bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibroblast Growth Factor-2 Selectively Stimulates Angiogenesis of Small Vessels in Arterial Tree
Authors: Parsons-Wingerter Patricia , Elliott Katherine E. , Clark John I. , Farr Andrew G.
Published: June 2011

Abstract
—There is a critical need for quantifiable models of angiogenesis in vivo, and in general, differential effects of angiogenic regulators on vascular morphology have not been measured. Because the potent angiogenic stimulators fibroblast growth factor (FGF)-2 (basic FGF) and vascular endothelial growth factor (VEGF) are reported to stimulate angiogenesis through distinct signaling pathways, we hypothesized that FGF-2 stimulates vascular morphology differently than does VEGF and that stimulation of angiogenesis by FGF-2 is directly correlated to FGF receptor density. FGF-2 was applied at embryonic day 7 (E7), E8, or E9 to the quail chorioallantoic membrane (CAM); subsequent response of the arterial tree was measured by the fractal dimension (D
f
), a mathematical descriptor of complex spatial patterns, and by several generational branching parameters that included vessel length density (L
v
). After application of FGF-2 at E7, arterial density increased according to D
f
as a direct function of increasing FGF-2 concentration, and FGF-2 stimulated the growth of small vessels, but not of large vessels, according to L
v
and other branching parameters. For untreated control specimens at E7, L
v
of small vessels and D
f
were 11.1±1.6 cm
−1
and 1.38±0.01, respectively; at E8, after treatment with 5 μg FGF-2/CAM for 24 hours, L
v
of small vessels and D
f
increased respectively to 22.8±0.7 cm
−1
and 1.49±0.02 compared with 16.3±0.9 cm
−1
and 1.43±0.02 for PBS-treated control specimens. Application of FGF-2 at E8 and E9 did not significantly increase arterial density. By immunohistochemistry, the expression of 4 high-affinity tyrosine kinase FGF receptors was significantly expressed at E7, when CAM vasculature responded strongly to FGF-2 stimulation, but FGF receptor expression decreased throughout the CAM by E8, when vascular response to FGF-2 was negligible. In conclusion, the “fingerprint” vascular pattern elicited by FGF-2 was distinct from vascular patterns induced by other angiogenic regulators that included VEGF
165
, transforming growth factor-β1, and angiostatin.
Assay for Human Matrix Gla Protein in Serum
Authors: Braam L. A. J. L. M. , Dissel P. , Gijsbers B. L. M. G. , Spronk H. M. H. , Hamulyák K. , Soute B. A. M. , Debie W. , Vermeer C.
Published: June 2011

Abstract
—Matrix Gla protein (MGP) is synthesized in a vitamin K–dependent way in smooth muscle cells of the healthy vessel wall, and its mRNA transcription is substantially upregulated in atherosclerotic lesions. Here we report the preparation of a monoclonal antibody against human MGP and its use in an enzyme-linked immunosorbent assay. The intra-assay and interassay coefficients of variation in serum samples were 5.4% and 12.6%, respectively, and the lower detection limit was 8.5% of the normal serum value. Individual within-day variations were <11% and did not show a distinct circadian pattern. Day-to-day variations in fasting morning samples were <8%. In a first explorative survey, serum MGP concentrations were found to be significantly increased in patients with severe atherosclerosis, whereas these values were normal in those with low bone mass and osteoporosis. This finding is consistent with the high MGP mRNA expression observed in atherosclerotic vessels and plaques. More elaborate studies are required to assess the potential clinical utility of this newly developed assay.
Mildly Oxidized LDL Induces Expression of Group IIa Secretory Phospholipase A
2
in Human Monocyte–Derived Macrophages
Authors: Anthonsen Marit W. , Stengel Dominique , Hourton Delphine , Ninio Ewa , Johansen Berit
Published: June 2011

Abstract
—Phospholipase A
2
s (PLA
2
s) constitute a family of enzymes that hydrolyze fatty acids of membrane phospholipids, thus initiating the synthesis of proinflammatory mediators. Various PLA
2
s have been detected in human atherosclerotic arteries (advanced lesions); however, only the secretory group of PLA
2
has been shown to specifically hydrolyze low density lipoprotein (LDL)–associated phospholipids and, as such, may play a potential role in atherogenesis. In the present study, we investigated the expression pattern of group IIa, IV, and V PLA
2
s in human macrophages, which are the key cells involved in the onset and perpetuation of atherosclerosis. Immunohistochemical staining by double labeling showed that the secretory nonpancreatic PLA
2
(snpPLA
2
) is detectable in macrophages in the intima of early atherosclerotic lesions. Reverse transcription–polymerase chain reaction analysis of RNA extracted from human monocytes clearly showed that expression of group IV PLA
2
was enhanced during differentiation into macrophages, with an onset of induction at days 2 to 3 of differentiation. Group V snpPLA
2
was constitutively expressed on differentiation, whereas the detection of group IIa snpPLA
2
was dependent on both differentiation and subsequent stimulation of macrophages. Indeed, the transcription of group IIa snpPLA
2
in macrophages was induced by treatment with minimally modified or mildly oxidized LDL, whereas native, extensively oxidized, or acetylated LDL had no effect. To our knowledge, this is the first report describing induction of group IIa snpPLA
2
expression in human monocyte–derived macrophages. The mRNA levels of cytosolic PLA
2
group IV and snpPLA
2
group V remained unchanged on LDL treatment. Thus, our results show that the expression of distinct PLA
2
enzymes is regulated not only during differentiation of monocytes into macrophages but also on exposure of macrophages to distinct LDL species. Consequently, our results indicate a potential role for both cytosolic and secretory PLA
2
enzymes in inflammation and in macrophage functions related to atherosclerosis, with a specific role for group IIa snpPLA2 in LDL scavenging.
Expression, Localization, and Activity of Tissue Factor Pathway Inhibitor in Normal and Atherosclerotic Human Vessels
Authors: Crawley James , Lupu Florea , Westmuckett Andrew D. , Severs Nicholas J. , Kakkar Vijay V. , Lupu Cristina
Published: June 2011

