bluuebunny/crossref_metadata_2025 huggingface dataset:
Smooth Muscle-Specific SM22 Protein Is Expressed in the Adventitial Cells of Balloon-Injured Rabbit Carotid Artery
Authors: Faggin Elisabetta , Puato Massimo , Zardo Lorena , Franch Rafaella , Millino Caterina , Sarinella Federica , Pauletto Paolo , Sartore Saverio , Chiavegato Angela
Published: June 2011

Abstract—During the “response-to-injury” process after a mechanical insult to the porcine coronary arteries, the adventitial cells acquire the structural characteristics of myofibroblasts before being incorporated into smooth muscle (SM) layer. We assessed whether the SM-specific SM22 protein can be used as a tracer of adventitial cell-myofibroblast differentiation in the mild balloon injury of rabbit carotid artery. To achieve this goal, we used 2 monoclonal anti-SM22 antibodies (E-11 and 1-B8) and a molecular probe for the SM22α mRNA isoform in immunocytochemical and in situ hybridization experiments. The differentiation profile and the migratory and proliferative ability of activated adventitial cells were evaluated by a panel of antibodies to some SM and nonmuscle antigens and pulse- and end-labeling with bromo-deoxyuridine, respectively. In adventitial cells, SM22 antigenicity and SM22α mRNA were detectable at days 2 and 4 and, to a lesser extent, at days 7 and 21 after injury, particularly near the adventitia-media interface and mostly colocalizing with bromo-deoxyuridine–positive cells. The pulse-labeling experiments showed that the large majority of these cells penetrated the outermost layer of the tunica media without migrating to the subendothelial region. The phenotypic features of activated migrating and nonmigrating adventitial cells resembled those of vimentin-actin myofibroblast subtype and fetal-type SM cells. These findings indicate that a direct exposure of adventitia to the lumen is not required for phenotypic changes and proliferation/migration of these cells. After comparison of the SM22 expression in arterial vessels during early stages of development, we hypothesize that in the injured carotid artery the mural incorporation of adventitial cells and the spatiotemporal activation of SM22 expression are reminiscent of the vascular morphogenetic process and suggest the existence of a stem cell-like reservoir in adventitia. The early adventitial upregulation of SM22 expression in the injured vessel might be related to a multistep transition process in which nonmuscle cells are converted to myofibroblasts and, possibly, to SM cells.
Differentiation of Human Monocytes to Monocyte-Derived Macrophages Is Associated With Increased Lipoprotein Lipase–Induced Tumor Necrosis Factor-α Expression and Production
Authors: Mamputu Jean-Claude , Renier Geneviève
Published: June 2011

Abstract—The aim of the present study was to (1) evaluate the responsiveness of human mononuclear cells to lipoprotein lipase (LPL), as assessed by tumor necrosis factor-α (TNFα) production, during the process of differentiation of monocytes to macrophages, and (2) determine the mechanisms by which LPL exerts its effect on these cells. Treatment of human monocytes with purified endotoxin-free bovine LPL (1 μg/mL) resulted in a 161±15% increase in TNFα production over control values (P<0.01). A further increase in TNFα production was observed after treatment of monocyte-derived macrophages (MDMs) with LPL (490±81% over control values,P<0.01). Increased TNFα mRNA expression and protein kinase C activity were also observed in LPL-treated human monocytes and MDMs. These LPL effects were abrogated by the specific protein kinase C inhibitor calphostin C (1 μmol/L). Although heparinase totally abolished LPL-induced TNFα production in human monocytes, this agent did not significantly inhibit LPL effect in human MDMs. In contrast, treatment of MDMs with chondroitinase suppressed LPL-induced TNFα production. Taken together, these data suggest that (1) differentiation of human monocytes to MDMs is associated with increased LPL-induced TNFα mRNA expression and production, (2) a protein kinase C–dependent pathway is involved in the induction of TNFα by LPL in these cells, and (3) LPL effect is mediated by cell surface proteoglycans. As MDMs secrete LPL in the vascular wall, we propose that LPL, by acting as an autocrine activator of MDM function, may contribute to the high level of TNFα found in the atheromatous lesion.
SREBP-1 Binds to Multiple Sites and Transactivates the Human ApoA-II Promoter In Vitro
Authors: Pissios Pavlos , Kan Horng-Yuan , Nagaoka Satoshi , Zannis Vassilis I.
Published: June 2011

Abstract
—Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (−64 to −48), AIICD (−178 to −154), AIIDE (−352 to −332), AIIHI (−594 to −574), and AIIK (−760 to −743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the −911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.
Atrophic Remodeling of the Artery-Cuffed Artery
Authors: Bayer Ilana M. , Adamson S. Lee , Langille B. Lowell
Published: June 2011

