bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk Factors for Abdominal Aortic Aneurysms in Older Adults Enrolled in the Cardiovascular Health Study
Authors: Alcorn Hope G. , Wolfson Sidney K. , Sutton-Tyrrell Kim , Kuller Lewis H. , O'Leary Daniel
Published: June 2011

B-mode ultrasound examinations of the abdominal aorta were performed from 1990 to 1992 to evaluate the prevalence of abdominal aortic aneurysm (AAA) in a subgroup of the Pittsburgh cohort (656 participants, aged 65 to 90 years) of the Cardiovascular Health Study (CHS). In this pilot study, we evaluated various definitions of aneurysm and the reproducibility of the measurements. In year 5 (1992 to 1993) of the CHS, the entire cohort (4741 participants) was examined. AAA was defined as an infrarenal aortic diameter of ≥3.0 cm, or a ratio of infrarenal to suprarenal diameter of ≥1.2, or a history of AAA repair. For the entire CHS cohort, prevalence of aneurysms was 9.5% (451/4741) overall, with a prevalence among men of 14.2% (278/1956) and prevalence among women of 6.2% (173/2785). Variables significantly related to AAA were older age; male sex; history of angina, coronary heart disease, and myocardial infarction; lower ankle-arm blood pressure ratio; higher maximum carotid stenosis; greater intima-media thickness of the internal carotid artery; higher creatinine; lower HDL levels and higher LDL levels; and cigarette smoking. The study has documented the strong association of cardiovascular risk factors and measures of clinical and subclinical atherosclerosis and cardiovascular disease and prevalence of aneurysms. We used a definition that is more sensitive than previously reported (diameter or ratio), which allowed the detection of smaller aneurysms and possibly those at an earlier stage of development. Follow-up of this cohort may lead to new criteria for determining the risk factors for progression of aneurysms.
Chronic α
1
-Adrenoreceptor Stimulation Increases DNA Synthesis in Rat Arterial Wall
Authors: deBlois D. , Schwartz S.M. , van Kleef E.M. , Su J.E. , Griffin K.A. , Bidani A.K. , Daemen M.J.A.P. , Lombardi D.M.
Published: June 2011

Despite indirect evidence from studies using adrenergic antagonists or sympathectomy, catecholamines have never been shown directly to stimulate vascular smooth muscle cell (SMC) DNA replication in vivo. We studied whether a chronic infusion of catecholamine stimulates SMC replication in vivo in both uninjured arteries and arteries with a neointima formed after vascular injury. Animals were killed after 2 weeks of continuous infusion of bromodeoxyuridine (to label replicating DNA) and either phenylephrine, norepinephrine, or vehicle solution, starting early (third week) or late (ninth week) after balloon injury to the left common carotid artery. In catecholamine-infused animals, the uninjured carotid artery or thoracic aorta showed a marked increase in cross-sectional area (>25%) and frequency of cells undergoing DNA synthesis among medial SMCs (4- to 10-fold) and endothelial cells (13-fold). With catecholamine infusion at 9 to 10 weeks after injury, the media or neointima of the injured carotid artery showed a smaller increase in SMC DNA replication (≤4-fold) than did the normal arterial media. In contrast, catecholamine infusion at 3 to 4 weeks did not cause significant SMC growth in the injured vessel. Catecholamine infusion caused labile elevations of systolic blood pressure. Taken together with our previous observation that α
1
-blockers suppress arterial SMC replication without preventing severe hypertension in the rat, the present data strongly suggest that α
1
-adrenoreceptors stimulate SMC DNA synthesis in vivo in arteries with or without intimal thickening, although not during the first weeks after balloon injury. The stimulation of DNA synthesis in vascular cells via the α
1
-adrenoreceptor pathway may contribute to the vascular remodeling that occurs in hypertension and atherosclerosis.
Human Brain Pericytes Differentially Regulate Expression of Procoagulant Enzyme Complexes Comprising the Extrinsic Pathway of Blood Coagulation
Authors: Bouchard Beth A. , Shatos Marie A. , Tracy Paula B.
Published: June 2011

After vascular injury, pericytes may function in blood coagulation events that lead to thrombin formation due to their subendothelial location in the microvasculature. Pericytes from human cerebral cortex microvessels were isolated and characterized, and their ability to express and regulate procoagulant enzyme complexes was determined. Tissue factor was detected on the cell surface of cultured human brain pericytes by immunocytochemistry and was shown to form a functional complex with factor (F) VIIa to effect both FIX and FX activation. Treatment of pericytes with the calcium ionophore A23187 increased the observed tissue factor activity twofold to fivefold, which was shown to be due to an enhancement of cofactor activity and not the release of endogenous antigen stores. Pericytes also provided the appropriate membrane surface required for the assembly of a functional prothrombinase complex, so that in the presence of FVa and FXa, they effected thrombin formation 50 to 100 times faster than any other cell examined to date. In marked contrast to observations in other cell systems, pericyte expression of prothrombinase activity remained unaltered after treatment with A23187. As has been shown for platelets, the membrane receptor on pericytes for FXa assembly into the prothrombinase complex appears to at least partially consist of the FXa receptor effector cell protease receptor-1. These combined data indicate that pericytes can activate and propagate the coagulant response through the extrinsic pathway and that the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation. These observations support the concept that pericytes may play an important role in regulating coagulation events after cerebrovascular injury.
Plasma Protein C Inhibitor Is Elevated in Survivors of Myocardial Infarction
Authors: Carroll Veronica A. , Griffiths Margaret R. , Geiger Margarete , Merlo Christoph , Furlan Miha , Lämmle Bernhard , Binder Bernd R.
Published: June 2011

