bluuebunny/crossref_metadata_2025 huggingface dataset:
Short-term consumption of a low-fat diet beneficially affects plasma lipid concentrations only when accompanied by weight loss. Hypercholesterolemia, low-fat diet, and plasma lipids.
Authors: Lichtenstein A H , Ausman L M , Carrasco W , Jenner J L , Ordovas J M , Schaefer E J
Published: June 2011

Study subjects (6 women and 5 men) over the age of 40 years with fasting low-density lipoprotein cholesterol concentrations > 130 mg/dL were studied during three 5-week diet phases and one 10-week phase: baseline (36% fat: 13% saturated fatty acids [SFA], 12% monounsaturated fatty acids [MUFA], 8% polyunsaturated fatty acids [PUFA], and 128 mg cholesterol/1000 kcal); reduced fat (29% fat: 7% SFA, 9% MUFA, 11% PUFA, and 85 mg cholesterol/1000 kcal); and two low fat (15% fat: 5% SFA, 5% MUFA, 3% PUFA, and 73 mg cholesterol/1000 kcal). Body weight was maintained during the first three 5-week phases (baseline, reduced fat, and low fat [-->energy]) and decreased during the last 10-week phase when the low-fat diet was provided such that the subjects determined, in part, their caloric intake (low fat [decreases energy]). Mean body weight declined by 0.62 +/- 0.47 kg/wk during the first 5 weeks and 0.43 +/- 0.43 kg/wk during the second 5 weeks of the 10-week low-fat (decreases energy) period. Relative to the baseline diet, plasma cholesterol concentrations decreased from 226 +/- 33 to 195 +/- 19 (-13%), 208 +/- 22 (-7%), and 190 +/- 19 (-15%) mg/dL when the subjects consumed the reduced-fat, low-fat (--> energy), and low-fat (decreases energy) diets, respectively. Low-density lipoprotein cholesterol concentrations decreased from 158 +/- 28 to 128 +/- 16 (-18%), 134 +/- 17 (-14%), and 119 +/- 15 (-23%) mg/dL when the subjects consumed the reduced-fat, low-fat (--> energy), and low-fat (decreases energy) diets, respectively. High-density lipoprotein cholesterol concentrations decreased from 48 +/- 11 to 42 +/- 9 (-10%), 35 +/- 7 (-25%), and 38 +/- 8 (-18%) mg/dL when the subjects consumed the reduced-fat, low-fat (--> energy), and low-fat (decreases energy) diets, respectively. Triglyceride concentrations increased from 110 +/- 32 to 115 +/- 31 (8%), 188 +/- 76 (75%), and 130 +/- 32 (22%) mg/dL when the subjects consumed the reduced-fat, low-fat (--> energy), and low-fat (decreases energy) diets, respectively. Maximal changes in plasma lipid concentrations were observed after the first 5 weeks of the low-fat (decreases energy) diet phase despite continued weight loss throughout the entire 10-week diet period.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of endotoxin and cytokines on lipoprotein lipase activity in mice.
Authors: Feingold K R , Marshall M , Gulli R , Moser A H , Grunfeld C
Published: June 2011

Endotoxin (lipopolysaccharide [LPS]) stimulates the production of cytokines, which mediate many of the metabolic effects associated with infection. In LPS-sensitive C57B1/6 mice, LPS doses as low as 0.01 micrograms per mouse decreased adipose tissue lipoprotein lipase (LPL) activity by greater than 50%. In LPS-resistant C3H/HeJ mice, which do not produce cytokines in response to LPS, doses of LPS as high as 10 micrograms per mouse did not affect LPL activity in adipose tissue. In muscle of C57Bl/6 mice, LPL activity was decreased by 27% after 10 micrograms of LPS, whereas in C3H/HeJ mice there was no effect. These results indicate that the LPS-induced decrease in both adipose and muscle LPL activity is mediated by cytokines. Tumor necrosis factor (TNF), interleukin (IL)-1, leukemia-inhibiting factor (LIF), interferon alfa, and interferon gamma all decreased adipose tissue LPL activity in intact mice. In skeletal and cardiac muscle, only IL-1 and interferon gamma decreased LPL activity, whereas TNF, LIF, and interferon alfa had no effect. Inhibition of TNF activity blocked the increase in serum triglycerides that is characteristically observed after LPS but did not affect the ability of LPS to decrease adipose tissue LPL activity. Inhibition of IL-1 activity with IL-1 receptor antagonist partially inhibited the increase in serum triglycerides; however, the ability of LPS to decrease LPL activity in either adipose or muscle tissue was not affected. These data indicate that although TNF and IL-1 play a role in mediating the increase in serum triglyceride levels, these cytokines do not play a crucial role in the inhibition of either adipose or muscle LPL activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Ultrasound-determined intima-media thickness and atherosclerosis. Direct and indirect validation.
Authors: Persson J , Formgren J , Israelsson B , Berglund G
Published: June 2011

