bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of Diabetes and Hypertension on Determinants of Endothelial Adhesiveness
Authors: Tsao Philip S. , Niebauer Josef , Buitrago Ricardo , Lin Patrick S. , Wang Bing-yin , Cooke John P. , Ida Chen Y-d. , Reaven Gerald M.
Published: June 2011

Abstract
—Epidemiological studies have established that diabetes mellitus and hypertension are independent risk factors for atherosclerosis. One of the earliest abnormalities seen in atherogenesis is enhanced monocyte adherence to the endothelium. The mechanisms by which diabetes mellitus or hypertension enhances monocyte–endothelial cell interactions are incompletely characterized. It is not known whether there are additive interactions between these risk factors on endothelial adhesiveness for monocytes. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were fed a normal or fructose-enriched diet. In some cases, animals were injected with streptozotocin (35 mg/kg body weight) to induce diabetes. After 2 weeks, plasma was drawn for biochemical measurements, and thoracic aortas were harvested, opened longitudinally, and exposed to fluorescently labeled mouse monocytoid cells (WEHI 78/24, 2×10
6
/mL) for 30 minutes on a rocking platform. Adherent cells were counted by epifluorescence microscopy. WEHI 78/24 binding to aortic segments from SHR animals was elevated compared with segments from WKYs. Fructose feeding alone had no effect on endothelial adhesiveness. When WKYs were made hyperglycemic by STZ injection, monocyte binding was 160% of the control value. Elevated monocyte binding was also observed in aortas derived from SHR animals injected with STZ, indicating an additive effect of hypertension and hyperglycemia. To determine whether alterations in oxidative state played a role in the endothelial adhesiveness, aortic segments were exposed to lucigenin (250 μmol/L) for measurement of superoxide anion. Aortic segments from SHR elaborated 120% more superoxide anion than did controls. Elevated free-radical production was also observed in aortas from diabetic WKYs. Furthermore, thoracic aortas derived from diabetic SHR animals elaborated more superoxide anion than did any of the other groups (374%,
P
<0.05). Immunohistochemical staining for monocyte chemotactic protein-1 demonstrated increased expression in aortas isolated from diabetic WKY and SHR compared with control vessels. These studies demonstrate that both diabetes and hypertension lead to increased monocyte adherence to the endothelium. This abnormality is associated with increased vascular superoxide production and monocyte chemotactic protein-1 expression. Furthermore, there appears to be an additive interaction between hyperglycemia and hypertension in their effects on endothelial adhesiveness and its determinants.
Dominant Negative Effect of TGF-β1 and TNF-α on Basal and IL-6–Induced Lipoprotein(a) and Apolipoprotein(a) mRNA Expression in Primary Monkey Hepatocyte Cultures
Authors: Ramharack Randy , Barkalow Derek , Spahr Mark A.
Published: June 2011

Abstract—Lipoprotein(a) [Lp(a)] consists of apolipoprotein(a) [apo(a)] disulfide linked to apolipoprotein B-100 of LDL. Elevated plasma Lp(a) is an independent risk factor for a variety of vascular diseases. Lp(a) has been reported to be an acute-phase reactant, suggesting that cytokines may regulate its levels. To determine whether Lp(a) expression was subject to modulation by cytokines, primary monkey hepatocytes that endogenously express Lp(a) were used. Hepatocytes were treated with interleukin (IL)-6, the major mediator of the acute-phase response, and several other cytokines. IL-6 treatment (0.3 to 10 ng/mL) resulted in a marked, dose-dependent, 2- to 4-fold enhancement of Lp(a) accumulation in the hepatocyte culture media that was highly correlated with changes in apo(a) mRNA levels (r>0.9). Several other cytokines, such as IL-2, IL-8, and hepatocyte growth factor, had no significant effect on Lp(a) levels; however, transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were very active in inhibiting Lp(a) accumulation in the culture media, with IC50s of ≈0.3 and 1 ng/mL, respectively. Both TGF-β1 and TNF-α also decreased the apo(a) transcript. Mixing experiments, in which hepatocytes were treated with 10 ng/mL of IL-6 and 0.3 to 10 ng/mL of TGF-β1 or TNF-α, demonstrated that the IL-6–mediated induction of Lp(a) and apo(a) mRNA was ablated with very low levels of either inhibitory cytokine, suggesting a dominant negative effect of TGF-β1 and TNF-α. These results show that Lp(a) and apo(a) mRNA expression in primary monkey hepatocytes is subject to both positive (IL-6) and negative (TGF-β1 and TNF-α) regulation by physiological levels of cytokines. Thus, in vivo Lp(a) levels may be dependent on the balance between stimulatory and inhibitory cytokines.
Relation of Lipoprotein Subclasses as Measured by Proton Nuclear Magnetic Resonance Spectroscopy to Coronary Artery Disease
Authors: Freedman David S. , Otvos James D. , Jeyarajah Elias J. , Barboriak Joseph J. , Anderson Alfred J. , Walker John A.
Published: June 2011