Abstract
—Increased arterial wall tension stimulates growth and remodeling of arteries, but little is known about the effects of decreased wall tension, despite its developmental and pathological significance. Consequently, we cuffed 1 carotid artery in rabbits with a portion of the contralateral artery to off-load circumferential wall tension. The model produced rapid and extensive atrophy of the cuffed artery that yielded decreases in the DNA content of the cuffed artery (a measure of cell number) from 8.0±0.5 μg/cm of in situ vessel length to 5.6±0.5 μg/cm at 21 days postoperatively. The elastin content of the cuffed artery was also significantly reduced, from 399±17 to 283±17 μg/cm, and collagen content was reduced from 468.0±59.0 to 154±24 μg/cm (
P
<0.05) at 21 days postoperatively. Detection of DNA oligonucleosomes by gel electrophoresis implicated apoptotic cell death in remodeling due to cuffing. Upregulation of matrix metalloproteinases (MMPs), including MMP-2, MMP-9, and unidentified gelatinases, indicated that these enzymes may also be involved in remodeling. No further changes in wall structure were seen between 3 weeks and 6 months, and the excised artery that was used as a cuff exhibited normal medial morphology for at least 6 months postoperatively. We infer from these experiments that off-loading of arterial wall tension induces rapid and extensive atrophy of the arterial media.
Localization of Lipoprotein Lipase in the Diabetic Heart
Authors: Sambandam Nandakumar , Abrahani Mohammed A. , St. Pierre Edith , Al-Atar Osama , Cam Margaret C. , Rodrigues Brian
Published: June 2011

Abstract
—Vascular endothelium–bound lipoprotein lipase (LPL) is rate limiting for free fatty acid (FFA) transport into tissues. In streptozotocin (STZ)-diabetic rats, we have previously demonstrated an increased heparin-releasable LPL activity from perfused hearts. Because heparin can traverse the endothelial barrier, conventional Langendorff retrograde perfusion of the heart with heparin could release LPL from both the capillary luminal and abluminal surfaces. To determine the precise location of the augmented LPL, a modified Langendorff retrograde perfusion was used to isolate the enzyme at the coronary lumen from that in the interstitial effluent. In response to heparin, a 4-fold increase in LPL activity and protein mass was observed in the coronary perfusate after 2 weeks of STZ diabetes. Release of LPL activity into the interstitial fluid of control hearts was slow but progressive, whereas in diabetic hearts, peak enzyme activity was observed within 1 to 2 minutes after heparin, followed by a gradual decline. Immunohistochemical studies of myocardial sections confirmed that the augmented LPL in diabetic hearts was mainly localized at the capillary endothelium. To study the acute effects of insulin on endothelial LPL activity, we examined rat hearts at various times after the onset of hyperglycemia. An increased heparin-releasable LPL activity in diabetic rats was demonstrated shortly (6 to 24 hours) after STZ injection or after withdrawal from exogenous insulin. Heparin-releasable coronary LPL activity was also increased after an overnight fast. These studies indicate that the intravascular heparin-releasable fraction of cardiac LPL activity is acutely regulated by short-term changes in insulin rather than glucose. Thus, during short periods (hours) of hypoinsulinemia, increased LPL activity at the capillary endothelium can increase the delivery of FFAs to the heart. The resultant metabolic changes could induce the subsequent cardiomyopathy that is observed in the chronic diabetic rat.
Effects of Lovastatin Therapy on Susceptibility of LDL to Oxidation During α-Tocopherol Supplementation
Authors: Palomäki Ari , Malminiemi Kimmo , Malminiemi Outi , Solakivi Tiina
Published: June 2011

Abstract
—A randomized, double-masked, crossover clinical trial was carried out to evaluate whether lovastatin therapy (60 mg daily) affects the initiation of oxidation of low density lipoprotein (LDL) in cardiac patients on α-tocopherol supplementation therapy (450 IU daily). Twenty-eight men with verified coronary heart disease and hypercholesterolemia received α-tocopherol with lovastatin or with dummy tablets in random order. The two 6-week, active-treatment periods were preceded by a washout period of at least 8 weeks. The oxidizability of LDL was determined by 2 methods ex vivo. The depletion times for LDL ubiquinol and LDL α-tocopherol were determined in timed samples taken during oxidation induced by 2,2-azobis(2,4-dimethylvaleronitrile). Copper-mediated oxidation of LDL isolated by rapid density-gradient ultracentrifugation was used to measure the lag time to the propagation phase of conjugated-diene formation. α-Tocopherol supplementation led to a 1.9-fold concentration of reduced α-tocopherol in LDL (
P
<0.0001) and to a 2.0-fold longer depletion time (
P
<0.0001) of α-tocopherol compared with determinations after the washout period. A 43% prolongation (
P
<0.0001) was seen in the lag time of conjugated-diene formation. Lovastatin decreased the depletion time of reduced α-tocopherol in metal ion–independent oxidation by 44% and shortened the lag time of conjugated-diene formation in metal ion–dependent oxidation by 7%. In conclusion, α-tocopherol supplementation significantly increased the antioxidative capacity of LDL when measured ex vivo, which was partially abolished by concomitant lovastatin therapy.
Influence of Antithrombin III on Coagulation and Inflammation in Porcine Septic Shock
Authors: Dickneite Gerhard , Leithäuser Boris
Published: June 2011

