Venous Thromboembolism
Authors: Salomon Ophira , Steinberg David M. , Zivelin Ariella , Gitel Sanford , Dardik Rima , Rosenberg Nurit , Berliner Shlomo , Inbal Aida , Many Amira , Lubetsky Aharon , Varon David , Martinowitz Uriel , Seligsohn Uri
Published: June 2011

Abstract
—The inherited thrombophilias—deficiencies of protein C, protein S, and antithrombin III—and the prothrombotic polymorphisms factor V G1691A and factor II G20210A predispose patients toward venous thromboembolism (VTE). The aim of this study was to determine the prevalence of single and combined prothrombotic factors in patients with idiopathic VTE and to estimate the associated risks. The study group consisted of 162 patients referred for work-up of thrombophilia after documented VTE. The controls were 336 consecutively admitted patients. In all subjects factor V G1691A, factor II G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were analyzed by specific polymerase chain reactions and restriction enzymes. Activities of antithrombin III and protein C, free protein S antigen, and lupus anticoagulant were determined in a subset of 109 patients who were not receiving oral anticoagulants. The prevalences of heterozygotes and homozygotes for factor V G1691A and factor II G20210A among patients and controls were 40.1% versus 3.9% and 18.5% versus 5.4%, respectively (
P
=0.0001). The prevalence of homozygotes for MTHFR C677T in patients was 22.8% and in controls, 14.3% (
P
=0.025). Heterozygous and homozygous factor V G1691A, factor II G20210A, and homozygous MTHFR C677T were found to be independent risk factors for VTE, with odds ratios of 16.3, 3.6, and 2.1, respectively. Two or more polymorphisms were detected in 27 of 162 patients (16.7%) and in 3 of 336 controls (0.9%). Logistic regression analysis disclosed odds ratios of 58.6 (confidence interval [CI], 22.1 to 155.2) for joint occurrence of factor V and factor II polymorphisms, of 35.0 (CI, 14.5 to 84.7) for factor V and MTHFR polymorphisms, and of 7.7 (CI, 3.0 to 19.6) for factor II and MTHFR polymorphisms. Among 109 patients in whom a complete thrombophilic work-up was performed, 74% had at least 1 underlying defect. These data indicate that in most patients referred for evaluation of thrombophilia due to idiopathic VTE, 1 or more underlying genetic predispositions were discernible. The presence of >1 of the prothrombotic polymorphisms was associated with a substantial risk of VTE.
Expression of Interleukin-10 in Advanced Human Atherosclerotic Plaques
Authors: Mallat Ziad , Heymes Christophe , Ohan Jeanny , Faggin Elisabetta , Lesèche Guy , Tedgui Alain
Published: June 2011

Abstract—Inflammation is a major feature of human atherosclerosis and is central to development and progression of the disease. A variety of proinflammatory cytokines are expressed in the atherosclerotic plaque and may modulate extracellular matrix remodeling, cell proliferation, and cell death. Little is known, however, about the expression and potential role of anti-inflammatory cytokines in human atherosclerosis. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine whose expression and potential effects in advanced human atherosclerotic plaques have not been evaluated. We studied 21 advanced human atherosclerotic plaques. IL-10 expression was analyzed by use of reverse transcription–polymerase chain reaction and immunohistochemical techniques. Inducible nitric oxide synthase expression was assessed by using immunohistochemistry, and cell death was determined by use of the TUNEL method. Reverse transcription–polymerase chain reaction identified IL-10 mRNA in 12 of 17 atherosclerotic plaques. Immunohistochemical staining of serial sections and double staining identified immunoreactive IL-10 mainly in macrophages, as well as in smooth muscle cells. Consistent with its anti-inflammatory properties, high levels of IL-10 expression were associated with significant decrease in inducible nitric oxide synthase expression (P<0.0001) and cell death (P<0.0001). Hence, IL-10, a potent anti-inflammatory cytokine, is expressed in a substantial number of advanced human atherosclerotic plaques and might contribute to the modulation of the local inflammatory response and protect from excessive cell death in the plaque.
Specific Interaction of Oxidized Low-Density Lipoprotein With Macrophage-Derived Foam Cells Isolated From Rabbit Atherosclerotic Lesions
Authors: de Vries Helga E. , Buchner Bas , van Berkel Theo J. C. , Kuiper Johan
Published: June 2011

Abstract
—Interaction of oxidized LDL (OxLDL) with macrophage-derived foam cells is one of the key events in the development and progression of atherosclerosis. To study this interaction, macrophage-derived foam cells were isolated from rabbit atherosclerotic lesions and the expression of scavenger receptors for OxLDL was examined. Atherosclerosis was induced in rabbits by denudation of the large arteries, followed by a hypercholesteremic diet. Macrophage-derived foam cells, characterized by immunostaining with an RAM-11 antibody (a macrophage marker), contained a high content of intracellular lipid. Maximal binding of radiolabeled OxLDL to isolated macrophage-derived foam cells (1652±235 ng
125
I-OxLDL/mg of cell protein) was 20-fold higher compared with B
max
values of monocytes. Levels of association of OxLDL to macrophage-derived foam cells isolated from atherosclerotic lesions 12 weeks after denudation were >3-fold higher compared with the levels expressed by macrophage-derived foam cells isolated after 6 weeks. Association of
125
I-OxLDL could be completely blocked by OxLDL, and partially by acetylated LDL and polyinosinic acid, indicating the presence of a specific binding site for OxLDL on macrophage-derived foam cells. The induction of scavenger receptors for OxLDL on macrophage-derived foam cells during the development of atherosclerosis, as described in this study, may facilitate the lipid accumulation in macrophage-derived foam cells, as observed in advanced atherosclerotic lesions.