Abstract
—Although each of the major lipoprotein fractions is composed of various subclasses that may differ in atherogenicity, the importance of this heterogeneity has been difficult to ascertain owing to the labor-intensive nature of subclass measurement methods. We have recently developed a procedure, using proton nuclear magnetic resonance (NMR) spectroscopy, to simultaneously quantify levels of subclasses of very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins; subclass distributions determined with this method agree well with those derived by gradient gel electrophoresis. The objective of the current study of 158 men was to examine whether NMR-derived lipoprotein subclass levels improve the prediction of arteriographically documented coronary artery disease (CAD) when levels of lipids and lipoproteins are known. We found that a global measure of CAD severity was positively associated with levels of large VLDL and small HDL particles and inversely associated with intermediate size HDL particles; these associations were independent of age and standard lipid measurements. At comparable lipid and lipoprotein levels, for example, men with relatively high (higher than the median) levels of either small HDL or large VLDL particles were three to four times more likely to have extensive CAD than were the other men; the 27 men with high levels of both large VLDL and small HDL were 15 times more likely to have extensive CAD than were men with low levels. In contrast, adjustment for levels of triglycerides or HDL cholesterol greatly reduced the relation of small LDL particles to CAD. These findings suggest that large VLDL and small HDL particles may play important roles in the development of occlusive disease and that their measurement, which is not possible with routine lipid testing, may lead to more accurate risk assessment.
Inhibition by Cholesterol Oxides of NO Release From Human Vascular Endothelial Cells
Authors: Deckert Valérie , Brunet Annie , Lantoine Frédérique , Lizard Gérard , Brussel Elisabeth Millanvoye-van , Monier Serge , Lagrost Laurent , David-Dufilho Monique , Gambert Philippe , Devynck Marie-Aude
Published: June 2011

Abstract
—Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas (
Circulation.
1997;95:723–731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin– and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7β-hydroxycholesterol, 5α,6α-epoxycholesterol, 19-hydroxycholesterol (60 μg/mL), and α-lysophosphatidylcholine (10 μg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5α,6α-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7β-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of
l
-arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.
Exercise and Cardiovascular Disease
Authors: Shern-Brewer Robin , Santanam Nalini , Wetzstein Carla , White-Welkley Jill , Parthasarathy Sampath
Published: June 2011

Abstract
—The oxidation of low density lipoprotein (LDL) has been suggested as a key event in atherogenesis. Paradoxically, exercise, which imposes an oxidative stress, is an important deterrent of cardiovascular disease. In study 1 the oxidizability of LDL was enhanced in exercisers compared with sedentary controls. The lag time of isolated LDL subjected to copper-induced in vitro oxidation was significantly shortened in the exercisers compared with sedentary subjects. This increased sensitivity was not due to a decreased presence of vitamin E. Instead, these findings suggested that the LDL of exercisers may contain increased amounts of preformed lipid peroxides, which account for the increased oxidizability. In study 2, a group×sex ANOVA revealed that male exercisers had a significantly longer mean lag time than male sedentary subjects and that females had similar mean lag times regardless of exercise group. This remained the case when statistical adjustment was made for age, body mass index, blood lipid levels, LDL, and plasma α-tocopherol levels. Study 1 exercisers had been in training for a shorter time (<1 year) than study 2 exercisers (>2 years). These findings suggest that truly “chronic” exercise (aerobic intensity over several months) decreases the susceptibility of a male exerciser’s LDL to undergo oxidation. Conversely, regular aerobic stress during an overall shorter time span creates a more oxidative environment in the body, thus increasing the susceptibility of LDL to undergo oxidation. The oxidative stress of aerobic exercise does not appear to adversely affect the oxidizability of LDL in women.
Adventitial Expression of Recombinant eNOS Gene Restores NO Production in Arteries Without Endothelium
Authors: Tsutsui Masato , Chen Alex F. Y. , O’Brien Timothy , Crotty Thomas B. , Katusic Zvonimir S.
Published: June 2011

Abstract
—The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the β-galactosidase reporter gene (AdCMVβ-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVβ-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3×10
−11
to 10
−9
mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVβ-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3×10
−11
to 10
−9
mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor,
N
G
-nitro-
l
-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.
Clinical Studies and Thrombin Generation in Patients Homozygous or Heterozygous for the G20210A Mutation in the Prothrombin Gene
Authors: Kyrle Paul A. , Mannhalter Christine , Béguin Suzette , Stümpflen Andreas , Hirschl Mirko , Weltermann Ansgar , Stain Milena , Brenner Brigitte , Speiser Wolfgang , Pabinger Ingrid , Lechner Klaus , Eichinger Sabine
Published: June 2011