To evaluate ultrasonographically determined intima-media thickness as a measure of early atherosclerosis, three studies were performed. Ultrasound measurements of intima-media thickness in the carotid artery were directly validated by comparing the same thickness measured by light microscopy. The values were closely correlated (r = .82, P < .001). Intima-media thickness determined by light microscopy was consistently smaller than that determined by ultrasound, probably due to shrinkage during histological preparation. As an indirect validation, mean intima-media thickness was calculated in three large groups of patients with no plaque (n = 224), one plaque (n = 105), and one circumferential or two or more plaques (n = 54) in the carotid bifurcation. Intima-media thickness increased significantly with increasing plaque score, indicating that diffuse intima-media thickening is more pronounced with more severe atherosclerosis. The intima-media thickness also increased with increasing multifactorial cardiovascular risk, reflecting a positive relation between signs of early atherosclerosis and the burden of known risk factors for the disease. Our studies support earlier findings that have found that ultrasonographically determined intima-media thickness is a valid way to study early atherosclerosis.
Structural organization of reconstituted human arterial smooth muscle tissue.
Authors: Björkerud S , Björkerud B , Joelsson M
Published: June 2011

We used human arterial smooth muscle cells (SMCs) that had been reorganized three-dimensionally into aggregates, so-called spheroids, as a model system that might more closely correspond to arterial smooth muscle in vivo than do conventional monolayer cultures. After reaggregation the presence of serum in the culture medium strongly promoted the maintenance of spheroidal SMCs. With access to fresh serum, the spheroids developed into highly organized structures with an outer laminated shell of spindle-shaped SMCs and a more porous core of rounded or polygonal SMCs. After several weeks in culture, extracellular matrix components appeared and the tissue assumed features characteristic of maturing intimal repair tissue. Many cells had features of programmed cell death (apoptosis). This feature may be important because it may indicate that regression of arterial smooth muscle tissue may be a much more strongly controlled process than hitherto realized. Without access to fresh serum, the spheroidal tissue showed degenerative features, much like those in atherosclerotic lesions, ie, the presence of foam cells, cellular debris, and some cell death. It is possible that this situation in vitro resembles that of atherosclerotic tissue in vivo, in which retention of plasma constituents is a conspicuous feature. In some respects, therefore, the small sample of human arterial tissue represented by the spheroid may represent an in vitro analogue of the arterial wall, which may undergo maturation or degenerative atherosclerosis-like changes depending on exogenous factors. The spheroidal SMC system may therefore also be a suitable model for in vitro studies of atherogenesis.
Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta. PGH2 does not contribute to impaired endothelium-dependent relaxation.
Authors: Naruse K , Shimizu K , Muramatsu M , Toki Y , Miyazaki Y , Okumura K , Hashimoto H , Ito T
Published: June 2011

We examined whether prostaglandin (PG) H2, as an endothelium-dependent contracting factor, or the disturbed production of endothelium-derived relaxing factor, impairs endothelium-dependent relaxation and whether long-term inhibition of nitric oxide (NO) synthesis aggravates atherosclerosis in hypercholesterolemic rabbits. Male New Zealand White rabbits were fed one of the following diets: (1) standard chow; (2) 2% cholesterol-supplemented chow; (3) standard chow with 80 micrograms/mL N omega-nitro-L-arginine methylester (L-NAME), an NO synthetase inhibitor, in their drinking water; or (4) 2% cholesterol-supplemented chow with 80 or 160 micrograms/mL L-NAME in their drinking water. The rabbits were fed these diets for 8 or 12 weeks. Then aortic rings were obtained, and changes in isometric tension were recorded. Intimal atherosclerotic areas of the thoracic aortas were subsequently measured by planimetry. The cholesterol-supplemented diet significantly impaired endothelium-dependent aortic relaxation to acetylcholine. Pretreatment with the thromboxane A2/PGH2 receptor antagonist ONO-3708 did not reverse this impaired response. Vessels from both normocholesterolemic and hypercholesterolemic rabbits given L-NAME showed more impaired endothelium-dependent relaxation than those from their dietary counterparts not given L-NAME. Morphometric analysis revealed marked enlargement of intimal atherosclerotic areas in aortas from L-NAME-treated hypercholesterolemic rabbits compared with those from untreated hypercholesterolemic rabbits. These findings suggest that PGH2 does not contribute to impaired endothelium-dependent relaxation and that long-term administration of L-NAME promotes atherosclerosis by inhibition of NO synthesis in the hypercholesterolemic rabbit thoracic aorta.
Morphological alterations in endothelial cells associated with the release of von Willebrand factor after thrombin generation in vivo.
Authors: Richardson M , Tinlin S , De Reske M , Webster S , Senis Y , Giles A R
Published: June 2011