Many studies have shown alterations of the hemostatic and fibrinolytic systems in patients with atherosclerotic disease, principally in levels of plasminogen activator inhibitor-1. However, in a large prospective study only fibrinogen, von Willebrand factor antigen, and tissue plasminogen activator antigen were found to be independent risk markers for acute coronary events. The present study evaluated the fibrinolytic system in coronary artery disease, paying particular attention to another inhibitor of fibrinolysis, plasminogen activator inhibitor-3, also called protein C inhibitor (PCI). One hundred fifteen nonanticoagulated male survivors of myocardial infarction were investigated for a range of hemostatic and fibrinolytic parameters that were compared with values in 87 age-matched healthy control male subjects. PCI active antigen was significantly (
P
<.03) elevated in the myocardial infarction group compared with the control group and was associated with the number of acute coronary events suffered (
P
=.005) but not with the severity of disease as determined by coronary angiography. Elevated PCI plasma levels can be considered as a risk marker for acute coronary events and might be of particular importance in the pathogenesis of this disease due to the interference of PCI in both the anticoagulant and fibrinolytic systems.
The Redox Status of Coenzyme Q10 in Total LDL as an Indicator of In Vivo Oxidative Modification
Authors: de Rijke* Yolanda B. , Bredie* Sebastian J.H. , Demacker Pierre N.M. , Vogelaar Janine M. , Hak-Lemmers Heidi L.M. , Stalenhoef Anton F.H.
Published: June 2011

Familial combined hyperlipidemia (FCH) is characterized by a familial occurrence of a multiple-type hyperlipidemia, associated with coronary risk. The latter may be related to increased levels of small, dense LDL particles that have been found to be more prone to oxidative modification. We isolated total LDL as fresh as possible from 12 normolipidemic relatives with a buoyant LDL subfraction profile (group 1), 7 normolipidemic subjects with a dense LDL subfraction profile (group 2), and 16 hyperlipidemic FCH subjects with a dense LDL subfraction profile (group 3). In these nonobese and normotensive men, we studied the resistance of total LDL against Cu
2+
-oxidation in vitro. In addition, we analyzed the α-tocopherol and the coenzyme Q10 contents of LDL and determined their relation to LDL oxidizability. LDL isolated from group 3 subjects was more susceptible to oxidative modification than LDL from group 1 subjects (lag time: 60.4±8.1 versus 70.4±11.4 minutes;
P
<.05). For the combined groups, the ratio of ubiquinol-10 to polyunsaturated fatty acids in LDL, together with the basal amount of dienes in LDL, were good predictors of the rate of LDL oxidation (
R
2
=.73,
P
=.0001). In groups 2 and 3, the redox status of coenzyme Q10 (ubiquinol-10/ubiquinone-10) and the ratio of ubiquinol-10 to α-tocopherol in LDL were reduced compared with group 1 (
P
<.05). The K-value, a measure of the LDL density, correlated with the the redox status (
r
=.37,
P
<.05). We conclude that in subjects with FCH total LDL is more prone to oxidation, due to the predominance of dense LDL particles. In addition, the decreased redox status of coenzyme Q10 in LDL from subjects with a dense LDL subfraction profile suggests that the LDL in the circulation has already undergone some oxidation.
Polymorphisms of the Genes Encoding Apoproteins A-I, B, C-III, and E and LDL Receptor, and Cholesterol and LDL Metabolism During Increased Cholesterol Intake
Authors: Gylling Helena , Kontula Kimmo , Koivisto Ulla-Maija , Miettinen Helena E. , Miettinen Tatu A.
Published: June 2011

Genetic and dietary factors regulate serum cholesterol level, but detailed investigations into their interactions have not been established. We assessed the effects of apoprotein (apo) E phenotype and polymorphic alleles of the apo A-I, apo B, apo C-III, and LDL receptor genes, separately and together, on regulation of serum LDL cholesterol level. The study group consisted of 29 middle-aged men, and cholesterol absorption, bile acid, and cholesterol synthesis and LDL apo B kinetics were studied in these men during low- and high-cholesterol diets. The six apo B alleles were identified on the basis ofXbaI,EcoRI, andMspI restriction fragment length polymorphism (RFLP), the apo A-I alleles with theMspI RFLP, and the apo C-III and LDL receptor alleles corresponded to theSstI andPvuII RPLPs of these genes, respectively. During low cholesterol intake, LDL cholesterol levels were similar in all of the genetic groups except for men with apo E2 phenotype. They had significantly (P<.05) lower levels of LDL apo B and cholesterol than men without the ε2 allele. The low values were caused by a significantly higher removal of LDL apo B (apo E2, 0.453±0.03 versus apo E3, 0.312±0.01 pools per day,P<.05). High cholesterol intake increased LDL cholesterol levels in all genetic categories except in the apo E2 phenotype irrespective of the combinations with other polymorphisms. Carriers of the apo B R+ allele (EcoRI site present) presented with the most prominent LDL cholesterol rise (from 2.71±0.14 to 3.37±0.29 mmol/L). In multiple stepwise regression analysis, apo BEcoRI RFLP and apo E phenotypes were the only variables that explained the variability of high cholesterol intake–induced change in LDL cholesterol levels. In summary, in any genetic combination, individuals with the ε2 allele had the lowest LDL cholesterol values and were nonresponders to dietary cholesterol, whereas subjects with the apo B R+ allele had marked LDL elevations, especially in combination with the ε4.
Possible Mechanisms of Collar-Induced Intimal Thickening
Authors: De Meyer Guido R.Y. , Van Put Dominica J.M. , Kockx Mark M. , Van Schil Paul , Bosmans Rosette , Bult Hidde , Buyssens Norbert , Vanmaele Rudolphe , Herman Arnold G.
Published: June 2011