Abstract
—A genetic variation in the prothrombin gene, the G→A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8±114.9 versus 387±50.1 nmol/L · min,
P
<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L · min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.
Inhibition of Ileal Na
+
/Bile Acid Cotransporter by S-8921 Reduces Serum Cholesterol and Prevents Atherosclerosis in Rabbits
Authors: Higaki Junko , Hara Seijiro , Takasu Nobuo , Tonda Kanya , Miyata Kenji , Shike Tsutomu , Nagata Kiyoshi , Mizui Takuji
Published: June 2011

Abstract
—The ileal Na
+
/bile acid cotransporter (IBAT) plays an important role in the enterohepatic circulation of bile acids. We investigated the effects of IBAT inhibition on the maintenance of serum cholesterol level by using a novel IBAT inhibitor, S-8921, in rabbits. Administration of S-8921 by its incorporation into the diet (0.01% to 0.1%) for 1 to 2 weeks in heterozygous Watanabe heritable hyperlipidemic rabbits decreased serum cholesterol by 29% to 37% and increased fecal excretion of measured bile acids by 60% to 180% compared with control rabbits. Liver microsomal cholesterol 7α-hydroxylase and 3-hydroxy-3-methylglutaryl coenzyme A reductase activities were increased by 75% to 84% and 84% to 89%, respectively, with S-8921 treatment. S-8921 administration (0.1% in the diet) to normal New Zealand White rabbits for 2 weeks resulted in increased hepatic low density lipoprotein receptor expression, which was assessed by Northern blot analysis. In cholesterol-fed New Zealand White rabbits, S-8921 treatment (0.003% to 0.1% in the diet) for 10 weeks dose-dependently inhibited the development of hypercholesterolemia. It also inhibited the accumulation of cholesterol in the aortic arch and reduced the severity of coronary atherosclerosis. These results indicate that IBAT inhibition by S-8921 affects serum cholesterol, liver enzymes, low density lipoprotein receptor activity, and atherosclerosis in the same manner as bile acid sequestrants. We suggest that an IBAT inhibitor such as S-8921 could be useful in the treatment of hypercholesterolemia.
Oxidized LDL Increases and Interferon-γ Decreases Expression of CD36 in Human Monocyte–Derived Macrophages
Authors: Nakagawa Tsutomu , Nozaki Shuichi , Nishida Makoto , Yakub Janabi Mohamed , Tomiyama Yoshiaki , Nakata Atsuyuki , Matsumoto Kengo , Funahashi Tohru , Kameda-Takemura Kaoru , Kurata Yoshiyuki , Yamashita Shizuya , Matsuzawa Yuji
Published: June 2011

Abstract
—CD36 is a glycoprotein with an
M
r
of 88 kDa that is expressed on platelets, monocytes/macrophages, capillary endothelial cells, and adipocytes. We previously demonstrated that CD36 is involved in the uptake of oxidized low density lipoprotein (OxLDL) by using CD36-deficient macrophages (
J Clin
Invest
. 1995;96:1859). However, the regulation of CD36 expression in human monocyte–derived macrophages has not been fully elucidated. The current study attempted to clarify the effect of OxLDL and cytokines, both of which are present in atherosclerotic lesions and may play an important role in atherogenesis, on the expression of CD36. A cell enzyme-linked immunosorbent assay and flow cytometry were used to detect CD36 protein. A ribonuclease protection assay was used to measure CD36 mRNA in human monocyte–derived macrophages. The expression of CD36 was increased during the differentiation of monocytes to macrophages. Incubation of macrophages with 25 μg/mL OxLDL for 24 hours increased the level of CD36 protein by 56% and that of CD36 mRNA by 58%. Lysophosphatidylcholine did not affect the expression of CD36. The effects of OxLDL were demonstrated in macrophages that had already differentiated to the point where CD36 expression was almost maximal. Interferon-γ (IFN-γ) reduced the expression of CD36 in a dose-dependent manner. A concentration of 1000 U/mL IFN-γ significantly reduced the expression of CD36 protein by 57% and that of CD36 mRNA by 30%. In conclusion, CD36 may be important in the formation of foam cells by induction through its ligand (OxLDL). Moreover, some local factors, such as IFN-γ, may suppress CD36 expression on macrophages in human atherosclerotic lesions.
Apolipoprotein(a) Enhances Platelet Responses to the Thrombin Receptor–Activating Peptide SFLLRN
Authors: Rand Margaret L. , Sangrar Waheed , Hancock Mark A. , Taylor Desirée M. , Marcovina Santica M. , Packham Marian A. , Koschinsky Marlys L.
Published: June 2011