von Willebrand factor (vWF) is synthesized by endothelial cells and stored in endothelium-specific granules, the Weibel-Palade (WP) bodies. The release of vWF from endothelial cells in vitro in response to secretagogues such as thrombin is considered to result in the loss of WP bodies through the fusion of the WP bodies with the plasma membrane. Biochemical and morphological techniques, including transmission (TEM) and scanning (SEM) electron microscopy, were used to examine the plasma profile of vWF in parallel with morphological alterations in endothelial cells associated with the generation of thrombin in vivo. There was a rapid loss of high-molecular-weight multimers of the circulating vWF, with full recovery within 1 hour. Simultaneously, TEM demonstrated that the endothelial cells lost WP bodies and became severely vacuolated; this was associated with the appearance of craters in the endothelial surface on SEM. Release of stored vWF in WP bodies seemed to follow the fusion of multiple rather than individual WP bodies, with the resulting vacuole fusing and rupturing through the plasmatic membrane. Within 1 hour there was increased morphological evidence of metabolic organelle activity associated with replacement of WP bodies, presumably due to de novo synthesis of the basic protomer and its packaging in high-molecular-weight multimeric form in the storage organelles.
Increased intima-media thickness of the common carotid artery in hypercholesterolemic children.
Authors: Pauciullo P , Iannuzzi A , Sartorio R , Irace C , Covetti G , Di Costanzo A , Rubba P
Published: June 2011

Common carotid intima-media thickness was measured by B-mode ultrasound imaging in 46 children (mean age, 7.4 years) with serum cholesterol > or = 6.4 mmol/L (mean, 8.25 mmol/L) and in 48 children (mean age, 6.4 years) with serum cholesterol < 6.4 mmol/L (mean, 4.60 mmol/L). Maximum thickness was significantly higher in hypercholesterolemic children than in control children (0.50 versus 0.47 mm, P = .007). Subgroup analysis showed that only in children > 6.2 years old (the median of all the children's ages) was maximum thickness significantly higher in hypercholesterolemic children than in control children (0.51 versus 0.48 mm, P = .014). The odds ratio (OR) of common carotid intima-media thickening (maximum thickness of the far wall higher than the 95th percentile of the control group, 0.51 mm) between patients and control subjects was statistically significant both in univariate analysis (OR, 6.39; 95% confidence interval, 1.19 to 32.3; P = .025) and after age (OR, 5.96; 95% confidence interval, 1.09 to 32.4; P = .039) and sex (OR, 7.54; 95% confidence interval, 1.38 to 41.2; P = .020) were controlled for. Children > 6 years old with serum cholesterol > or = 6.4 mmol/L show increased thickness of the common carotid intima-media.
Susceptibility of human LDL to oxidative modification. Effects of variations in beta-carotene concentration and oxygen tension.
Authors: Reaven P D , Ferguson E , Navab M , Powell F L
Published: June 2011