Abstract
—Tissue factor (TF) pathway inhibitor (TFPI) is the major downregulator of the procoagulant activity of the TF–factor VIIa (FVIIa) complex (TF · FVII). The active TF present in the atherosclerotic vessel wall is proposed to be responsible for the major complication of primary atherosclerosis, namely, acute thrombosis after plaque rupture, but our knowledge of the sites of TFPI expression in relation to TF remains fragmentary. The aim of this study was to investigate the expression, localization, and activity of TFPI and its relation to the activity and distribution of TF in the normal and atherosclerotic vessel wall. We applied a novel approach in which serial cross sections of human vascular segments were used to perform a complete set of assays: immunolabeling for TFPI and/or TF, in situ hybridization for the expression of TFPI mRNA, ELISA for the determination of TFPI antigen, and functional assay for the activity of TFPI and TF. In healthy vessels, TFPI protein and mRNA are present in luminal and microvascular endothelial cells (ECs) and in the medial smooth muscle cells (SMCs). In atherosclerotic vessels, TFPI protein and mRNA frequently colocalized with TF in ECs overlying the plaque and in microvessels, as well as in the medial and neointimal SMCs, and in macrophages and T cells in areas surrounding the necrotic core. At the ultrastructural level, immunogold electron microscopy confirmed the localization of TFPI in ECs, macrophages/foam cells, and SMCs. In ECs and SMCs, the gold particles decorated the plasmalemma proper and the caveolae. ELISA on cross sections revealed that atherosclerotic tissues contain more TFPI than do the healthy vessels. TFPI was functionally active against TF · FVIIa-induced coagulation, and its activity was higher in those tissues that display less TF. The largest amount of TFPI and TF were detected in complicated arterial plaques. By immunofluorescence, TFPI colocalized with platelet- and fibrin-rich areas within the organized thrombi. Atherosclerotic vessel sections promote activation of factor X, which is dependent on the presence of TF and enhanced by preincubation of the sections with anti-TFPI IgG. Taken altogether, our results suggest that TFPI is largely expressed in the normal vessel wall and enhanced in the atherosclerotic vessel, in a manner suggesting a significant role of TFPI in the regulation of TF activity.
Vascular Endothelial Growth Factor Production by Fibroblasts in Response to Factor VIIa Binding to Tissue Factor Involves Thrombin and Factor Xa
Authors: Ollivier V. , Chabbat J. , Herbert J. M. , Hakim J. , de Prost D.
Published: June 2011

Abstract
—Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti–effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.
Development of Endothelial Cell Lines From Embryonic Stem Cells
Authors: Balconi Giovanna , Spagnuolo Raffaella , Dejana Elisabetta
Published: June 2011

Abstract
—Totipotent embryonic stem cells can be induced to differentiate to endothelium in vitro. This may be a useful tool for obtaining cultures of genetically manipulated endothelial cells because embryonic stem cells are relatively easy to transfect and are commonly used for gene inactivation experiments in mice. However, embryonic stem cell–derived endothelial cells could not be easily separated from embryoid bodies and maintained in culture. In this study, we describe the isolation and characterization of immortalized endothelial cell lines obtained from embryonic stem cells differentiated in vitro. The cell lines were analyzed for expression of endothelial cell markers, including growth factor receptors and adhesion molecules, and compared with endothelial cells obtained from the yolk sac, the embryo proper, or the heart microcirculation of the adult. We propose that this approach may be useful for obtaining endothelial cells carrying gene mutations that are lethal at very early stages of development.
Adenovirus-Mediated Human Tissue Kallikrein Gene Delivery Inhibits Neointima Formation Induced by Interruption of Blood Flow in Mice
Authors: Emanueli Costanza , Salis Maria Bonaria , Chao Julie , Chao Lee , Agata Jun , Lin Kuei-Fu , Munaò Antonella , Straino Stefania , Minasi Alessandra , Capogrossi Maurizio C. , Madeddu Paolo
Published: June 2011