Abstract
The positioning of a soft silicone collar around the rabbit carotid artery induces intimal thickening. We investigated to which extent occlusion of the vasa vasorum, damage of the perivascular nerve network, and/or changes in blood flow velocity contribute to intimal thickening. To this end, collars with different bores (diameter of inlet and outlet) were positioned around the carotid artery of male rabbits for 14 days. In another experiment, 75% of the wall of fitting collars was removed (open collar). In the midcollar region, the cross-sectional area of the intima reached a maximum (72±14 mm
2
/1000) when the endings of the collar fitted the artery closely. Removal of the side wall of these fitting collars reduced intimal thickening by 90%. Examination of unoperated carotid arteries never showed penetration of the adventitia or the media by vasa vasorum. The perivascular neuronal network in the region surrounded by a closed or an open collar was almost completely lost as compared with the zones outside the collar. Both the closed and open collar slightly bent the artery and increased the peak systolic velocity, measured with pulsed color Doppler after 6 hours, to a similar extent as compared with the proximal zone outside the collar. After 2 weeks, the peak systolic velocity within both the closed and open collar was partly normalized and was statistically not different from the proximal zone outside the collar. In conclusion, the geometry of the collar influenced the extent of intimal thickening, whereby more intimal thickening was obtained with a collar whose endings fit the carotid artery, rather than with a loose collar. Moreover, a closed structure was essential. The results obtained with the open collar exclude occlusion of vasa vasorum, damage of the perivascular neuronal network, kinking of the artery, and changes in blood flow velocity as major factors in the collar-induced intimal thickening. Our findings are consistent with the possibility that intimal thickening is the consequence of the combination of both vascular injury and hindrance of transmural flow by the collar. The obstruction of transmural fluid transport may then lead to retention of toxic metabolites, and/or cytokines within the segment enclosed by the collar.
Folic Acid Deficiency Enhances Oral Contraceptive-Induced Platelet Hyperactivity
Authors: Durand Philippe , Prost Michel , Blache Denis
Published: June 2011

Abstract
In previous studies conducted in female rats and in women, oral contraceptives (OC) were found to induce a platelet hyperactivity that was related to an oxidative stress. Because cases of megaloblastic anemia have been reported to occur in women taking OC, these treatments are suspected of depleting folate stores. In the study presented herein, which was conducted in rats, we sought to determine the influence of dietary folic acid deficiency (FD) on the thrombogenicity of OC. Animals were fed for 6 weeks with either a folic acid-deficient diet (250 μg/kg folic acid) or a control diet (750 μg/kg). One-half of the animals in each group were treated with OC (ethinyl estradiol plus lynestrenol). FD and OC individually potentiated platelet aggregation in response to thrombin and ADP and the release and metabolism of arachidonic acid, in particular, the biosynthesis of thromboxane. These platelet activities were further enhanced in animals given both the folic acid-deficient diet and the OC treatment. In addition, FD enhanced the pro-oxidant state in OC-treated rats characterized by (1) a fall in platelet and plasma n-3 fatty acids, (2) an increase in plasma lipid peroxidation products such as conjugated dienes, lipid peroxides, and thiobarbituric reactive substances, (3) a rise in ex vivo erythrocyte susceptibility to free radicals. Moreover, we found that OC treatment led to a reduction of plasma and erythrocyte folate concentrations associated with a moderate hyperhomocysteinemia. Under our experimental conditions, we did not find significant synergistic effects between OC and FD. We propose that, although the untoward effects associated with the OC treatment may not primarily be dependent on FD, the folic acid deficiency magnified OC-induced oxidative stress, which resulted in platelet hyperactivity by elevating the pro-oxidant homocysteine plasma concentration. Despite the limitations of this animal model, the data of the present study suggest that in addition to cigarette smoking, inadequate folic acid intake might predispose those taking OC to vascular thrombosis.
High Concentration of Glucose Increases Mitogenic Responsiveness to Heparin-Binding Epidermal Growth Factor–like Growth Factor in Rat Vascular Smooth Muscle Cells
Authors: Fukuda Kazuto , Kawata Sumio , Inui Yoshiaki , Higashiyama Shigeki , Matsuda Yukihiko , Igura Takumi , Tamura Shinji , Taniguchi Naoyuki , Matsuzawa Yuji
Published: June 2011