Abstract
—The physiological inhibitor of thrombin, antithrombin III (ATIII, Kybernin P) was investigated for its antiinflammatory and anticoagulant effects in a pig model of septic shock. Pigs were infused with a dose of 0.25 μg · kg
−1
· h
−1
of lipopolysaccharide (LPS) over a period of 3 hours. Animals developed systemic inflammation, disseminated intravascular coagulation (DIC), organ failure and cardiovascular abnormalities, namely pulmonary hypertension and systemic hypotension. Twenty septic pigs were allocated to 2 study groups, treated either with ATIII (n=10) or placebo (n=10). ATIII was administered as a 250-U/kg IV bolus infusion for 30 minutes (−60 to −30 minutes) followed by a single IV bolus of 125 U/kg (t=0) and a second 30-minute infusion of 250 U/kg (120 to 150 minutes). ATIII significantly prevented the development of a DIC; the increase in fibrin monomers (placebo, 11.4±9.1 reciprocal titers, at 6 hours) was completely overcome by ATIII (
P
<0.05). ATIII significantly prevented the increase in thromboxane (TXB
2
) levels, which were 809±287 pg/mL in the placebo and 420±174 pg/mL in the verum group after 6 hours (
P
<0.02). On the other hand, ATIII had no influence on TNF levels. In a lethal study with an increased dose of LPS (0.5 μg · kg
−1
· h
−1
). A significant reduction in mortality was observed in the ATIII group (0 of 7) compared with the placebo group (4 of 6) (
P
<0.05, χ
2
test) a significant reduction of pulmonary hypertension (placebo, 42.0±11.1 mm Hg; ATIII, 23.6±7.5 mm Hg,
P
<0.05), but no effect on systemic hypotension, was noted in the ATIII group. It was thus concluded that modulation of the procoagulatory state by substitution of ATIII results in a late beneficial antiinflammatory effect in this model of septic shock.
Impact of Adipose Tissue on Plasma Plasminogen Activator Inhibitor-1 in Dieting Obese Women
Authors: Mavri Alenka , Stegnar Mojca , Krebs Michael , Sentoc̆nik Joz̆ica T. , Geiger Margarethe , Binder Bernard R.
Published: June 2011

Abstract
—The increased incidence of cardiovascular diseases in obese subjects could be partially attributed to impaired fibrinolysis due to elevated plasma levels of tissue plasminogen activator inhibitor 1 (PAI-1). The associations between changes in plasma PAI-1, metabolic variables, and adipose tissue during weight loss and regain were studied in 52 healthy, premenopausal, obese women participating in a weight reduction program with a hypocaloric diet. PAI-1, insulin, triglyceride, leptin, and adipsin levels were determined at entry, after the first week, after completion of the program, and after 5 months of follow-up. In the 33 obese women who completed the program, decreases in PAI-1 antigen (−54%), PAI activity (−74%), and leptin (−51%), but not of adipsin, were observed. Changes in PAI-1 were associated with changes in body mass index (BMI), body fat, leptin, and insulin. The decreased level of PAI-1 remained low after follow-up in the 14 women who maintained their reduced weight but increased in the 16 women who regained weight. This increase in PAI-1 was correlated with an increase in body fat and leptin. On multivariate analysis, BMI was the major determinant of PAI-1 level. In conclusion, during weight reduction with a hypocaloric diet, the decrease in PAI-1 is more closely related to changes in adipose tissue than to changes in metabolic variables, suggesting a significant role for adipose tissue in regulating plasma levels of PAI-1.
Oxidized LDL-Containing Immune Complexes Induce Fc Gamma Receptor I–Mediated Mitogen-Activated Protein Kinase Activation in THP-1 Macrophages
Authors: Huang Yan , Jaffa Ayad , Koskinen Sinikka , Takei Akira , Lopes-Virella Maria F.
Published: June 2011

Abstract—Our previous studies have shown that Fc gamma receptor (FcγR)-mediated uptake of LDL-containing immune complexes (oxLDL-ICs) by human monocyte-derived macrophages leads to not only transformation of macrophages into foam cells but also macrophage activation and release of cytokines. It has been shown that cross-linking of FcγR triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we determined whether engagement of FcγR by oxLDL-ICs leads to activation of mitogen-activated protein (MAP) kinase pathway, a signaling cascade serving many important functions, including the regulation of gene expression, in THP-1 macrophage-like cells. Our results from immunoblotting, using specific anti-phosphorylated MAP kinase antibodies, showed that oxLDL-ICs induced extracellular signal regulated kinase 2 (ERK2) MAP kinase phosphorylation in THP-1 macrophage-like cells in time- and dose-dependent manners. Cholesterol loading before stimulation led to a longer phosphorylation of ERK2. Nuclear translocation of phosphorylated ERK was markedly increased after the stimulation. Moreover, our data showed that oxLDL-IC induction of MAP kinase was prevented by human monomeric IgG1, suggesting that the specific engagement of type I FcγR by oxLDL-IC is responsible for the MAP kinase activation. Finally, we showed that human anti-oxLDL autoantibody-containing immune complexes immobilized on type I collagen induced MAP kinase activation in THP-1 cells. These results strongly suggest that oxLDL-IC, which has been detected in atherosclerotic plaques, may play an important role in macrophage activation and atherogenesis.
Versican/PG-M Isoforms in Vascular Smooth Muscle Cells
Authors: Lemire Joan M. , Braun Kathleen R. , Maurel Patrice , Kaplan Elizabeth D. , Schwartz Stephen M. , Wight Thomas N.
Published: June 2011