Abstract
—Tissue kallikrein cleaves kininogen to produce vasoactive kinin peptides. Binding of kinins to bradykinin B
2
receptors on vascular endothelial cells stimulates the release of nitric oxide and prostacyclin, thus activating the cGMP and cAMP pathways. In this study, we evaluated the effects of adenovirus-mediated human tissue kallikrein gene (Ad.CMV-cHK) delivery in a mouse model of arterial remodeling induced by permanent alteration in shear stress conditions. Mice underwent ligature of the left common carotid artery and were injected intravenously with saline or 1.8×10
9
plaque-forming units of Ad.CMV-cHK or control virus (Ad.CMV-LacZ). Fourteen days after surgery, morphometric analysis revealed that Ad.CMV-cHK reduced neointima formation by 52% (
P
<0.05) compared with Ad.CMV-LacZ. Expression of human tissue kallikrein (HK) mRNA was detected in mouse carotid artery, aorta, kidney, heart, and liver, and recombinant HK was present in the urine and plasma of mice receiving HK gene. Kallikrein gene transfer resulted in increases in urinary kinin, cGMP, and cAMP levels. The protective action of Ad.CMV-cHK on neointima formation was significantly reduced (
P
<0.05) in mice with knockout of the kinin B
2
receptor gene compared with wild-type control mice (J129Sv mice). In contrast, the effect of Ad.CMV-cHK was amplified (
P
<0.05) in transgenic mice overexpressing human B
2
receptor compared with wild-type control mice (c57/Bl6 mice). Thus, the inhibitory effect of recombinant kallikrein on structural alterations caused by the interruption of blood flow appears to be mediated by the B
2
receptor. These results provide new insight into the role of the tissue kallikrein-kinin system in vascular remodeling and suggest the application of HK gene therapy to treat restenosis and atherosclerosis.
Peripheral Vascular Endothelial Dysfunction and Apoptosis in Old Monkeys
Authors: Asai Kuniya , Kudej Raymond K. , Shen You-Tang , Yang Gui-Ping , Takagi Gen , Kudej Amelia B. , Geng Yong-Jian , Sato Naoki , Nazareno Jerome B. , Vatner Dorothy E. , Natividad Filipinas , Bishop Sanford P. , Vatner Stephen F.
Published: June 2011

Abstract
—To determine the effects of aging on vasoactivity in a primate model (
Macaca fascicularis
), 13 young male monkeys (aged 7.1±0.4 years) and 9 old male monkeys (aged 19.8±0.6 years) were chronically instrumented for measurement of left ventricular and aortic pressures and cardiac output. Total cholesterol, triglyceride, and fasting blood sugar levels were not different between the 2 groups. There were no significant differences in baseline mean aortic pressure and total peripheral resistance (TPR) in the young monkeys versus the old monkeys. TPR fell less (
P
<0.05) with acetylcholine (1 μg/kg) in old monkeys (−25±1%) than in young monkeys (−34±2%), whereas decreases in TPR with sodium nitroprusside were similar in old and young monkeys. There was no evidence of atherosclerosis, but apoptosis of endothelial cells was enhanced (
P
<0.05) in the aortas and femoral arteries, but not in the media, of the old monkeys. There was a relationship (
r
=0.62,
P
=0.013) between the incidence of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)-positive endothelial cells and endothelial cell density in the femoral artery. The reduced endothelial cell density was also correlated (
r
=0.82,
P
<0.01) with depressed TPR responses to acetylcholine. Thus, vascular endothelial dysfunction was present in old monkeys without evidence of atherosclerosis, which may be due to endothelial apoptosis and reduced endothelial cell density.
Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells
Authors: Hu Wen-Yang , Fukuda Noboru , Satoh Chikara , Jian Teng , Kubo Atsushi , Nakayama Mari , Kishioka Hirobumi , Kanmatsuse Katsuo
Published: June 2011

Abstract
—We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II–generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0.2% calf serum for 48 hours. Immunofluorescence and protein levels of α-smooth muscle (SM) actin and expression of SM22α mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 μg/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II–like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II–generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-β1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.
Cafestol Increases Serum Cholesterol Levels in Apolipoprotein E*3-Leiden Transgenic Mice by Suppression of Bile Acid Synthesis
Authors: Post Sabine M. , de Roos Baukje , Vermeulen Martijn , Afman Lydia , Jong Miek C. , Dahlmans Vivian E. H. , Havekes Louis M. , Stellaard Frans , Katan Martijn B. , Princen Hans M. G.
Published: June 2011