Abstract
The effect of a high extracellular glucose concentration on the mitogenic response of rat vascular smooth muscle cells (SMCs) to heparin-binding epidermal growth factor–like growth factor (HB-EGF) was investigated. The mitogenic effect of HB-EGF was significantly greater in SMCs cultured in high glucose (25 mmol/L) than in cells cultured in low glucose (5.5 mmol/L) or at high osmolarity (5.5 mmol/L glucose plus 19.5 mmol/L mannitol). The mitogenic effect of epidermal growth factor (EGF), which shares the EGF receptor with HB-EGF, was not affected by glucose concentration. The mitogenic effect of HB-EGF was greater when incubated with heparan sulfate (HS) isolated from SMCs cultured in high glucose than with HS from cells cultured in low glucose. HS synthesized by cells in high glucose was of smaller molecular size and less sulfated than HS synthesized by cells in low glucose. The abundance of mRNA encoding HS-
N
-deacetylase/
N
-sulfotransferase (HS-NdAc/NST), a regulatory enzyme in the biosynthesis of HS, was decreased by high glucose in a protein kinase C–independent manner. These observations suggest that the enhanced mitogenic response to HB-EGF in SMCs cultured in high glucose may be attributable to changes in cell-associated HS. Downregulation of HS-NdAc/NST gene expression by high glucose may be related to the altered HS biosynthesis.
Androgen Deprivation Is Associated With Enhanced Endothelium-Dependent Dilatation in Adult Men
Authors: Herman S. Mark , Robinson Jacqui T.C. , McCredie Robyn J. , Adams Mark R. , Boyer Michael J. , Celermajer David S.
Published: June 2011

Abstract
Male gender is an independent risk factor for coronary artery disease, and androgen administration has been associated with increased atherosclerosis in experimental animals. Since endothelial dysfunction is an important event in the atherogenic process, we hypothesized that androgen deprivation in adult men might be associated with enhanced arterial endothelial function. Using external vascular ultrasound, brachial artery diameter was measured at rest, after flow increase (causing endothelium-dependent dilatation) and after nitroglycerin (an endothelium-independent dilator). We studied 30 adult males aged 40 to 70 years: 10 had had bilateral orchidectomy and/or maximal androgen blockade for ≥6 months for treatment of prostate cancer, and all were in complete remission (group 1). Ten healthy controls (group 2) and 10 controls who had remission from nonprostate cancers (group 3) were matched for age and smoking history. Testosterone levels were lower in men in group 1 versus groups 2 or 3 (0.8±0.1 versus 19.2±8.4 or 16.1±4.9 nmol/L,
P
<.001). By contrast, endothelium-dependent dilatation was markedly higher in group 1 than in groups 2 or 3 (6.2±3 versus 2.7±2 or 2.0±1.9%,
P
<.001). The nitroglycerin response was similar in all three groups (
P
=.92). On multivariate analysis, increased endothelium-dependent dilatation was significantly associated with low serum testosterone levels (
P
=.001) but not with cholesterol levels or with a past history of malignancy (
P
>.25). The withdrawal of male sex hormones may be associated with enhanced endothelial function in adult men. This is consistent with a deleterious effect of physiologic levels of male sex steroids on the arterial wall.
Niacin Decreases Removal of High-Density Lipoprotein Apolipoprotein A-I But Not Cholesterol Ester by Hep G2 Cells
Authors: Jin Fu-You , Kamanna Vaijinath S. , Kashyap Moti L.
Published: June 2011

Abstract
Niacin (nicotinic acid) is the most potent clinically used agent for increasing plasma HDL and apolipoprotein (apo) A-I. The mechanism by which niacin increases apoA-I is not clearly understood. We have examined the effect of niacin on the hepatic production and removal of apoA-I using Hep G2 cells as an in vitro model. Incubation of Hep G2 cells with niacin resulted in increased accumulation of apoA-I in the medium in a dose-dependent manner. Incorporation of [
3
H]leucine and [
35
S]methionine into apoA-I and apoA-I mRNA expression was unchanged by niacin, suggesting that it did not affect apoA-I de novo synthesis. Uptake of radiolabeled HDL protein and HDL apoA-I by Hep G2 cells was significantly reduced to as much as 82.9±2.2% (
P
=.04) and 84.2±2.8% (
P
=.02), respectively, of the baseline with increasing concentrations of niacin (0 to 3.0 mmol/L). Specific
125
I-HDL protein uptake measured with a 50-fold excess of unlabeled HDL was reduced to as much as 78.3±4.8% (
P
=.005) in niacin-treated cells. The uptake of labeled cholesterol esters in HDL was unaffected by niacin. Niacin also effected a similar decrease in HDL protein uptake, but not cholesterol esters, from apoA-I–containing HDL particles isolated by immunoaffinity. The conditioned medium obtained from Hep G2 cells incubated with niacin significantly (
P
=.002) increased cholesterol efflux from cultured human fibroblasts. These data indicate a novel mechanism whereby niacin selectively decreases hepatic removal of HDL apoA-I but not cholesterol esters, thereby increasing the capacity of retained apoA-I to augment reverse cholesterol transport.
Response of Vascular Smooth Muscle Cells to the Neuropeptide Secretoneurin
Authors: Kähler Christian M. , Schratzberger Peter , Wiedermann Christian J.
Published: June 2011