Abstract
—The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.
Impaired Anticoagulant Response to Infusion of Thrombin in Atherosclerotic Monkeys Associated With Acquired Defects in the Protein C System
Authors: Lentz Steven R. , Fernández José A. , Griffin John H. , Piegors Donald J. , Erger Rochelle A. , Malinow M. René , Heistad Donald D.
Published: June 2011

Abstract
—To examine the effects of atherosclerosis on the protein C anticoagulant pathway in vivo, we measured anticoagulant responses to intravenous administration of human α-thrombin or activated protein C (APC) in cynomolgus monkeys. Two groups of monkeys were fed either a control diet (n=18) or an atherogenic diet (n=12) that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. A third group (n=8) was fed an atherogenic diet for 15 months, and then fed the atherogenic diet supplemented with B vitamins for 6 months to correct the hyperhomocyst(e)inemia. The plasma homocyst(e)ine level was higher in monkeys fed the atherogenic diet (9.6±1.0 μmol/L) than in monkeys fed the control diet (3.7±0.2 μmol/L) or the atherogenic diet with B vitamins (3.6±0.2 μmol/L) (
P
<0.001). Infusion of thrombin produced a much greater prolongation of the activated partial thromboplastin time in monkeys fed the control diet (52±10 seconds) than in monkeys fed the atherogenic diet either with (24±4 seconds) or without (27±5 seconds) supplemental B vitamins (
P
<0.02). Thrombin-dependent generation of circulating APC was higher in control (294±17 U/mL) than in atherosclerotic (240±14 U/mL) monkeys (
P
<0.05), although levels of fibrinogen, plasminogen, D-dimer, and thrombin-antithrombin complexes were similar in each group. Injection of human APC produced a similar prolongation of the activated partial thromboplastin time in control (31±3 seconds) and atherosclerotic (29±2 seconds) monkeys. These findings provide evidence for impaired anticoagulation, due partly to decreased formation of APC, in atherosclerosis. The blunted anticoagulant response to thrombin in hypercholesterolemic monkeys was not corrected by supplementation with B vitamins.
Captopril Administration Reduces Thrombus Formation and Surface Expression of Platelet Glycoprotein IIb/IIIa in Early Postmyocardial Infarction Stage
Authors: Zurbano María J. , Anguera Ignasi , Heras Magda , Roig Eulàlia , Lozano Miguel , Sanz Ginés , Escolar Ginés
Published: June 2011

Abstract
—Long-term administration of the angiotensin-converting enzyme inhibitor captopril in survivors of myocardial infarction (MI) reduces the risk of cardiovascular death, recurrence of MI, and unstable angina, suggesting that captopril may posses antithrombotic properties that have not been clearly elucidated. We assessed the short-term antithrombotic effects of captopril on platelet aggregation, platelet-subendothelium interaction, and the expression of major glycoproteins on platelet surface. A double-blind study was carried out in 25 patients with MI. Blood samples were taken before (baseline) and 12 days after treatment in both the control and captopril groups. Platelet aggregation was tested by conventional aggregometry using common activating agents. Platelet interaction with deeply damaged subendothelial surface was evaluated in a perfusion model, with blood maintained under flow conditions. Deposition of platelets was quantified by using computer-assisted morphometric techniques on histological sections, and it was expressed as a percentage of total vessel surface covered by platelets (CS) and as a ratio between large aggregates (T) and surface covered by platelets (100×T/CS). Glycoprotein expression was measured using flow cytometric techniques. Aggregometric responses showed no significant variations; however, in the captopril group, 100×T/CS decreased after 12 days of treatment (100×T/CS: 36±12.1% captopril versus 64±8.0% baseline;
P
=0.05). This parameter was also significantly decreased from that found in control group patients (100×T/CS: 67±4.5%;
P
=0.008). Flow cytometry showed a 30% reduction in glycoprotein IIb/IIIa expression (
P
=0.02). Captopril reduced the formation of large aggregates in a perfusion system, which might be related to a down-regulation of glycoprotein IIb/IIIa complex on the platelet surface. These results suggest that captopril exerts an antiplatelet effect that may contribute to its beneficial action in MI.
Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells
Authors: Rosnoblet Corinne , Vischer Ulrich M. , Gerard Robert D. , Irminger Jean-Claude , Halban Philippe A. , Kruithof Egbert K. O.
Published: June 2011