Abstract
—Cafestol, a diterpene present in unfiltered coffee, potently increases serum cholesterol levels in humans. So far, no suitable animal model has been found to study the biochemical background of this effect. We determined the effect of cafestol on serum cholesterol and triglycerides in different mouse strains and subsequently studied its mechanism of action in apolipoprotein (apo) E*3-Leiden transgenic mice. ApoE*3-Leiden, heterozygous low density lipoprotein–receptor (LDLR+/−) knockout, or wild-type (WT) C57BL/6 mice were fed a high- (0.05% wt/wt) or a low- (0.01% wt/wt) cafestol diet or a placebo diet for 8 weeks. Standardized to energy intake, these amounts are equal to 40, 8, or 0 cups of unfiltered coffee per 10 MJ per day in humans. In apoE*3-Leiden mice, serum cholesterol was statistically significantly increased by 33% on the low- and by 61% on the high-cafestol diet. In LDLR+/− and WT mice, the increases were 20% and 24%, respectively, on the low-cafestol diet and 55% and 46%, respectively, on the high-cafestol diet. These increases were mainly due to a rise in very low density lipoprotein (VLDL) and intermediate density lipoprotein cholesterol in all 3 mouse strains. To investigate the mechanism of this effect, apoE*3-Leiden mice were fed a high-cafestol or a placebo diet for 3 weeks. Cafestol suppressed enzyme activity and mRNA levels of cholesterol 7α-hydroxylase by 57% and 58%, respectively. mRNA levels of enzymes involved in the alternate pathway of bile acid synthesis, ie, sterol 27-hydroxylase and oxysterol 7α-hydroxylase, were reduced by 32% and 48%, respectively. The total fecal bile acid output was decreased by 41%. Cafestol did not affect hepatic free and esterified cholesterol, but it decreased LDLR mRNA levels by 37%. The VLDL apoB and triglyceride production rates, as measured after Triton injection, were 2-fold decreased by cafestol, indicating that the number of particles secreted had declined and that there was no change in the amount of triglycerides present in the VLDL particle during cafestol treatment. However, the VLDL particles contained a 4-times higher amount of cholesteryl esters, resulting in a net 2-fold increased secretion of cholesteryl esters. The decrease in triglyceride production was the result of a reduction in hepatic triglyceride content by 52%. In conclusion, cafestol increases serum cholesterol levels in apoE*3-Leiden mice by suppression of the major regulatory enzymes in the bile acid synthesis pathways, leading to decreased LDLR mRNA levels and increased secretion of hepatic cholesterol esters. We suggest that suppression of bile acid synthesis may provide an explanation for the cholesterol-raising effect of cafestol in humans.
Plasma Concentration of Asymmetric Dimethylarginine, an Endogenous Inhibitor of Nitric Oxide Synthase, Is Elevated in Monkeys With Hyperhomocyst(e)inemia or Hypercholesterolemia
Authors: Böger Rainer H. , Bode-Böger Stefanie M. , Sydow Karsten , Heistad Donald D. , Lentz Steven R.
Published: June 2011

Abstract
—Hyperhomocyst(e)inemia is associated with endothelial dysfunction. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inemia may involve impaired bioavailability of endothelium-dependent nitric oxide. We tested the hypothesis that hyperhomocyst(e)inemia is associated with an elevated plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. One group of adult cynomolgus monkeys was fed either a control or hyperhomocyst(e)inemic diet for 4 weeks in a randomized crossover design. The second group was fed an atherogenic diet that produces both hyperhomocyst(e)inemia and hypercholesterolemia for 17 months, followed by an atherogenic diet supplemented with B vitamins for 6 months to decrease plasma homocyst(e)ine concentration. Human endothelial cells were used to study the effects of methionine and homocysteine in the presence or absence of B vitamins or the methylation inhibitor
S
-adenosylhomocysteine on the formation of ADMA and its inactive stereoisomer, symmetric dimethylarginine. The hyperhomocyst(e)inemic diet produced 2- to 3-fold increases in plasma levels of homocyst(e)ine and ADMA (both
P
<0.05). The atherogenic diet also produced elevated plasma levels of homocyst(e)ine and ADMA (both
P
<0.05). Supplementation of the atherogenic diet with B vitamins decreased the plasma levels of homocyst(e)ine but did not affect the plasma levels of ADMA or endothelial function. There was a strong correlation between plasma ADMA and homocyst(e)ine and a strong inverse correlation between ADMA and carotid artery relaxation to acetylcholine. ADMA release by cultured endothelial cells was significantly increased in the presence of methionine or homocysteine. This effect was blocked by
S
-adenosylhomocysteine but not by B vitamins. We conclude that plasma levels of ADMA are elevated in hyperhomocyst(e)inemia. Because ADMA acts as a competitive inhibitor of endothelial nitric oxide synthase, these findings suggest a novel mechanism for impaired endothelial function in hyperhomocyst(e)inemia.
LDL Receptor–Related Protein Mediates Uptake of Aggregated LDL in Human Vascular Smooth Muscle Cells
Authors: Llorente-Cortés Vicenta , Martínez-González José , Badimon Lina
Published: June 2011