AbstractMesenchymal cell migration and replication are central biologic events involved in atherosclerosis and lung and hepatic fibrosis. Tissue repair and fibrosis are thought to be regulated by growth regulatory molecules, comprising both stimulators and inhibitors of mesenchymal cell functions, including platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), fibroblast growth factors, and several neuropeptides such as substance P. Secretoneurin (SN), a novel 33-amino acid neuropeptide derived from secretogranin II (chromogranin C), is widely distributed in the central and peripheral nervous and neuroendocrine systems, including afferent C-fibers, and can be released in the periphery by capsaicin. Recently, we reported that SN triggers the selective migration of human monocytes and fibroblasts in vitro, implicating its involvement in inflammatory responses. We report herein that SN stimulates specific migration (maximal response at 10−10M) of cultured arterial smooth muscle cells (SMCs), originating from rat thoracic aorta, and initiates DNA synthesis and SMC growth (BrdU incorporation, MTT test) with a maximum at 10−8M SN to a similar extent as observed by PDGF. Both functional activities of SN were inhibited by specific anti-SN immunoglobulins (dilution, 1:1000), and furthermore, a trypsinized SN peptide (10−8M) was unable to provoke biologic effects. Our studies suggest that SN functions as a regulatory peptide to modulate SMC migration and proliferation, which in conjunction with other factors could serve to aggravate and accelerate the development of atherosclerotic or restenotic lesions at sites of vascular injury.
Plasminogen Activator Inhibitor-1 (PAI-1) Antigen Plasma Levels in Subjects Attending a Metabolic Ward: Relation to Polymorphisms of PAI-1 and Angiontensin Converting Enzyme (ACE) Genes
Authors: Margaglione Maurizio , Grandone Elvira , Vecchione Gennaro , Cappucci Giuseppe , Giuliani Nicola , Colaizzo Donatella , Celentano Egidio , Panico Salvatore , Di Minno Giovanni
Published: June 2011

Abstract
Plasminogen activator inhibitor 1 (PAI-1) is a determinant of vascular events. Subjects in metabolic wards are at high risk for these events. The renin-angiotensin system modulates plasma PAI-1 levels. An insertion (4G)/deletion (5G) polymorphism is involved in the regulation of the circulating levels of PAI-1. We have evaluated the levels of plasma PAI-1 in 208 individuals from our metabolic ward and correlated these levels with the 4G/5G genotype as well as with a genotype (homozygosity for a deletion polymorphism, DD genotype) of the angiotensin-converting enzyme (ACE) gene. Homozygosity for the insertion genotype (5G/5G) was associated with PAI-1 levels lower than those associated with the deletion genotype (4G/4G) (26.2x/:1.6 versus 33.7x/:1.7 ng/mL,
P
=.036). Plasma PAI-1 levels appeared to depend on the genotype (
P
=.014) as much as on age (
P
=.044), t-PA (
P
=.0001), or triglyceride levels (
P
=.005). The association between triglycerides and PAI-1 was significant in subjects carrying the 4G/4G and the 4G/5G genotypes (
P
=.013 and .036, respectively) but not in those with the 5G/5G genotype. When stratified according to PAI-1 and ACE genotypes, individuals homozygous for both deletions (4G/4G-DD genotypes) exhibited higher plasma PAI-1 levels compared with those of individuals without such homozygosities. However, this difference did not reach statistical significance. We conclude that in a group of subjects from a metabolic ward, a 4G/5G polymorphism of the PAI-1 gene exerts effects on plasma PAI-1 antigen levels comparable to those of established determinants. The association between triglycerides and plasma PAI-1 levels is genotype dependent. A trend to a positive interaction between ACE DD and PAI-1 4G/4G in the regulation of circulating plasma PAI-1 levels is present in this setting.
Involvement of Calcium and G Proteins in the Acute Release of Tissue-Type Plasminogen Activator and von Willebrand Factor From Cultured Human Endothelial Cells
Authors: van den Eijnden-Schrauwen Y. , Atsma D. E. , Lupu F. , de Vries R. E. M. , Kooistra T. , Emeis J. J.
Published: June 2011

Abstract
In this study, we investigated the role of Ca
2+
and G proteins in thrombin-induced acute release (regulated secretion) of tissue-type plasminogen activator (TPA) and von Willebrand factor (vWF), using a previously described system of primary human umbilical vein endothelial cells (HUVECs). The acute release of TPA and vWF, as induced by α-thrombin, was almost zero after chelation of Ca
2+
i
, showing that an increase in [Ca
2+
]
i
was required. It did not matter whether the increase in [Ca
2+
]
i
came from an intracellular or extracellular Ca
2+
source. Thrombin-induced release of TPA and vWF already started at low [Ca
2+
]
i
, around 100 nmol/L. Half-maximal release was found at a [Ca
2+
]
i
of 261 nmol/L for TPA and at 222 nmol/L for vWF. The Ca
2+
signal was transduced to calmodulin, as calmodulin inhibitors inhibited TPA and vWF release. The Ca
2+
ionophore ionomycin dose dependently released vWF; half-maximal vWF release occurred at a [Ca
2+
]
i
of 311 nmol/L. In contrast, no TPA release was found at all below a [Ca
2+
]
i
of 500 nmol/L. Thus, below 500 nmol/L [Ca
2+
]
i
, an increase in [Ca
2+
]
i
alone was sufficient to induce vWF release but not sufficient to induce TPA release. Protein kinase C did not appear to be involved in TPA or vWF release, as neither an activator nor an inhibitor of protein kinase C significantly influenced release. Inhibition of phospholipase A
2
also did not reduce thrombin-induced TPA and vWF release. The involvement of G proteins was studied by using both saponin-permeabilized and intact cells. GDP-β-S, which inhibits heterotrimeric and small G proteins, significantly inhibited thrombin-induced vWF and TPA release from permeabilized cells. AlF
4
−
, which activates heterotrimeric G proteins, induced TPA and vWF release in both intact and permeabilized HUVECs. Preincubation of HUVECs with pertussis toxin significantly inhibited thrombin-induced vWF release, due to inhibition of thrombin-induced Ca
2+
influx. Pertussis toxin did not affect ionomycin-induced release. The inhibitory effect of pertussis toxin was less obvious in thrombin-induced TPA release, because it was counterbalanced by a positive effect of the toxin on TPA release. Thus, both inhibitory and stimulatory (pertussis toxin–sensitive) G proteins were involved in TPA release. Therefore, thrombin-induced acute release of TPA and vWF differed in two respects. First, below a [Ca
2+
]
i
of 500 nmol/L, an increase in Ca
2+
was sufficient for vWF release but not for TPA release. Second, pertussis toxin–sensitive G proteins were differentially involved in acute TPA and vWF release.
Egr-1 is Activated in Endothelial Cells Exposed to Fluid Shear Stress and Interacts With a Novel Shear-Stress-Response Element in the PDGF A-Chain Promoter
Authors: Khachigian Levon M. , Anderson Keith R. , Halnon Nancy J. , Gimbrone Michael A. , Resnick Nitzan , Collins Tucker
Published: June 2011