Abstract—Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone–containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.
Platelet Deposition on Eroded Vessel Walls at a Stenotic Shear Rate Is Inhibited by Lipid-Lowering Treatment With Atorvastatin
Authors: Alfon José , Royo Teresa , Garcia-Moll Xavier , Badimon Lina
Published: June 2011

Abstract
—Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase are widely used in the treatment of dyslipemias and have shown beneficial effects in the primary and secondary prevention of cardiovascular diseases. However, regression studies with lipid-lowering drugs have not shown significant lesion reduction associated with the improvement in clinical events. Therefore, our objective has been to study whether treatment with a lipid-lowering drug of this family, atorvastatin, could reduce platelet deposition on the damaged vessel wall at different shear stress conditions, simultaneously with retardation of the development of atherosclerotic lesions. Using cholesterol-fed swine as the model, we found that atorvastatin significantly diminished platelet deposition on the mildly damaged vessel wall at high shear rates (50%,
P
<0.01), but it did not have any effect in preventing platelet deposition triggered by a severely injured vessel wall. Development of coronary lesions was also reduced by treatment. These findings suggest that atorvastatin may prevent platelet attachment to eroded vessels and hence, contribute to reducing the thrombotic risk associated with the erosions of the luminal surface and the platelet-dependent progression of atherosclerotic plaques.
LDL Stimulates Mitogen-Activated Protein Kinase Phosphatase-1 Expression, Independent of LDL Receptors, in Vascular Smooth Muscle Cells
Authors: Metzler Bernhard , Li Chaohong , Hu Yanhua , Sturm Gertraud , Ghaffari-Tabrizi Nassim , Xu Qingbo
Published: June 2011

Abstract
—Low density lipoprotein (LDL) is a well-established risk factor for atherosclerosis, stimulating vascular smooth muscle cell (SMC) differentiation and proliferation, but the signal transduction pathways between LDL stimulation and cell proliferation are poorly understood. Because mitogen-activated protein kinases (MAPKs) play a crucial role in mediating cell growth, we studied the effect of LDL on the induction of MAPK phosphatase-1 (MKP-1) in human SMCs and found that LDL stimulated induction of MKP-1 mRNA and proteins in a time- and dose-dependent manner. Heparin, inhibiting LDL-receptor binding, did not influence LDL-stimulated MKP-1 mRNA expression, and human LDL also induced MKP-1 expression in rat SMCs and fibroblasts derived from LDL receptor–deficient mice, indicating an LDL receptor–independent process. Pretreatment of SMCs with pertussis toxin markedly inhibited LDL-induced MKP-1 expression. Depletion of protein kinase C (PKC) by phorbol 12-myristate 13 acetate or inhibition of PKC by calphostin C blocked MKP-1 induction, but the phospholipase C inhibitor U73122 had no effect. Pretreatment of SMCs with genistein or herbimycin A abrogated LDL-stimulated MKP-1 induction. The MAPK kinase inhibitor PD98059 abolished LDL-stimulated activation of extracellular signal–regulated protein kinases (ERKs) but not MKP-1 induction. Furthermore, constitutive expression of MKP-1 in vivo reduced LDL-induced expression of Elk-1–dependent reporter genes, and SMC lines overexpressing recombinant MKP-1 exhibited decreased ERK activities and retarded proliferation in response to LDL. Our findings demonstrate that LDL induces MKP-1 expression in SMCs via activation of PKC and tyrosine kinases, independent of LDL receptors and ERK-MAPKs, and that MKP-1 plays an important role in the regulation of LDL-initiated signal transductions leading to SMC proliferation.
Crystallization of Free Cholesterol in Model Macrophage Foam Cells
Authors: Kellner-Weibel G. , Yancey P. G. , Jerome W. G. , Walser T. , Mason R. P. , Phillips M. C. , Rothblat G. H.
Published: June 2011

Abstract
—The present study examined free cholesterol (FC) crystallization in macrophage foam cells. Model foam cells (J774 or mouse peritoneal macrophages [MPMs]) were incubated with acetylated low density lipoprotein and FC/phospholipid dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incubated for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of FC. FC crystals of various shapes and sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the crystals were externalized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal formation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibition of cell growth. In addition, 2 separate cholesterol phases (liquid-crystalline and cholesterol monohydrate) in the plane of the membrane bilayer were detected after 31 hours of ACAT inhibition by the use of small-angle x-ray diffraction of J774 macrophage foam cells treated with CPT-cAMP. Other compounds reported to inhibit ACAT, namely progesterone (20 μg/mL) and
N
-acetyl-
d
-sphingosine (c
2
-ceramide, 10 μg/mL), induced cellular toxicity in J774 macrophage foam cells and FC crystallization when coincubated with CPT-cAMP. Addition of the extracellular FC acceptors apolipoproteins (apo) E and A-I (50 μg/mL) reduced FC crystal formation. In MPMs, lower cell density and frequent changes of medium were conducive to crystal formation. This may be due to “dilution” of apoE secreted by the MPMs and is consistent with our observation that the addition of exogenous apoE or apoA-I inhibits FC crystal formation in J774 macrophage foam cells cotreated with CP-113,818 plus CPT-cAMP. These data demonstrate that FC crystals can form from the hydrolysis of cytoplasmic stores of CEs in model foam cells. FC crystal formation can be modulated by the addition of extracellular FC acceptors or by affecting the cellular rate of CE hydrolysis. This process may contribute to the formation of FC crystals in atherosclerotic plaques.
Aortic Plaque Size and Endometrial Response in Cholesterol-Fed Rabbits Treated With Estrogen Plus Continuous or Sequential Progestin
Authors: Brehme Ute , Bruck Birgit , Gugel Natalia , Wehrmann Manfred , Hanke Sybille , Finking Gerald , Schmahl Friedrich W. , Hanke Hartmut
Published: June 2011