Abstract
—Foam cell formation is a key event in the onset and progression of atherosclerotic lesions. We have previously reported that internalization of aggregated low density lipoproteins (agLDLs) by vascular smooth muscle cells (VSMCs) produces cholesteryl ester (CE) accumulation in these cells. The aim of this study was to analyze whether the low density lipoprotein receptor–related protein (LRP) mediates the uptake of agLDL by VSMCs. First, immunocytochemistry and fluorescence microscopic analysis with the use of anti-LRP antibodies indicated that there was a high expression of LRP in VSMCs. Confocal microscopic analysis with the use of agLDLs labeled with fluorochrome 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine and anti-LRP antibodies showed the colocalization of agLDL and LRP. The second approach was to analyze the effect of LRP ligands on agLDL internalization; lactoferrin strongly inhibited CE accumulation from agLDLs (85.0±5.7% at 25 μg/mL) by impairing agLDL binding. Coincubation of agLDL with anti-LRP antibodies decreased in a dose-dependent manner agLDL-derived CE accumulation (from 20% at 12.5 μg/mL to 80% at 50 μg/mL). The third approach was to evaluate whether antisense LRP oligodeoxynucleotides were able to block agLDL internalization. Treatment of VSMCs with 5 μmol/L antisense LRP oligodeoxynucleotides reduced agLDL-derived CE accumulation by 84±2%. In conclusion, these results from immunologic, biochemical, and molecular interventions demonstrate that LRP mediates the binding and internalization of agLDL in human VSMCs. Because LRP is highly expressed in VSMCs and the uptake of 1 LDL aggregate amounts to the deposition of several hundreds of LDL particles, the uptake of agLDL through LRP could have a crucial role for lipid deposition in VSMCs.
Common Carotid Intima-Media Thickness Predicts Occurrence of Carotid Atherosclerotic Plaques
Authors: Zureik Mahmoud , Ducimetière Pierre , Touboul Pierre-Jean , Courbon Dominique , Bonithon-Kopp Claire , Berr Claudine , Magne Christine
Published: June 2011

Abstract
—The role of the increase in the common carotid artery (CCA) intima-media wall thickness (IMT) in the atherosclerotic process is questionable. This longitudinal study examined the predictive value of CCA-IMT measured at baseline examination (at sites free of plaques) on the occurrence of atherosclerotic plaques in the extracranial carotid arteries during 4 years of follow-up study in a sample of 1010 subjects aged 59 to 71 years. Ultrasound examinations were performed at baseline and 2 years and 4 years later. The occurrence of carotid plaques during follow-up was defined as the appearance of ≥1 plaque in previously normal carotid segments and/or the appearance of new plaques in the carotid segments that previously had plaques. Carotid plaque occurrence was observed in 185 subjects (18.3%). Age- and sex- adjusted odds ratios of carotid plaque occurrence were 2.66 (95% CI 1.58 to 4.46,
P
<0.001) in subjects having intermediate baseline CCA-IMT values (quartiles 2 and 3) and 3.67 (CI 2.09 to 6.44,
P
<0.001) in those having the highest baseline CCA-IMT values (quartile 4) compared with those having the lowest baseline CCA-IMT values (quartile 1). Multivariate adjustment for major cardiovascular risk factors did not alter the results. These findings were observed for men and women as well as for subjects with and without carotid plaques at baseline. This 4-year longitudinal study shows that CCA-IMT predicts carotid plaque occurrence in a large sample of relatively old subjects. It extends the findings obtained from cross-sectional studies and suggests that increased intima-media thickness might occur in an earlier phase of the atherosclerotic process.
Progressive and Transient Expression of Tissue Plasminogen Activator During Fetal Development
Authors: Levin Eugene G. , Banka Carole L. , Parry Graham C. N.
Published: June 2011

Abstract
—In previous studies of the role of tissue plasminogen activator (tPA) in the lung inflammatory response, we observed that tPA expression was present exclusively in the small arteries and arterioles within the lung and absent from the capillaries, veins, and large pulmonary arteries. To define more completely the expression pattern of tPA, we evaluated the distribution of this protein during prenatal and postnatal development. tPA was first observed in the rat fetus at day 13 in the large arteries of both the thoracic and cranial cavities, including the dorsal aortas and pulmonary arteries in the former and the internal carotid and middle cerebral arteries in the latter. By day 15, tPA was no longer detectable in the aortas but appeared throughout the pulmonary, subclavian, vertebral, and basilar arteries. At day 17, tPA had disappeared from the subclavian artery and the proximal portion of the vertebral artery but was found in the smaller arterial branches of these 2 large vessels. By the end of gestation, tPA had also disappeared from the main pulmonary arteries but remained in the branches at the hilus of the lung. At birth, tPA was concentrated in the endothelia of arteries within the pia mater, the basilar and superficial cerebral arteries, and the lung arterial system. As the animals reached maturity, tPA disappeared from the larger cerebral arteries and their cortical branches but continued to be expressed in the vessels of the pia mater and lung. This study indicates that tPA expression is a dynamic process that responds to a changing arterial environment during vascular development.
Role of Cytokines in the Regulation of Plasminogen Activator Inhibitor-1 Expression and Secretion in Newly Differentiated Subcutaneous Human Adipocytes
Authors: Birgel M. , Gottschling-Zeller H. , Röhrig K. , Hauner H.
Published: June 2011