Abstract
Exposure of vascular endothelial cells to fluid mechanical forces can modulate the expression of many genes involved in vascular physiology and pathophysiology. Here, we report that platelet-derived growth factor (PDGF) A-chain gene expression is induced at the level of transcription in cultured bovine aortic endothelial cells exposed to a physiologic level of steady laminar shear stress (10 dyn/cm
2
). 5′ Deletion analysis of the human PDGF-A promoter revealed that a GC-rich region near the TATA box was required for shear-inducible reporter gene expression. This element conferred shear inducibility onto a heterologous promoter-reporter construct that was otherwise unresponsive to shear stress. The induction of PDGF-A expression by shear was preceded by rapid and transient induction in the expression of the immediate-early gene,
egr-1
, which binds to GC-rich sequences. Gel shift studies indicated that shear-induced Egr-1 bound to the proximal PDGF-A promoter in a specific and time-dependent manner, displacing Sp1 from their overlapping recognition elements. Overlapping consensus binding sites for Egr-1 and Sp1 also appear in the proximal promoters of several other endothelial genes, including transforming growth factor-β
1
and tissue factor, whose expression is modulated by shear stress. These findings define the Egr-1 binding site in the proximal PDGF-A promoter as a shear-stress-responsive element and suggest that shear-stimulated Egr-1 gene expression may be a unifying theme in the induction of various other endothelial genes exposed to biomechanical forces.
Adipose Tissue Lipoprotein Lipase and Hormone-Sensitive Lipase
Authors: Reynisdottir Signy , Angelin Bo , Langin Dominique , Lithell Hans , Eriksson Mats , Holm Cecilia , Arner Peter
Published: June 2011

Abstract
The metabolism of free fatty acids (FFA) is altered in two common atherosclerosis-promoting disorders: familial combined hyperlipidemia (FCHL) and insulin resistance syndrome (IRS). It has been suggested that these two conditions may have a common etiology. The enzymes lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) are rate-limiting steps for the turnover of fatty acids in adipose tissue, because they hydrolyze extracellular triglycerides in lipoproteins (LPL) and intracellular triglycerides in adipocytes (HSL). The present study was undertaken to simultaneously determine the activities of LPL and HSL in subcutaneous adipose tissue from male patients with FCHL and IRS. LPL and HSL activity was investigated in 10 nonobese FCHL patients and compared with 10 matched healthy nonobese subjects, and in 8 essentially normolipidemic IRS patients (who did not have overt diabetes mellitus) and compared with 9 nonobese matched control subjects. LPL activity was 43% lower in patients with IRS (
P
<.0005), as compared with control subjects, but HSL activity was not significantly different in the two groups. On the other hand, HSL activity was decreased by 45% in FCHL patients (
P
<.01), as compared with control subjects, but LPL activity was not significantly different in FCHL patients and the control group. In conclusion, triglyceride metabolism in adipose tissue is altered in both FCHL and IRS. However, the abnormalities observed involve impaired function of LPL in IRS and impaired function of HSL in FCHL, suggesting separate etiologies for the altered lipolysis in these conditions, at least in male subjects.
Effect of Phosphorothioated Oligonucleotides on Neointima Formation in the Rat Carotid Artery
Authors: Bennett M. R. , Lindner V. , DeBlois D. , Reidy M. A. , Schwartz S. M.
Published: June 2011