Abstract
—ERT is associated with a reduced incidence of coronary risk and cardiac events in postmenopausal women, but increases the risk of endometrial hyperplasia and carcinoma. Combined estrogen and progestin therapy protects the endometrium; however, its effects on heart disease risk factors are not completely known. In our study, 56 ovariectomized New Zealand White rabbits in 7 groups received a 0.5% cholesterol diet for 12 weeks. Controls were not treated with hormones. All other animals received (per kilogram body weight per week) intramuscular injections of either 0.3 mg estrogen (estradiol valerate) alone, 8.3 mg progestin (hydroxyprogesterone caproate) alone, estrogen and progestin continuously in 3 different dosages (0.3 and 8.3 mg; 1 and 8.3 mg; or 1 and 2.8 mg; estrogen and progestin, respectively), or 1 mg estrogen with 25 mg progestin sequentially in 2-week cycles. Eight non-ovariectomized animals served as further controls for endometrial analysis. Morphometric analysis of plaque size in the aortic arch showed that estrogen monotherapy, and the 3 combined therapies with 1 mg estrogen, significantly reduced intimal thickening (
P
<0.05). The application of progestin alone had no effect on plaque size. The endometrium was enlarged by 3-fold after estrogen treatment, and was decreased by half after progestin treatment, compared with control uteri (
P
<0.05). In all groups with combined hormone regimens, endometrial size was not significantly different from control uteri. However, these uteri showed more inflammatory reactions, especially when higher doses of hormones were given. In this animal model, doses of progestin that are able to successfully reduce the proliferative effect of estrogen on endometrium do not diminish the desirable antiatherosclerotic properties of estrogen.
Plasma Glutathione Peroxidase Deficiency and Platelet Insensitivity to Nitric Oxide in Children With Familial Stroke
Authors: Kenet Gili , Freedman Jane , Shenkman Boris , Regina Eskaraev , Brok-Simoni Frida , Holzman Fanny , Vavva Fotini , Brand Nathan , Michelson Alan , Trolliet Maria , Loscalzo Joseph , Inbal Aida
Published: June 2011

Abstract—In a previous report by Freedman et al (J Clin Invest.1996;97:979–987), plasma from 2 brothers with stroke or transient ischemic attack inactivated the antiplatelet effects of nitric oxide (NO), and this effect was found to be a consequence of a deficiency of plasma glutathione peroxidase (GSH-Px). In this study, we attempted to define the generalizability of this deficiency by studying NO-mediated antiplatelet effects in 7 families with familial childhood stroke. Seven families with familial childhood stroke that consecutively presented to a large referral center were included in the study. We monitored ADP-induced aggregation of normal gel-filtered platelets (GFP) in platelet-poor plasma (PPP) from normal individuals and from patients in the presence or absence of an NO donor (S-nitroso–glutathione). Surface P-selectin expression of normal GFP in patients’ PPP was analyzed by flow cytometry after incubation with a P-selectin–specific monoclonal antibody in the presence or absence of the NO donor. We also measured GSH-Px activity in plasmas from family members and normal controls using standard methods. In 6 of 7 families, NO failed to inhibit platelet P-selectin expression and platelet aggregation in PPP from the affected family members and some of their relatives. Of 4 families studied, 3 probands and their corresponding affected parent had 50% decrease in plasma GSH-Px activity. In some patients with childhood stroke, impaired metabolism of reactive oxygen species as a result of reduced GSH-Px activity results in NO insufficiency that affects normal platelet inhibitory mechanisms and predisposes to arterial thrombosis.
Peroxisome Proliferator–Activated Receptor Activators Target Human Endothelial Cells to Inhibit Leukocyte–Endothelial Cell Interaction
Authors: Jackson Simon M. , Parhami Farhad , Xi Xiao-Ping , Berliner Judith A. , Hsueh Willa A. , Law Ronald E. , Demer Linda L.
Published: June 2011