Abstract—Elevated levels of lipoprotein(a) [Lp(a)] are correlated with an increased risk of atherosclerotic disease. We examined the effect of recombinant apolipoprotein(a) [r-apo(a)] and Lp(a) on responses of washed human platelets, prelabeled in the dense granules with [14C]serotonin and suspended in Tyrode’s solution, to ADP and the thrombin receptor–activating peptide SFLLRN. No effect of the 17 kringle (K), 12K, or 6K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 μmol/L) or Lp(a) (up to 0.1 μmol/L) on primary ADP-induced platelet aggregation was observed. In contrast, weak platelet responses stimulated by 7.5 μmol/L SFLLRN were significantly enhanced by the r-apo(a) derivatives; eg, 0.7 μmol/L 17K r-apo(a) increased aggregation from 15±4% to 58±6%, release of [14C]serotonin from 9±3% to 36±6%, and formation of thromboxane A2, measured as its stable metabolite thromboxane B2, from 7±1 to 29±5 ng/109platelets (n=3;P<0.04 to 0.015). Significant enhancement of aggregation and release of granule contents was observed at a concentration of 17K r-apo(a) as low as 0.175 μmol/L. Purified Lp(a) (0.25 to 0.1 μmol/L) also enhanced SFLLRN-induced aggregation and release in a dose-dependent manner. Although plasminogen (0.7 and 1.5 μmol/L) and low density lipoprotein (0.025 to 0.1 μmol/L) both exhibited potentiating effects on SFLLRN-mediated platelet aggregation, the magnitude of the responses was less than that observed with either the r-apo(a) derivatives or Lp(a). The enhanced responses of platelets via the protease-activated receptor-1 thrombin receptor in the presence of Lp(a) may contribute to the increased risk of thromboembolic complications of atherosclerosis associated with this lipoprotein.
Increased Angiotensin II Type 1 Receptor Expression in Hypercholesterolemic Atherosclerosis in Rabbits
Authors: Yang Baichun C. , Phillips M. Ian , Mohuczy Dagmara , Meng Hongbin , Shen Leping , Mehta Paulette , Mehta Jawahar L.
Published: June 2011

Abstract
—Angiotensin II (Ang II) promotes vascular smooth muscle growth and may be involved in the initiation and progression of atherosclerosis. To examine whether Ang II receptor expression in vascular tissues is altered in atherosclerosis, male New Zealand White rabbits were fed a high-cholesterol diet (1% cholesterol+4% coconut oil mixed with regular chow; hypercholesterolemic group, n=12) or regular chow (control group, n=8) for 10 weeks. At the end of this period, the serum cholesterol level in the rabbits fed the high-cholesterol diet was higher than that in the control group (3616±144 versus 30±1 mg/dL,
P
<0.001). There was no atherosclerosis in the aortas of the control group, whereas 51±6% of the aorta was covered with atherosclerosis in the hypercholesterolemic group. Total Ang II receptor expression in the atherosclerotic aortic tissues was increased 5-fold in the hypercholesterolemic rabbits (292±28 versus 51±32 fmol/mg tissue, mean±SE,
P
<0.001), and the increased Ang II receptor expression was entirely due to enhanced Ang II type 1 (AT
1
) receptor expression (289±38 versus 38±18 fmol/mg,
P
<0.001), as Ang II type 2 receptor expression was unaltered (7±5 versus 3±2 fmol/mg,
P
=NS). AT
1
receptors were localized primarily in the media and to some extent in the intima of the atherosclerotic aorta, as determined by immunohistochemistry with specific monoclonal and polyclonal AT
1
receptor antibodies. Increased synthesis of AT
1
receptor mRNA in atherosclerotic tissues was confirmed by reverse transcription–polymerase chain reaction. To evaluate the functional significance of increased AT
1
receptor expression, the constrictor response of aortic rings to Ang II was examined and found to be markedly enhanced in atherosclerotic aortic rings (
P
<0.01 versus control aortic rings). The endothelium-dependent relaxation of aortic rings from hypercholesterolemic rabbits was markedly attenuated (
P
<0.001). This study shows that hypercholesterolemia in rabbits results in atherosclerosis, loss of endothelium-dependent relaxation, and increased Ang II receptor (entirely AT
1
receptor) expression in aortic tissues, which may result in altered vasoreactivity.
Hypocoagulant and Lipid-Lowering Effects of Dietary n-3 Polyunsaturated Fatty Acids With Unchanged Platelet Activation in Rats
Authors: Nieuwenhuys Cécile M. A. , Béguin Suzette , Offermans René F. G. , Emeis Jef J. , Hornstra Gerard , Heemskerk Johan W. M.
Published: June 2011