Abstract
—Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic for obesity and are associated with increased risk of thromboembolic complications. PAI-1 recently was reported to be expressed and secreted by human adipocytes, but little is known about regulation of PAI-1 in human adipose tissue. Therefore, we examined the effects of selected cytokines present in adipose tissue on expression and secretion of PAI-1 in in vitro, differentiated subcutaneous human adipocytes in primary culture. Transforming growth factor-β1 (TGF-β1) increased PAI-1 secretion in a dose- and time-dependent manner. PAI-1 protein increased by 3.2-fold and PAI-1 mRNA by 1.9-fold after a 6-hour exposure to 400 pmol/L TGF-β1. This effect is probably mediated by TGF-β1 type 2 and 3 receptors, which were found to be expressed in cultured human adipocytes. Moreover, TNF-α and interkeukin-1β (IL-1β) also exerted a stimulatory effect on PAI-1 release and increased PAI-1 mRNA levels. As assessed by a semiquantitative reverse transcription—polymerase chain reaction technique, TGF-β1 mRNA is expressed by differentiation of human preadipocytes and is moderately upregulated by TNF-α and IL-1β. In conclusion, our results clearly indicate that TGF-β1 is a potent inducer of PAI-1 production in subcutaneous human adipocytes. In addition, data suggest that TNF-α and IL-1β also have stimulatory effects on PAI-1 protein secretion and may contribute to the elevated PAI-1 levels observed in obesity.
Nitric Oxide Regulation of Free Radical– and Enzyme-Mediated Lipid and Lipoprotein Oxidation
Authors: Bloodsworth Allison , O’Donnell Valerie B. , Freeman Bruce A.
Published: June 2011

Abstract
—The regulation of nonenzymatic and enzymatic lipid oxidation reactions by nitric oxide (·NO) is potent and pervasive and reveals novel non–cGMP-dependent reactivities for this free radical inflammatory and signal transduction mediator. ·NO and its metabolites stimulate and inhibit lipid peroxidation reactions, modulate enzymatically catalyzed lipid oxidation, complex with lipid-reactive metals, and alter proinflammatory gene expression. Through these mechanisms, ·NO can regulate nonenzymatic lipid oxidation and the production of inflammatory and vasoactive eicosanoids by prostaglandin endoperoxide synthase and lipoxygenase. The accumulation of macrophages and oxidized low density lipoprotein within the vascular wall can also be modulated by ·NO. A key determinant of the pro-oxidant versus oxidant-protective influences of ·NO is the underlying oxidative status of tissue. When ·NO is in excess of surrounding oxidants, lipid oxidation and monocyte margination into the vascular wall are attenuated, producing antiatherogenic effects. However, when endogenous tissue rates of oxidant production are accelerated or when tissue oxidant defenses become depleted, ·NO gives rise to secondary oxidizing species that can increase membrane and lipoprotein lipid oxidation as well as foam cell formation in the vasculature, thus promoting proatherogenic effects. In summary, ·NO is a multifaceted molecule capable of reacting via multiple pathways to modulate lipid oxidation reactions, thereby impacting on tissue inflammatory reactions.
Monoclonal Antibody Against Vascular Cell Adhesion Molecule-1 Inhibits Neointimal Formation After Periadventitial Carotid Artery Injury in Genetically Hypercholesterolemic Mice
Authors: Oguchi Sumito , Dimayuga Paul , Zhu Jenny , Chyu Kuang-Yuh , Yano Juliana , Shah Prediman K. , Nilsson Jan , Cercek Bojan
Published: June 2011

Abstract
—Vascular cell adhesion molecule (VCAM)-1 is induced in smooth muscle cells after arterial injury, in which it has been implicated in the recruitment of inflammatory cells to the site of injury. To investigate the effect of hypercholesterolemia on VCAM-1 induction after injury and the role of VCAM-1 in neointimal response to injury, we injured the carotid artery of wild-type and apolipoprotein E null (KO) mice fed normal and high cholesterol chow. We demonstrate a graded response of VCAM-1 induction as well as monocyte/macrophage infiltration by immunohistochemistry 3 days after injury that correlated with increasing circulating cholesterol levels. Three weeks after injury, KO mice fed high cholesterol chow (KO HC group) had a significantly greater neointimal formation compared with wild-type and KO mice fed normal chow (
P
<0.05). Inhibition of VCAM-1 function in the KO HC group by monoclonal antibody treatment significantly reduced monocyte/macrophage infiltration and neointimal formation. There was reduced α-actin expression in KO HC mice 7 days after injury that was partially inhibited by VCAM-1 antibody treatment. Cell migration in an in vitro injury model was partially inhibited by monoclonal VCAM-1 antibody treatment. We propose an additional role for VCAM-1 in smooth muscle cell activation and neointimal formation after injury.
Enrichment of Acyl Coenzyme A:Cholesterol
O
-Acyltransferase Near
Trans
-Golgi Network and Endocytic Recycling Compartment
Authors: Nadia Khelef  , Soe T. T. , Quehenberger Oswald , Beatini Nanda , Tabas Ira , Maxfield Frederick R.
Published: June 2011

Abstract
—Acyl coenzyme A:cholesterol
O
-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent α
2
-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the
trans
-Golgi network (TGN), but farther from Gos28, a marker of
cis
, medial, and
trans
Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.
Apolipoprotein E2 (Lys146→Gln) Causes Hypertriglyceridemia due to an Apolipoprotein E Variant–Specific Inhibition of Lipolysis of Very Low Density Lipoproteins–Triglycerides
Authors: de Beer Femke , van Dijk Ko Willems , Jong Miek C. , van Vark Leonie C. , van der Zee Andre , Hofker Marten H. , Fallaux Frits J. , Hoeben Rob C. , Smelt Augustinus H. M. , Havekes Louis M.
Published: June 2011