Abstract—An early event in acute and chronic inflammation and associated diseases such as atherosclerosis and rheumatoid arthritis is the induced expression of specific adhesion molecules on the surface of endothelial cells (ECs), which subsequently bind leukocytes. Peroxisome proliferator–activated receptors (PPARs), members of the nuclear receptor superfamily of transcription factors, are activated by fatty acid metabolites, peroxisome proliferators, and thiazolidinediones and are now recognized as important mediators in the inflammatory response. Whether PPAR activators influence the inflammatory responses of ECs is unknown. We show that the PPAR activators 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2), Wyeth 14643, ciglitazone, and troglitazone, but not BRL 49653, partially inhibit the induced expression of vascular cell adhesion molecule-1 (VCAM-1), as measured by ELISA, and monocyte binding to human aortic endothelial cells (HAECs) activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide. The “natural” PPAR activator 15d-PGJ2had the greatest potency and was the only tested molecule capable of partially inhibiting the induced expression of E-selectin and neutrophil-like HL60 cell binding to PMA-activated HAECs. Intracellular adhesion molecule-1 induction by PMA was unaffected by any of the molecules tested. Both PPAR-α and PPAR-γ mRNAs were detected in HAECs by using reverse transcription–polymerase chain reaction and a ribonuclease protection assay; however, we have yet to determine which, if any, of the PPARs are mediating this process. These results suggest that certain PPAR activators may help limit chronic inflammation mediated by VCAM-1 and monocytes without affecting acute inflammation mediated by E-selectin and neutrophil binding.
Dexamethasone Enhances In Vitro Vascular Calcification by Promoting Osteoblastic Differentiation of Vascular Smooth Muscle Cells
Authors: Mori Katsuhito , Shioi Atsushi , Jono Shuichi , Nishizawa Yoshiki , Morii Hirotoshi
Published: June 2011

Abstract—Vascular calcification is often associated with atherosclerotic lesions. Moreover, the process of atherosclerotic calcification has several features similar to the mineralization of skeletal tissue. Therefore, we hypothesized that vascular smooth muscle cells might acquire osteoblastic characteristics during the development of atherosclerotic lesions. In the present study, we investigated the effect of dexamethasone (Dex), which is well known to be a potent stimulator of osteoblastic differentiation in vitro, on vascular calcification by using an in vitro calcification model. We demonstrated that Dex increased bovine vascular smooth muscle cell (BVSMC) calcification in a dose- and time-dependent manner. Dex also enhanced several phenotypic markers of osteoblasts, such as alkaline phosphatase activity, procollagen type I carboxy-terminal peptide production, and cAMP responses to parathyroid hormone in BVSMCs. We also examined the effects of Dex on human osteoblast-like (Saos-2) cells and compared its effects on BVSMCs and Saos-2 cells. The effects of Dex on alkaline phosphatase activity and the cAMP response to parathyroid hormone in BVSMCs were less prominent than those in Saos-2 cells. Interestingly, we detected that Osf2/Cbfa1, a key transcription factor in osteoblastic differentiation, was expressed in both BVSMCs and Saos-2 cells and that Dex increased the gene expression of both transcription factors. These findings suggest that Dex may enhance osteoblastic differentiation of BVSMCs in vitro.
Selective Activation of the Prostanoid EP
3
Receptor Reduces Myocardial Infarct Size in Rodents
Authors: Zacharowski Kai , Olbrich Antje , Piper Julie , Hafner Gerd , Kondo Kigen , Thiemermann Christoph
Published: June 2011

Abstract
—The cardioprotective effects of E-type prostaglandins (EPs) have been attributed to vasodilatation, inhibition of platelet and neutrophil function (EP
2
mediated), and an unknown “cytoprotective effect.” We have hypothesized that selective activation of EP
3
receptors may cause cardioprotection. The prostanoid derivative ONO-AE-248 selectively binds to murine EP
3α
receptors expressed in Chinese hamster ovary (CHO) cells (
K
i
, 15 nmol/L) and prevents the rise in cAMP caused by forskolin in CHO cells (IC
50
≈1 nmol/L) in which the EP
3α
receptor had been expressed. In anesthetized rats subjected to regional myocardial ischemia for 25 or 45 minutes and 2 hours of reperfusion, infusion of ONO-AE-248 (5 μg · kg
−1
· min
−1
IV) caused a significant reduction in infarct size, from 60±3% (n=8) to 36±6% (n=7) and from 78±2% (n=11) to 58±4% (n=9), respectively. The reduction in infarct size caused by ONO-AE-248 in rats subjected to 25 minutes of ischemia and reperfusion was abolished by a selective inhibitor of ATP-sensitive potassium (K
ATP
) channels, 5-hydroxydecanoate (n=6), and the protein kinase C inhibitors staurosporine (n=6) and chelerythrine (n=6). In anesthetized rabbits subjected to coronary artery occlusion for 45 or 60 minutes and 2 hours of reperfusion, infusion of ONO-AE-248 (5 μg · kg
−1
· min
−1
IV) caused a significant reduction in infarct size, from 61±2% (n=10) to 36±4% (n=8) and from 63±4% (n=7) to 42±4% (n=7), respectively. The reduction in infarct size caused by ONO-AE-248 in the rabbit was also abolished by 5-hydroxydecanoate. The cardioprotective effect of ONO-AE-248 in rats or rabbits was not associated with any hemodynamic effects. Selective activation of the prostanoid EP
3
receptor reduces myocardial infarct size in rodents by a mechanism(s) that may involve the activation of protein kinase C and the opening of K
ATP
channels.
Tissue Factor Pathway Inhibitor Attenuates Procoagulant Activity and Upregulation of Tissue Factor at the Site of Balloon-Induced Arterial Injury in Pigs
Authors: St. Pierre James , Yang Lu-Ying , Tamirisa Kamala , Scherrer David , De Ciechi Pamela , Eisenberg Paul , Tolunay Eser , Abendschein Dana
Published: June 2011