Abstract
—We investigated the effects of dietary polyunsaturated fatty acids (PUFAs) on blood lipids and processes that determine hemostatic potential: platelet activation, coagulation, and fibrinolysis. For 8 to 10 weeks, Wistar rats were fed a high-fat diet containing various amounts (2% to 16%) of n-3 PUFAs derived from fish oil (FO) or a diet enriched in n-6 PUFAs from sunflower seed oil (SO). Only the FO diets caused a reduction in mean platelet volume, platelet arachidonate level, and formation of thromboxane B
2
by activated platelets, but neither of the diets had a measurable effect on platelet activation. The FO-rich diets decreased the plasma concentrations of triglycerides and cholesterol, whereas the SO diet reduced triglycerides only. Parameters of fibrinolysis and standard coagulation times, ie, activated partial thromboplastin time and prothrombin time, were only marginally influenced by these diets. In contrast, dietary FO, but not SO, led to decreased levels of the vitamin K–dependent coagulation factors prothrombin and factor VII, while the level of antithrombin III was unchanged. The endogenous thrombin potential (ETP) was measured with an assay developed to detect the hypocoagulable state of plasma. After activation with tissue factor and phospholipids, the ETP was reduced by 23% or more in plasma from animals fed a diet with >4% FO. No significant effect of the SO diet on ETP was observed. Control experiments with plasma from warfarin-treated rats indicated that the ETP was more sensitive to changes in prothrombin concentration than in factor VII concentration. Taken together, these results indicate that in rats, prolonged administration of n-3 but not n-6 PUFAs can lead to a hypocoagulable state of plasma through a reduced capacity of vitamin K–dependent thrombin generation, with unchanged thrombin inactivation by antithrombin III.
Immunohistochemical Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor in Atherosclerotic Intimas of Human Coronary Arteries
Authors: Chen Yong-Xiang , Nakashima Yutaka , Tanaka Kenichiro , Shiraishi Sachiko , Nakagawa Kazunori , Sueishi Katsuo
Published: June 2011

Abstract
—Neovascularization is well known to occur in human atherosclerotic plaques; however, its pathophysiological roles, mechanisms, and stimuli in atherogenesis still remain unclear. In this study, 525 tissue blocks of coronary artery tissue obtained at autopsy from 48 patients ranging in age from 20 to 93 years old (mean±SD, 71±15 years) were immunohistochemically examined for vascular endothelial growth factor (VEGF) expression in the atherosclerotic intimas. The atherosclerotic lesions were histopathologically classified into types I through VI, as proposed by the American Heart Association Committee, and the numbers of intimal blood vessels and VEGF-positive cells were then morphometrically counted in sections that were immunohistochemically examined with anti-CD34 and human VEGF antibodies, respectively. The more the atherosclerotic lesion type advanced, the more often the lesion contained intimal blood vessels, which were expressed as percentages of the intimal section with intimal microvessels, viz, diffuse intimal thickening (DIT): 0% (0/111); type I, 31% (32/104); II, 42% (10/24); III, 66% (77/117); IV, 72% (48/67); V, 79% (70/89); and VI, 100% (13/13),
P
<0.0001. The number of VEGF-positive cells per intimal section was also positively correlated with the number of intimal blood vessels (
P
<0.0001). The VEGF-positive cells were scattered in the fibrous caps as well as the shoulders and deeper areas of the plaques, and the double-immunostaining method revealed that the VEGF-positive cells were largely spindle-shaped, smooth muscle cells with some macrophage-derived foam cells. These findings thus suggest the possibility that the VEGF expressed by the smooth muscle cells and foamy macrophages in the atherosclerotic intimas can act as a local and endogenous regulator of endothelial cell functions, including intimal neovascularization, in atherosclerotic lesions of human coronary arteries.
Natriuretic Peptides and Nitric Oxide Induce Endothelial Apoptosis via a cGMP–Dependent Mechanism
Authors: Suenobu Noriko , Shichiri Masayoshi , Iwashina Masatora , Marumo Fumiaki , Hirata Yukio
Published: June 2011

Abstract—Apoptosis is a mode of cell death in which the cell participates in its own demise. We studied whether endothelium-derived relaxing factor, nitric oxide (NO), and natriuretic peptides affect apoptosis of rat vascular endothelial cells via a cGMP-dependent pathway and whether such effects are antagonized by an endothelium-derived vasoconstrictor, endothelin-1 (ET-1). Three natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) induced endothelial apoptosis as demonstrated by nucleosomal laddering on agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase–mediated dUTP biotin nick end labeling method. This dose-dependent relation was assessed by quantifying the fragmented and intact DNA contents by the diphenylamine method. The atrial natriuretic peptide–induced endothelial apoptosis was completely blocked by a guanylate cyclase–coupled receptor antagonist (HS-142-1) and an inhibitor of cGMP-dependent protein kinase (KT5823). An NO donor, NOR3 {(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide; FK409} also induced endothelial apoptosis; the effect of this compound was abrogated by KT5823 and an inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). A cGMP derivative, 8-bromo-cGMP, but not the cAMP derivative 8-bromo-cAMP, caused endothelial apoptosis; the effect of ODQ was also abrogated by KT5823. Endothelial apoptosis induced by ANP, NOR3, and 8-bromo-cGMP was similarly antagonized by ET-1. ANP, NOR3, and 8-bromo-cGMP caused marked accumulations of the tumor suppressor gene product p53 but not of bcl-2, as determined by Western blot analysis. These results demonstrate for the first time that endothelium-derived NO and natriuretic peptides are proapoptotic factors for endothelial cells, whereas the endothelium-derived vasoconstrictor ET-1 is an antiapoptotic factor, suggesting that the countervailing balance between these vasodilators and vasoconstrictors, in addition to regulation of vascular tonus, may contribute to endothelial cell integrity.
Cellular Cholesterol Transport and Efflux in Fibroblasts Are Abnormal in Subjects With Familial HDL Deficiency
Authors: Marcil Michel , Yu Lu , Krimbou Larbi , Boucher Betsie , Oram John F. , Cohn Jeffrey S. , Genest Jacques
Published: June 2011