Epidemiological studies suggest that beta-carotene supplementation may decrease atherosclerotic events. Because beta-carotene is transported in low-density lipoprotein (LDL), one mechanism by which this protective effect may occur is through direct inhibition of LDL oxidation. Addition of beta-carotene to LDL in vitro inhibits the susceptibility of LDL to oxidation. In contrast, we have shown that feeding large doses of beta-carotene results in beta-carotene-enriched plasma LDL (16- to 35-fold), but such LDL does not show increased resistance to oxidation. Potential criticisms of our initial study relate to the unique antioxidant properties of beta-carotene. Beta-Carotene provides better quenching of some free radicals, such as singlet oxygen, than others. Additionally, since beta-carotene can easily autooxidize, forming metabolites that can also generate free radicals, it has been argued that at sufficient concentrations in vivo the antioxidant effect of beta-carotene may be diminished. It has thus been suggested that in our initial study we may have failed to properly assess the unique antioxidant effects of beta-carotene by our selection of oxidizing conditions and/or failed to achieve antioxidant activity because of excessive enrichment of LDL with beta-carotene. We now report the effects of feeding lower doses of beta-carotene on the susceptibility of LDL to oxidation and test whether any antioxidant activity of beta-carotene can be detected when different modes of initiating oxidation are used. Because the antioxidant activity of beta-carotene is reputedly most pronounced in low-oxygen environments, we also investigated its effect on LDL oxidation under conditions of reduced oxygen tension.(ABSTRACT TRUNCATED AT 250 WORDS)
Pathology of atheromatous lesions in inbred and genetically engineered mice. Genetic determination of arterial calcification.
Authors: Qiao J H , Xie P Z , Fishbein M C , Kreuzer J , Drake T A , Demer L L , Lusis A J
Published: June 2011

We report comprehensive pathological studies of atheromatous lesions in various inbred mouse strains fed a high-fat, high-cholesterol diet and in two genetically engineered strains that develop spontaneous lesions on a low-fat chow diet. Coronary and aortic lesions were studied with respect to anatomic locations, lesion severity, calcification, and lipofuscin deposition. Surprisingly, the genetic determinants for coronary fatty lesion formation differed in part from those for aortic lesion development. This suggests the existence of genetic factors acting locally as well as systematically in lesion development. We used immunohistochemical analyses to determine the cellular and molecular compositions of the lesions. The aortic lesions contained monocyte/macrophages, lipid, apolipoprotein B, serum amyloid A proteins, and immunoglobulin M and showed expression of vascular cell adhesion molecule-1 and tumor necrosis factor-alpha, all absent in normal arteries. In certain strains, advanced lesions developed in which smooth muscle cells were commonly observed. The lesions in mice targeted for a null mutation in the apolipoprotein E gene were much larger, more widely dispersed, and more fibrous, cellular, and calcified in nature than the lesions in laboratory inbred strains. When apolipoprotein A-II transgenic mice were maintained on a low-fat chow diet, the lesions in these mice were relatively small and located in the very proximal regions of the aorta. There were clear differences in the occurrence of arterial wall calcification among genetically distinct inbred mouse strains, indicating for the first time a genetic component in this clinically significant trait. Analysis of a genetic cross indicated a complex pattern of calcification inheritance with incomplete penetrance.
Extracellular Mast Cell Granules Carry Apolipoprotein B-100–Containing Lipoproteins Into Phagocytes in Human Arterial Intima
Authors: Kaartinen Maija , Penttilä Antti , Kovanen Petri T.
Published: June 2011

Abstract
In experimental studies in vitro, mast cells have induced uptake of apolipoprotein B-100 (apoB-100)–containing low-density lipoproteins by macrophages, with the subsequent formation of foam cells, the hallmarks of atherosclerosis. Recently, increased numbers of activated, ie, degranulated, mast cells were found to be present in human coronary fatty streaks and atheromas. We therefore sought evidence of a connection between mast cells and foam cell formation in vivo. In electron microscopic studies of human aortic and coronary fatty streaks and atheromas, exocytosed cytoplasmic secretory granules of mast cells were detected in the vicinity of their parent cells. These exocytosed granules had bound apoB-100–containing lipoproteins, as indicated by their positive staining with MB 47, a monoclonal antibody against apoB-100. A smooth muscle cell was observed to be in the process of phagocytosing one such exocytosed granule, and in the vicinity of a degranulated mast cell a foam cell contained an ingested mast cell granule. Therefore, the micrographs show that exocytosed granules of intimal mast cells may contribute to intimal foam cell formation and suggest a role for mast cells in human atherogenesis. More generally, the findings provide evidence that phagocytosis of apoB-100–carrying particles is one mechanism by which lipoproteins enter human arterial intimal cells.
Increases in Dietary Cholesterol Are Associated With Modest Increases in Both LDL and HDL Cholesterol in Healthy Young Women
Authors: Ginsberg Henry N. , Karmally Wahida , Siddiqui Maliha , Holleran Steve , Tall Alan R. , Blaner William S. , Ramakrishnan Rajasekhar
Published: June 2011