Abstract
—The apolipoprotein E2 (Lys146→Gln) variant is associated with a dominant form of familial dysbetalipoproteinemia. Heterozygous carriers of this variant have elevated levels of plasma triglycerides, cholesterol, and apolipoprotein E (apoE). It was hypothesized that the high amounts of triglycerides in the very low density lipoprotein (VLDL) fraction are due to a disturbed lipolysis of VLDL. To test this hypothesis, apoE knockout mice were injected with an adenovirus containing the human APOE*2 (Lys146→Gln) gene, Ad-E2(146), under the control of the cytomegalovirus promoter. ApoE knockout mice injected with an adenovirus vector encoding human apoE3 (Ad-E3) were used as controls. Five days after adenovirus injection, plasma cholesterol levels of mice injected with a high dose of Ad-E2(146) (2×10
9
plaque-forming units) were not changed compared with preinjection levels, whereas in the group who received a low dose of Ad-E2(146) (5×10
8
plaque-forming units) and in the groups injected with a low or a high dose of Ad-E3, plasma cholesterol levels were decreased 5-, 6-, and 12-fold, respectively. Plasma triglycerides were not affected in mice injected with Ad-E3. In contrast, a 7-fold increase in plasma triglycerides was observed in mice injected with the low dose of Ad-E2(146) compared with mice injected with Ad-E3. Injection with the high dose of Ad-E2(146) resulted in a dramatic increase of plasma triglycerides (50-fold compared with Ad-E3 injection). In vitro lipolysis experiments showed that the lipolysis rate of VLDLs containing normal amounts of apoE2 (Lys146→Gln) was decreased by 54% compared with that of VLDLs containing comparable amounts of apoE3. The in vivo VLDL-triglyceride production rate of Ad-E2(146)–injected mice was not significantly different from that of Ad-E3–injected mice. These results demonstrate that expression of apoE2 (Lys146→Gln) causes hypertriglyceridemia due to an apoE variant–specific inhibition of the hydrolysis of VLDL-triglycerides.
Expression of Class A Scavenger Receptor Inhibits Apoptosis of Macrophages Triggered by Oxidized Low Density Lipoprotein and Oxysterol
Authors: Liao Hai-Sun , Kodama Tatsuhiko , Geng Yong-Jian
Published: June 2011

Abstract
—The class A macrophage scavenger receptor (MSR-A) is a multifunctional trimeric glycoprotein involved in innate immune response as well as the development of lipid-laden foam cells during atherosclerosis. The MSR ligand, oxidized low density lipoprotein (oxLDL), is known to be cytotoxic to macrophages and other cell types. This study examined whether MSR mediates or modulates oxLDL-induced apoptosis. Treatment with oxLDL and its cytotoxic oxysterol, 7-ketocholesterol (7-KC), reduced viability and increased DNA fragmentation in human THP-1 cells, Chinese hamster ovary cells, and mouse peritoneal macrophages. However, cell death and DNA fragmentation were markedly diminished in the phorbol ester–differentiated MSR-expressing THP-1 cells and Chinese hamster ovary cells, with stable expression of MSR-AI after cDNA transfection when exposed to the same concentrations of oxLDL and 7-KC. Moreover, treatment with oxLDL and 7-KC induced much greater death and DNA fragmentation in MSR-A–deficient peritoneal macrophages compared with wild-type macrophages. Thus, MSR-A does not act as a receptor responsible for the apoptotic effect of oxLDL, and instead, expression of this receptor confers resistance of macrophages to the apoptotic stimulation by oxLDL and its cytotoxic lipid component. These results suggest that by preventing apoptosis, MSR-A may contribute to the long-term survival of macrophages and macrophage-derived lipid-laden foam cells in atherosclerotic lesions.
Common and Rare
ABCA1
Variants Affecting Plasma HDL Cholesterol
Authors: Wang Jian , Burnett John R. , Near Susan , Young Kue , Zinman Bernard , Hanley Anthony J. G. , Connelly Philip W. , Harris Stewart B. , Hegele Robert A.
Published: June 2011

Abstract
—Mutations in
ABCA1
, a member of the ATP-binding cassette family, have been shown to underlie Tangier disease (TD) and familial hypoalphalipoproteinemia (FHA), which are genetic disorders that are characterized by depressed concentrations of plasma high density lipoprotein (HDL) cholesterol. An important question is whether common variants within the coding sequence of
ABCA1
can affect plasma HDL cholesterol in the general population. To address this issue, we developed a screening strategy to find common
ABCA1
variants. This strategy involved long-range amplification of genomic DNA by using coding sequences only, followed by deep sequencing into the introns. This method helped us to characterize a new set of amplification primers, which permitted amplification of virtually all of the coding sequence of
ABCA1
and its intron-exon boundaries with a single DNA amplification program. With these new sequencing primers, we found 3 novel
ABCA1
mutations: a frameshift mutation (4570insA, A1484S→X1492), a missense mutation (A986D) in a TD family, and a missense mutation (R170C) in aboriginal subjects with FHA. We also used these sequencing primers to characterize 4 novel common amino acid variants in
ABCA1
, in addition to 5 novel common silent variants. We tested for association of the
ABCA1
I/M823 variant with plasma HDL cholesterol in Canadian Inuit and found that M823/M823 homozygotes had significantly higher plasma HDL cholesterol compared with subjects with the other genotypes. The results provide proof of principle of the effectiveness of this approach to identify both rare and common
ABCA1
genomic variants and also suggest that common amino acid variation in
ABCA1
is a determinant of plasma HDL cholesterol in the general population.
Functional Effects of the ABO Locus Polymorphism on Plasma Levels of von Willebrand Factor, Factor VIII, and Activated Partial Thromboplastin Time
Authors: Souto Juan Carlos , Almasy Laura , Muñiz-Diaz Eduardo , Soria José Manuel , Borrell Montserrat , Bayén Laia , Mateo José , Madoz Pedro , Stone William , Blangero John , Fontcuberta Jordi
Published: June 2011