Abstract
—Intravenous infusion of recombinant tissue factor pathway inhibitor (rTFPI) for 24 hours decreases neointimal thickening and luminal stenosis 1 month after balloon-induced injury to the carotid arteries in minipigs. This study was designed to determine whether the effect of rTFPI is accounted for by early decreases in procoagulant activity and thrombosis on the injured vessel wall. Vascular injury was induced by balloon hyperinflations in both carotid arteries of anesthetized pigs given no anticoagulant as a control (n=16), an intravenous infusion for 24 hours of rTFPI (0.5 mg/kg bolus and 25 μg · kg
−1
· min
−1
, n=14), or an intravenous infusion of unfractionated heparin (100 U · kg
−1
· h
−1
, n=19). Accumulation of radiolabeled autologous platelets was markedly decreased over 24 hours on injured arteries from animals given rTFPI (0.6×10
6
/cm
2
) compared with controls (2.5×10
6
/cm
2
,
P
=0.0004). Deposition of radiolabeled fibrin was also decreased in rTFPI-treated animals (269±266 μg/cm
2
) compared with controls (2389±1673 μg/cm
2
,
P
=0.04). Similar effects were observed with heparin. However, factor Xa activity, assayed after 24 hours by incubation of the injured arterial segments with the chromogenic substrate S-2222, was decreased more markedly on arteries from rTFPI-treated animals (0.14±0.13 OD) than those from heparin-treated animals (0.29±0.18 OD) compared with controls (0.47±0.24 OD,
P
=0.0007). In addition, arteries from rTFPI-treated animals showed a 4-fold lower induction of tissue factor protein compared with controls (
P
=0.0002). Attenuation of procoagulant activity and tissue factor–mediated thrombin generation in response to injury may account for the promising results with rTFPI in the porcine angioplasty model.
Neonatal umbilical cord blood lipoproteins. Isolation and characterization of intermediate density and low density lipoproteins.
Authors: Davis P A , Forte T M
Published: June 2011

Intermediate density lipoproteins (IDL) (d = 1.006 to 1.019 g/ml) and low density lipoproteins (LDL) (d = 1.019 to 1.063 g/ml) were isolated from human umbilical cord blood plasma by sequential ultracentrifugation. The concentration, chemical and apolipoprotein composition, size and size distribution of the neonatal IDL and LDL for both sexes were determined. The IDL and LDL from the neonates showed no sex-related differences in composition or concentration. The IDL and LDL were lower in concentration and differed in composition of concentration. The IDL and LDL were lower in concentration and differed in composition with regard to each other and with regard to the comparable adult fractions. The apolipoprotein (apo) composition showed only the high molecular weight form of apo B present in the IDL, while the LDL showed the presence of two lower molecular weight forms of apo B in addition to the high molecular weight form, along with appreciable amounts of apo E and apo A-l. The size distribution of the neonatal IDL and LDL showed a constant pattern, with peaks at approximately 300 A for IDL and 257 and 244 A for neonatal LDL. The alterations in composition, size and size distribution, as well as the lower concentrations present in neonate, point to differences between the neonate and the adult in the metabolism of lipoproteins with a density of 1.006 to 1.063 g/ml.
Factors affecting the extent and severity of coronary artery disease in patients enrolled in the coronary artery surgery study.
Authors: Vlietstra R E , Kronmal R A , Frye R L , Seth A K , Tristani F E , Killip T
Published: June 2011

This study was designed to test the relationship between each of nine risk variables and the extent and severity of coronary artery disease in 15,298 patients with coronary artery disease proven by arteriography who were enrolled in the Coronary Artery Surgery Study. The extent and severity of the disease were highly significantly (p less than 0.001), but modestly, correlated with age, sex, cholesterol levels, history of diabetes, and history of hypertension. Interestingly, no positive correlation (indeed, in some subgroups, a negative correlation) occurred between the arteriographic measures of disease and the cigarette smoking history (ever or never, number of pack-years of smoking, duration of cigarette smoking, and peak daily cigarette consumption). These results suggest that the risk factors for presence of disease may differ from those influencing angiographic extent and severity.