Abstract
We studied the effects of dietary cholesterol intake on lipid and lipoprotein levels in healthy young women (n=13) who were otherwise eating an American Heart Association (AHA) diet. The study used a randomized, three-way crossover design to determine the effects of 0, 1, or 3 eggs added per day (dietary cholesterol range, 108 to 667 mg/d). Each of the three diets was eaten for 8 weeks, with a washout period between diets. Three fasting blood samples were obtained during the last 3 weeks of each diet period to observe changes in fasting plasma lipid levels associated with the menstrual cycle. We also obtained blood just before and 4 and 8 hours after the subjects ingested a standard high-fat formula. During the menstrual cycle, total cholesterol and LDL cholesterol levels fell by 0.051 mmol/L (1.99 mg/dL) and 0.064 mmol/L (2.48 mg/dL) per week, respectively. HDL cholesterol concentrations increased by 0.060 mmol/L (2.3 mg/dL) per week during the first half of the cycle and then fell by 0.050 mmol/L (1.94 mg/dL) per week during the second half. Therefore, all statistical analyses were performed on values adjusted to midcycle. Total fasting cholesterol concentrations increased by 0.073 mmol/L (2.81 mg/dL) per 100 mg dietary cholesterol added to the diet per day (
P
=.001). LDL cholesterol increased by 0.054 mmol/L (2.08 mg/dL) per 100 mg/d dietary cholesterol (
P
=.003); this accounted for about 75% of the rise in total cholesterol. HDL cholesterol concentrations increased by 0.015 mmol/L (0.57 mg/dL) per 100 mg/d dietary cholesterol (
P
<.04). There was a wide range of responses among the women. Plasma apoB levels increased significantly, 0.93 mg/dL per 100 mg/d dietary cholesterol (
P
=.025), whereas apoA-I levels tended to rise (1.35 mg/dL per 100 mg/d,
P
=.056). Increases in dietary cholesterol did not produce any observable effects on fasting plasma cholesteryl ester transfer protein levels and had no effect on the response to a standard high-fat formula. Although menstrual-cycle changes in plasma total, LDL, and HDL cholesterol levels were observed, the effects of the diets were similar in the follicular and luteal phases of the menstrual cycle. Additionally, despite changes associated with the menstrual cycle, within-subject variation in plasma total cholesterol was actually smaller in this study than in our study of young men.
Characterization of Serum Amyloid P Component From Human Aortic Atherosclerotic Lesions
Authors: Li X. A. , Hatanaka K. , Ishibashi-Ueda H. , Yutani C. , Yamamoto A.
Published: June 2011

AbstractSerum amyloid P component (SAP) is a glycoprotein in human plasma. We recently showed the localization of SAP in human atherosclerotic lesions by immunohistochemical staining. In this study, the presence of SAP in atherosclerotic lesions was confirmed, and the biochemical character of SAP in atherosclerotic intima was investigated and compared with that of native SAP. Atherosclerotic intima was sequentially extracted with 2 mmol/L CaCl2–Tris-buffered saline (TBS), 10 mmol/L EDTA-TBS, 3 mol/L guanidine-TBS, and collagenase digestion. The character of SAP in each extract was studied with double immunodiffusion, electroimmunoassay, crossed immunoelectrophoresis, and Western immunoblotting. The total amount of SAP in atherosclerotic intima was 190±64 μg/g wet tissue with an SAP-albumin ratio of 1:22.7, which is 44 times higher than the relative plasma ratio of 1:1000. This suggests that SAP is specifically localized in atherosclerotic lesions. SAP from the intima was indistinguishable from plasma or purified SAP with respect to immunological character and molecular weight. However, electrophoretic mobility and the binding of SAP to atherosclerotic intima appeared heterogeneous. Of total extractable SAP, about 43% appeared in the CaCl2-TBS fraction, 25% in the EDTA-TBS fraction, and 32% in the collagenase digestion fraction. SAP is one of the two pentraxins in human plasma; the other is C-reactive protein, which has also been reported to locate in atherosclerotic lesions. Our findings suggest a role for SAP in atherogenesis and encourage efforts to determine more precisely the physiological contributions of the pentraxin family to the development of atherosclerosis.
Supplementation With Low Doses of Vitamin E Protects LDL From Lipid Peroxidation in Men and Women
Authors: Princen Hans M.G. , van Duyvenvoorde Wim , Buytenhek Rien , van der Laarse Arnoud , van Poppel Geert , Leuven Jan A. Gevers , van Hinsbergh Victor W.M.
Published: June 2011