Abstract
—Familial high density lipoprotein (HDL) deficiency (FHD) is a genetic lipoprotein disorder characterized by a severe decrease in the plasma HDL cholesterol (-C) level (less than the fifth percentile). Unlike Tangier disease, FHD is transmitted as an autosomal dominant trait. FHD subjects have none of the clinical manifestations of Tangier disease (lymphoid tissue infiltration with cholesteryl esters and/or neurological manifestations). Plasmas from FHD subjects contain pre–β-migrating HDLs but are deficient in α-migrating HDLs. We hypothesized that a reduced HDL-C level in FHD is due to abnormal transport of cellular cholesterol to the plasma membrane, resulting in reduced cholesterol efflux onto nascent HDL particles, leading to lipid-depleted HDL particles that are rapidly catabolized. Cellular cholesterol metabolism was investigated in skin fibroblasts from FHD and control subjects. HDL
3
- and apolipoprotein (apo) A-I–mediated cellular cholesterol and phosphatidylcholine efflux was examined by labeling cells with [
3
H]cholesterol and [
3
H]choline, respectively, during growth and cholesterol loading during growth arrest. FHD cells displayed an ≈25% reduction in HDL
3
-mediated cellular cholesterol efflux and an ≈50% to 80% reduction in apoA-I–mediated cholesterol and phosphatidylcholine efflux compared with normal cells. Cellular cholesterol ester levels were decreased when cholesterol-labeled cells were incubated with HDL
3
in normal cells, but cholesterol ester mobilization was significantly reduced in FHD cells. HDL
3
binding to fibroblasts and the possible role of the HDL binding protein/vigilin in FHD were also investigated. No differences were observed in
125
I-HDL
3
binding to LDL-loaded cells between FHD and control cells. HDL binding protein/vigilin mRNA levels and its protein expression were constitutively expressed in FHD cells and could be modulated (≈2-fold increase) by elevated cellular cholesterol in normal cells. In conclusion, FHD is characterized by reduced HDL
3
- and apoA-I–mediated cellular cholesterol efflux. It is not associated with abnormal cellular HDL
3
binding or a defect in a putative HDL binding protein.
Eicosapentaenoic Acid and Docosahexaenoic Acid Block Serotonin-Induced Smooth Muscle Cell Proliferation
Authors: Pakala Rajbabu , Pakala Rajashree , Sheng Wen Lu , Benedict Claude R.
Published: June 2011

Abstract
—Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) present in fish oils have been ascribed as having significant antithrombotic and antiatherosclerotic effects. Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have indicated that serotonin at concentrations present at sites of vascular injury stimulates SMC proliferation and may contribute to the restenotic process. In the present study we demonstrate that among the fatty acids tested, only EPA and DHA could block the mitogenic effect of serotonin on vascular SMC. Further, when added together these fatty acids act synergistically in blocking the mitogenic effect of serotonin. EPA and DHA blocked the 5HT-induced increase in the 5-HT
2
receptor mRNA. This antimitogenic effect of EPA and DHA may partially explain some of the beneficial effects of fish oils.
Interleukin-6 Exacerbates Early Atherosclerosis in Mice
Authors: Huber S. A. , Sakkinen P. , Conze D. , Hardin N. , Tracy R.
Published: June 2011