Abstract
Several studies have shown that single-dose administration of agents that inhibit medial cell replication, such as antisense oligonucleotides to cell replication genes, can inhibit neointima formation after arterial injury. However, the precise mechanism of action of these agents is unknown. We analyzed the effect of phosphorothioated oligonucleotides delivered periadventitially on the response to injury in the balloon-injured rat carotid artery. Antisense oligonucleotides to c-
myc
suppressed medial replication 2 days after injury, but this effect was not present at 4 or 14 days. Endothelial cell proliferation was not affected by antisense oligonucleotides. There was, however, a significant suppression of intimal area and intima/media ratio at 14 days and an increase in lumen area in the antisense-treated group. Indeed, an increase in the number of medial cells at 14 days in the antisense group indicated that most of the effect of the agent was due to the suppression of cell migration. No effect was noted on expression of two genes, osteopontin and tropoelastin, used as markers of modulation of smooth muscle cells to a “neonatal” phenotype at 4 days after injury. Because no effect on cell proliferation could be demonstrated after 2 days, our data indicate that an early effect of the antisense agent mediates its longer-term effects. We suggest that this effect may be due to the suppression of migration of medial smooth muscle cells rather than the suppression of medial or intimal cell proliferation.
Calreticulin, a Potential Vascular Regulatory Protein, Reduces Intimal Hyperplasia After Arterial Injury
Authors: Dai E. , Stewart M. , Ritchie B. , Mesaeli N. , Raha S. , Kolodziejczyk D. , Hobman M. Lundstrom , Liu L. Y. , Etches W. , Nation N. , Michalak M. , Lucas A.
Published: June 2011

Abstract
Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks’ follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca
2+
-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037±0.007 mm
2
, calsequestrin: 0.042±0.021 mm
2
, calreticulin: 0.003±0.002 mm
2
, n=46,
P
<.04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N+P domain: 0.038±0.012 mm
2
, C domain: 0.003±0.002 mm
2
, n=45 rats,
P
<.0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N+P: 4.06±1.42, calreticulin: 0.273±0.02; n=26,
P
<.009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23±2.04 seconds, calreticulin: 113.5±1.95 seconds; n=5,
P
<.002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.
Local Application of LDL Promotes Intimal Thickening in the Collared Carotid Artery of the Rabbit
Authors: Matthys Katelijne E. , Van Hove Cor E. , Kockx Mark M. , Andries Luc J. , Van Osselaer Nancy , Herman Arnold G. , Bult Hidde
Published: June 2011

Abstract
Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 μg h
−1
, n=16 arteries), oxLDL (Cu
2+
oxidized, 7 μg h
−1
, n=16), or phosphate-buffered saline (5 μL h
−1
, n=16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of α-SMC actin–immunoreactive cells in the intima, thereby increasing the intimal thickness from 5±1 to 26±5 μm. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5±2% in the discrete collar-induced intima to ≈20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.
Adenovirus-Mediated Expression of the Secreted Form of Basic Fibroblast Growth Factor (FGF-2) Induces Cellular Proliferation and Angiogenesis In Vivo
Authors: Ueno Hikaru , Li Jian-Jun , Masuda Satoko , Qi Zhe , Yamamoto Hiroaki , Takeshita Akira
Published: June 2011

Abstract
Blood supply through collateral arteries is of critical importance in occlusive arterial diseases such as coronary atherosclerosis. Induction of angiogenic growth factor within either the narrowing arteries or jeopardized myocardium may promote angiogenesis in vivo, leading to salvage of ischemic myocardium. We constructed a replication-defective adenovirus (AdCAsFGF-2) coding for human basic fibroblast growth factor (FGF)-2 that is modified, so that its secretion will be facilitated, by tagging a signal sequence derived from FGF-4. A large quantity of FGF-2 was detected in both the cell lysate and culture medium of COS cells infected with AdCAsFGF-2, indicating that FGF-2 was secreted at least partly from the infected cells. The conditioned medium from the infected COS cells stimulated DNA synthesis in and induced cellular proliferation of arterial smooth muscle cells. These effects were eliminated by adenovirus-mediated overexpression of a dominant-negative truncated FGF-receptor type 1. Implantation of a gel of basement membrane proteins containing fibroblasts infected with AdCAsFGF-2 into the ventral subcutaneous space of mice induced extensive cellular proliferation and the formation of functional arterioles. Cells surrounding the vessels were positively immunostained with antibodies recognizing either smooth muscle–specific α-actin or factor VIII antigen as a marker for endothelium. These results suggest that AdCAsFGF-2 may be useful for delivering functional FGF-2 into tissues and may lead to therapeutic angiogenesis in vivo.
Inhibition of Protein Tyrosine Kinases Attenuates Increases in Expression of Transforming Growth Factor-β Isoforms and Their Receptors Following Arterial Injury
Authors: Ward Michael R. , Agrotis Alex , Kanellakis Peter , Dilley Rodney , Jennings Garry , Bobik Alex
Published: June 2011