Abstract
—Lower levels of factor VIII and von Willebrand factor (vWF) have been reported in individuals with blood type O compared with individuals with other ABO blood types. However, this relationship has been demonstrated only by association studies and not by linkage studies. Also, it is not clear whether the ABO locus exerts a functional effect directly on these plasma factors or whether the ABO locus is in linkage disequilibrium with another locus that controls these factors. To distinguish between these 2 possibilities, we applied new statistical methods combining linkage and association tests in a pedigree-based sample. In contrast to most previous studies that used the ABO phenotypes, our study used the ABO genotypes, permitting us to distinguish AO from AA and BO from BB. Our results clearly showed significant linkage between the ABO locus and vWF antigen (
P
=0.00075). In addition, factor VIII coagulant activity and activated partial thromboplastin time showed suggestive linkage with the ABO locus (
P
=0.10 and
P
=0.13). All 3 plasma phenotypes showed significant differences between OO and non-OO genotypes. In addition, vWF antigen exhibited significant differences between O heterozygotes and non-OO homozygotes. This study is unique because it used a combined linkage and association test, which indicated that the ABO locus itself has a functional effect on these plasma phenotypes.
C-Reactive Protein in the Arterial Intima
Authors: Torzewski Michael , Rist Carsten , Mortensen Richard F. , Zwaka Thomas P. , Bienek Magda , Waltenberger Johannes , Koenig Wolfgang , Schmitz Gerd , Hombach Vinzenz , Torzewski Jan
Published: June 2011

Abstract
—Infiltration of monocytes into the arterial wall is an early cellular event in atherogenesis. Recent evidence shows that C-reactive protein (CRP) is deposited in the arterial intima at sites of atherogenesis. In this study, we demonstrate that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions. CRP is chemotactic for freshly isolated human blood monocytes. A specific CRP receptor is demonstrated on monocytes in vitro as well as in vivo, and blockage of the receptor by use of a monoclonal anti-receptor antibody completely abolishes CRP-induced chemotaxis. CRP may play a major role in the recruitment of monocytes during atherogenesis.
In Vivo Thrombin Generation and Activity During and After Intravenous Infusion of Heparin or Recombinant Hirudin in Patients With Unstable Angina Pectoris
Authors: Merlini Piera Angelica , Ardissino Diego , Rosenberg Robert D. , Colombi Elisabetta , Agricola Pietro , Oltrona Luigi , Ottani Filippo , Galvani Marcello , Bauer Kenneth A. , Bottasso Bianca , Bertocchi Federico , Mannucci Pier Mannuccio
Published: June 2011

Abstract
—In patients with unstable angina, intravenous heparin reduces thrombin activity but does not influence thrombin generation. Recombinant hirudin, a direct thrombin inhibitor, may be more effective in inhibiting both thrombin generation and activity. We measured the plasma levels of prothrombin fragment 1+2 (a marker of thrombin generation) and fibrinopeptide A (a marker of thrombin activity) in 67 patients with unstable angina enrolled in the GUSTO (Global Use of Strategies to Open Occluded Coronary Arteries) IIb trial who were receiving either recombinant hirudin (31 patients) or heparin (36 patients). Blood samples were obtained at baseline (before any treatment), after 3 to 5 days of study drug infusion (immediately before discontinuation), and 1 month later. In the patients receiving recombinant hirudin, the prothrombin fragment 1+2 levels measured immediately before drug discontinuation were significantly lower than at baseline (
P
=0.0014), whereas they had not changed in the patients receiving heparin; at this time point, the difference between patients receiving hirudin and those receiving heparin was statistically significant (
P
=0.032). One month later, the prothrombin fragment 1+2 levels in both groups were similarly persistently high and did not differ from baseline. Fibrinopeptide A plasma levels at the end of infusion were significantly lower than at baseline in both treatment groups (
P
=0.0005 for hirudin and
P
=0.042 for heparin) and remained lower after 1 month (
P
=0.0001 for both hirudin and heparin). The fibrinopeptide A plasma levels were not different between patients treated with hirudin versus heparin at baseline, at the end of infusion, and after 1 month. Thus, in patients with unstable angina, in vivo thrombin generation and activity are reduced during intravenous infusion of recombinant hirudin. However, the inhibition of thrombin generation is not sustained, and after 1 month, the majority of patients have biochemical signs of increased thrombin generation.