Abstract—Acute-phase proteins, which respond to systemic proinflammatory cytokines such as interleuken-6, are elevated in cardiovascular disease and are predictive markers of future ischemic events, even over decades. This suggests a role for proinflammatory cytokines and/or acute phase proteins in early lesion development. To explore this issue, we fed C57Bl/6 and nonobese diabetic male mice high-fat (20% total fat, 1.5% cholesterol) diets and ApoE-deficient male mice both high-fat and normal chow diets for 6 to 21 weeks, injecting them weekly with either 5000 U recombinant interleukin-6 (rIL-6) or saline buffer. Blood was collected when animals were euthanized and assayed for cytokines, acute-phase proteins, and cholesterol. Across all mice, IL-6 injection resulted in significant increases in proinflammatory cytokines (IL-6, 4.6-fold; IL-1β, 1.6-fold; and tissue necrosis factor-α, 1.7-fold) and fibrinogen (1.2-fold) and with decreased concentrations of albumin (0.9-fold) in plasma. Total cholesterol levels were unchanged between rIL-6–treated and nontreated groups. Serial sections through the aortic sinus were stained with oil red O to detect fatty streaks, and area of the lesions was determined by image analysis. Although no fatty streaks were detected in the nonobese diabetic mice with or without rIL-6 treatment, rIL-6 treatment increased lesion size in C57Bl/6 and ApoE-deficient mice 1.9- to 5.1-fold over lesions in saline-treated animals. These results suggest that under the appropriate circumstances changes in circulating proinflammatory cytokines and acute-phase proteins may be more than just markers of atherosclerosis but actual participants in early lesion development.
Gender Difference in Postprandial Lipemia
Authors: Couillard Charles , Bergeron Nathalie , Prud’homme Denis , Bergeron Jean , Tremblay Angelo , Bouchard Claude , Mauriège Pascale , Després Jean-Pierre
Published: June 2011

Abstract
—Insulin resistance, hyperinsulinemia, hypertriglyceridemia, and low HDL-cholesterol concentrations are common features of a plurimetabolic syndrome, which increases the risk of coronary artery disease. Although it has been proposed that the development of atherosclerosis through alterations in plasma lipid levels could be a postprandial phenomenon, most studies on gender differences in plasma lipoprotein-lipid concentrations have reported fasting levels. Therefore, the aim of our study was to examine the response of postprandial triglyceride-rich lipoproteins to a standardized meal in 63 men and 25 women. In addition to the measurement of fasting and postprandial plasma lipid levels, numerous physical and metabolic variables were assessed, including body composition by underwater weighing and body fat distribution by computed tomography. Although no gender difference was noted in total body fat mass, men were characterized by a preferential accumulation of abdominal adipose tissue as revealed by an increased waist circumference and a greater visceral adipose tissue accumulation (50% difference) compared with women (
P
<0.001). Men also showed a greater plasma triglyceride response (
P
<0.005) as well as increased postprandial insulin and free fatty acid levels compared with women (
P
<0.01). Visceral adipose tissue was significantly associated with the postprandial triglyceride response in both genders (men:
r
=0.49,
P
<0.0001; women:
r
=0.43,
P
<0.05). Finally, when men and women were matched for visceral adipose tissue accumulation, the gender difference in postprandial plasma triglyceride response was eliminated. Thus results of the present study suggest that the well known gender difference in visceral adipose tissue accumulation is an important contributing factor involved in the exaggerated postprandial triglyceride-rich lipoprotein response noted in men compared with women.
Endotoxin-Induced Activation of the Coagulation Cascade in Humans
Authors: Pernerstorfer T. , Stohlawetz P. , Hollenstein U. , Dzirlo L. , Eichler H.-G. , Kapiotis S. , Jilma B. , Speiser W.
Published: June 2011

Abstract
—During Gram-negative septic shock, lipopolysaccharide (LPS, endotoxin) induces tissue factor (TF) expression. TF expression is mediated by nuclear factor κB and amplified by activated platelets. TF forms a highly procoagulant complex with activated coagulation factor VII (FVIIa). Hence, we hypothesized that aspirin, which inhibits LPS-induced, nuclear factor κB–dependent TF expression in vitro and platelet activation in vivo, may suppress LPS-induced coagulation in humans. Therefore, we studied the effects of aspirin on systemic coagulation activation in the established and controlled setting of the human LPS model. Thirty healthy volunteers were challenged with LPS (4 ng/kg IV) after intake of either placebo or aspirin (1000 mg). Acetaminophen (1000 mg) was given to a third group to control for potential effects of antipyresis. Neither aspirin nor acetaminophen inhibited LPS-induced coagulation. However, LPS increased the percentage of circulating TF
+
monocytes by 2-fold. This increase was associated with a decrease in FVIIa levels, which reached a minimum of 50% 24 hours after LPS infusion. Furthermore, LPS-induced thrombin generation increased plasma levels of circulating polymerized, but not cross-linked, fibrin (ie, thrombus precursor protein), whereas levels of soluble fibrin were unaffected. In summary, a single 1000-mg dose of aspirin did not decrease LPS-induced coagulation. However, our study showed, for the first time, that LPS increases TF
+
monocytes, substantially decreases FVIIa levels, and enhances plasma levels of thrombus precursor protein, which may be a useful marker of fibrin formation in humans.
Activation of the Contact System of Coagulation Does Not Contribute to the Hemostatic Imbalance in Hypertriglyceridemia
Authors: Minnema M. C. , Wittekoek M. E. , Schoonenboom N. , Kastelein J. J. P. , Hack C. E. , Cate H. ten
Published: June 2011