AbstractTransforming growth factor-β1(TGF-β1) has been implicated in neointima formation in mechanically injured vessels and in restenosis after angioplasty. To further understand the significance of TGF-βs in neointima formation, we examined the temporal expression of three TGF-β isoforms (-β1, -β2, and -β3), their receptors (ALK-2, ALK-5, and TβRII), and two putative TGF-β responses (elevations in αvand β3integrin mRNAs) in balloon catheter–injured rat carotid arteries and their dependency on tyrosine kinase activity. Using a standardized reverse transcriptase–polymerase chain reaction assay optimized to estimate mRNA levels, we observed distinct patterns of mRNA regulation for TGF-β1, -β2, and -β3during the 48 hours immediately after injury, which were localized to the vessel’s media. TGF-β1mRNA increased 10-fold during this time while TGF-β3mRNA also increased almost 2-fold. There were also increases in mRNAs encoding the TGF-β type I receptors ALK-5 and ALK-2, as well as the type II receptor (TβRII). Eight hours after the injury, mRNA levels for ALK-2 and ALK-5 were on average 2-fold higher; mRNA encoding the type II receptor increased approximately 3-fold by 24 hours. There were also associated increases in TGF-β1, TGF-β3, ALK-5, and TβRII immunoreactive peptide levels. Peak increases in mRNAs for integrins αvand β3averaged approximately 2-fold and 2.5-fold, respectively. Perivascular administration of the tyrosine kinase inhibitor genistein at the time of vessel injury markedly (>85%) inhibited elevations in mRNAs encoding TGF-β1, TGF-β3, TβRII, and the two integrins αvand β3, while application of its inactive chemically similar homologue daidzein did not prevent the injury-induced elevations in mRNA levels. Since the increases in integrins αvand β3mRNA could be theoretically attributed to TGF-β actions despite being dependent on tyrosine kinase activity, we examined whether the observed elevations in integrins αvand β3were due to TGF-β1secretion, using cultured rat carotid artery smooth muscle cells. TGF-β1neutralizing antibodies specifically inhibited elevations in integrins αvand β3mRNAs due to platelet-derived growth factor-BB and fibroblast growth factor-2. We conclude that multiple components of the TGF-β system in vessels are activated following injury and influence expression of integrin receptors important for smooth muscle cell migration. Activation of the TGF-β system appears to be highly dependent on tyrosine kinases.
Basic Fibroblast Growth Factor (bFGF) Regulates the Expression of the CC Chemokine Monocyte Chemoattractant Protein-1 (MCP-1) in Autocrine-Activated Endothelial Cells
Authors: Wempe Frank , Lindner Volkhard , Augustin Hellmut G.
Published: June 2011

AbstractThe CC chemokine monocyte chemoattractant protein (MCP)-1 is induced by inflammatory cytokines and acts as a potent regulator of monocyte trafficking. Monocytes adhere preferentially to migrating endothelial cells in vitro and to endothelial cells at the migration front in vivo after aortic balloon denudation injury. Based on these findings, we analyzed MCP-1 expression in migrating and resting bovine aortic endothelial (BAE) cells and identified prominently upregulated levels of MCP-1 expression in migrating BAE cells. Stimulation of resting BAE cells with 5 ng/mL bFGF resulted in a fourfold induction of MCP-1 mRNA expression. The time course of bFGF-induced MCP-1 mRNA expression indicated a rapid and direct stimulation of MCP-1 expression that was detectable 30 minutes after stimulation. Levels of basal MCP-1 expression, as well as upregulated levels of MCP-1 in migrating BAE cells, were downregulated by addition of a neutralizing anti-bFGF monoclonal antibody (1.0 μg/mL). Digestion of conditioned media of resting BAE cells with collagenase led to a dose-dependent induction of MCP-1 expression in resting BAE cells, which was inhibited >50% by addition of neutralizing anti-bFGF antibody. Confirmation of the Northern blot experiments by ELISA-based quantitation of MCP-1 protein levels identified threefold to sixfold higher levels of MCP-1 in the supernatants of bFGF-stimulated BAE cells than in unstimulated resting BAE cells. Finally, analysis of MCP-1 expression by in situ hybridization carried out on en face preparations of aortas demonstrated that MCP-1 expression is dramatically upregulated in regenerating endothelial cells in vivo after balloon denudation. Though not establishing a direct causal relation between the preferential adhesion of monocytes to migrating endothelial cells, these findings strongly suggest that autocrine-activated endothelial cell–derived MCP-1 may play a critical role in recruiting monocytes. They furthermore support the concept that bFGF acts as an autocrine regulator of endothelial cell activity and may imply an involvement of bFGF as a mediator of inflammatory cell trafficking.
Expression of Multiple Isoforms of Nitric Oxide Synthase in Normal and Atherosclerotic Vessels
Authors: Wilcox Josiah N. , Subramanian Romesh R. , Sundell Cynthia L. , Tracey W. Ross , Pollock Jennifer S. , Harrison David G. , Marsden Philip A.
Published: June 2011

Abstract
Atherosclerosis is associated with reduced endothelium-derived relaxing factor bioactivity. To determine whether this is due to decreased synthesis of nitric oxide synthase (NOS), we examined normal and atherosclerotic human vessels by in situ hybridization and immunocytochemistry by using probes specific for endothelial (ecNOS), inducible (iNOS), and neuronal (nNOS) NOS isoforms. ecNOS was detected in endothelial cells overlying normal human aortas, fatty streaks, and advanced atherosclerotic lesions. A comparison of the relative expression of ecNOS to von Willebrand factor on serial sections of normal and atherosclerotic vessels indicated that there was a decrease in the number of endothelial cells expressing ecNOS in advanced lesions. iNOS and nNOS were not detected in normal vessels, but widespread production of these isoforms was found in early and advanced lesions associated with macrophages, endothelial cells, and mesenchymal-appearing intimal cells. These data suggest that there is (1) a loss of ecNOS expression by endothelial cells over advanced atherosclerotic lesions and (2) a significant increase in overall NOS synthesis by other cell types in advanced lesions composed of the ecNOS, nNOS, and iNOS isoforms. We hypothesize that the increased expression of NOS and presumably NO in atherosclerotic plaques may be related to cell death and necrosis in these tissues.