Abstract
—In vitro, triglyceride-rich lipoproteins may act as a surface to initiate the contact system of coagulation. Therefore, we studied the activation of factor XII (FXII), prekallikrein, and FXI and the generation of thrombin in 52 hypertriglyceridemic patients before and after 12 weeks of triglyceride-lowering treatment with gemfibrozil or n-3 polyunsaturated fatty acids. Thrombin generation was assessed by measuring the levels of prothrombin fragment F1+2 and thrombin-antithrombin (TAT) complexes. Contact activation was assessed by measuring FXIIa, kallikrein, and FXIa in complex with their major inhibitor, C1 inhibitor, and FXIa was also determined as part of a complex with α
1
-antitrypsin. Triglyceride and cholesterol levels decreased equally in both treatment groups. In the gemfibrozil group, there was a significant decrease in F1+2, while TAT complexes did not change. FXIIa- and kallikrein-C1 inhibitor complexes were elevated in 13% and 9% of the patients before treatment, respectively, and no changes were observed on triglyceride-lowering therapy. Also, no significant changes in regard to FXIa–C1 inhibitor and FXIa–α
1
-antitrypsin complexes were seen. FXIa–α
1
-antitrypsin complexes were present in 70% of the patients before therapy and were positively correlated with the level of TAT complexes. In conclusion, we did not detect an effect on activation markers of the contact coagulation system in hypertriglyceridemic patients after triglyceride-lowering therapy. Therefore, contact activation is not likely to contribute to the hypercoagulability seen in these patients.
Antithrombotic Efficacy of a Novel Murine Antihuman Factor IX Antibody in Rats
Authors: Feuerstein Giora Z. , Patel Arun , Toomey John R. , Bugelski Peter , Nichols Andrew J. , Church William R. , Valocik Richard , Koster Paul , Baker Audrey , Blackburn Michael N.
Published: June 2011

Abstract
—A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg · kg
−1
· h
−1
), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl
3
-patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U · kg
−1
· min
−1
), whereas the latter rendered the blood incoagulable (aPTT>1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18±0.49 to 1.80±0.3 mg,
P
<0.001), increasing vessel patency time (from 14.9±0.9 minutes to 58.3±1.7 minutes,
P
<0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle- versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (
P
<0.01), extended vessel patency time (
P
<0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8±0.7 to 3.3±0.6 mL (
P
<0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.
Structural Requirements for TFPI-Mediated Inhibition of Neointimal Thickening After Balloon Injury in the Rat
Authors: Han Xin , Girard Thomas J. , Baum Pamela , Abendschein Dana R. , Broze George J.
Published: June 2011

Abstract
—The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK
1
, a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI
WT
, the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI
K36I
, an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI
13–161
, a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK
1
, TFPI
WT
, and high levels of TFPI
K36I
begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI
13–161
was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK
1
≈TFPI
WT
>TFPI
K36I
≫TFPI
13–161
. The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action.
Protection by Shear Stress From Collar-Induced Intimal Thickening
Authors: Marano Giuseppe , Palazzesi Sergio , Vergari Alessandro , Ferrari Alberto U.
Published: June 2011

Abstract
—Nitric oxide (NO) has potent relaxant and antiproliferative effects on vascular smooth muscle cells, which may represent an important antiatherosclerotic mechanism. Since one of the major stimuli for NO release is flow-related shear stress, we have investigated (1) the effect of increased shear stress on neointimal formation induced in the rabbit carotid artery by enclosing the vessel in a nonconstrictive silicone soft collar and (2) the role of NO in the antiproliferative effect of increased shear stress. Forty-three New Zealand White rabbits were used. High shear stress in the left common carotid artery (CCA) was induced by ligature of the contralateral right internal carotid artery; intimal thickening was produced by the positioning a nonconstrictive silicone soft collar around the left CCA. To evaluate the role of NO,
N
G
-nitro-
l
-arginine methyl ester (L-NAME) was orally administered at a subpressor dose. In all rabbits, arterial blood pressure, heart rate, arterial diameters, and blood flow velocities of both CCAs were determined at days 0, 3, 7, and 14. At the end of the study, all rabbits were euthanized, and histological analyses were performed on both CCAs of each animal. The presence of the collar was associated with a marked degree of intimal hyperplasia (intimal/medial area ratio 29±3.0% in collared arteries compared with 3±0.7% in sham control [noncollared] arteries,
P
<0.001). The increase in blood flow almost completely inhibited neointimal formation and induced an increase in arterial diameter of ≈30%. The effects of increased blood flow were reversed by the administration of L-NAME. In conclusion, we demonstrate that in collar-induced intimal thickening, a chronic increase in shear stress (1) almost completely inhibits intimal thickening, and (2) this protective effect is mediated by NO production.