Abstract
There is accumulating evidence that oxidative modification of LDL is an important step in the process of atherogenesis and that antioxidants may protect LDL from oxidation. We and others have previously shown that ingestion of pharmacological doses of the antioxidant
d,l
-α-tocopherol (vitamin E), far above the recommended daily intake (ie, 12 to 15 IU/d for adults), increases the oxidation resistance of LDL. In this study, we ascertained the minimal supplementary dose of vitamin E necessary to protect LDL against oxidation in vitro. Twenty healthy volunteers (10 men and 10 women, aged 21 to 31 years) ingested consecutively 25, 50, 100, 200, 400, and 800 IU/d
d,l
-α-tocopherol acetate during six 2-week periods. No changes were observed in LDL triglyceride content, fatty acid composition of LDL, or LDL size during the intervention. Concentrations of α-tocopherol in plasma and LDL were both 1.2 times the baseline values after the first period (25 IU/d) and 2.6 and 2.2 times, respectively, after the last period (800 IU/d). There was a linear increase in LDL α-tocopherol levels up to an intake of 800 IU/d (
r
=.79,
P
<.0001) and a good correlation between α-tocopherol in plasma and LDL (
r
=.66,
P
<.0001). Simultaneously, the resistance of LDL to oxidation was elevated dose-dependently (+28% after the last period) and differed significantly from the baseline resistance time even after ingestion of only 25 IU/d. Correlation between α-tocopherol content of LDL and resistance time for all data was
r
=.57 (
P
<.0001), whereas the correlation between plasma α-tocopherol and resistance time was
r
=.69 (
P
<.0001). The rate of oxidation was decreased significantly at 400 and 800 IU/d (−13% and −17%, respectively). Baseline resistance time was not significantly different between men and women, but propagation rate was higher with LDL from men at baseline and after intake of 25 and 50 IU/d. Minor differences in LDL vitamin E levels and resistance time were found between men and women in response to vitamin E intake. Statistical evaluation of the relations between α-tocopherol content of LDL and resistance time in each of the 20 individual subjects showed strong and significant correlations for 14 individuals, indicating that vitamin E was the most important parameter that determined the oxidation resistance of LDL in these subjects. ANOVA indicated that LDL α-tocopherol content (47%) and interindividual variation (39%) were the most prominent parameters that contributed to the total variance in resistance time.
Relation of Vascular Oxidative Stress, α-Tocopherol, and Hypercholesterolemia to Early Atherosclerosis in Hamsters
Authors: Parker Rex A. , Sabrah Talal , Cap Michael , Gill Brian T.
Published: June 2011

Abstract
A model of early atherosclerosis in hamsters with moderate hypercholesterolemia (217 to 271 mg/dL) was established that was highly responsive to exogenous antioxidants. A key feature of this model was elevation of vascular oxidative stress by use of a diet deficient in nutritional antioxidants and supplemented with corn oil (10%) and cholesterol (0.2%, 0.4%, or 0.8%). After 10 weeks on the 0.4% cholesterol diet, mean plasma α-tocopherol levels declined from 5.68±0.30 to 1.27±0.15 μg/mL, while monocyte-macrophage foam cell lesions in the aortic arch, as assayed by video microscopy/image analysis of oil red O–stained histological specimens, increased from undetectable at week 0 to 60 900±5400 μm
2
per specimen at week 10 (mean±SEM, n=36). α-Tocopherol or probucol administered for 10 weeks markedly suppressed LDL oxidation ex vivo and profoundly inhibited aortic foam cell formation. However, the effects of antioxidants on aortic lesions were attenuated at higher plasma cholesterol levels, although LDL oxidation ex vivo was effectively inhibited. With a plasma cholesterol level at ≈250 mg/dL, the maximum effect of α-tocopherol on lesion size reached ≈36% of control value, and the dose for half-maximal effect was ≈10 mg · kg
−1
· d
−1
, which resulted in a plasma α-tocopherol value of ≈20 μg/mL. Under these conditions a linear, inverse correlation of aortic lesion size and plasma α-tocopherol concentration was observed (n=68,
r
=−0.581,
P
<.001). The data demonstrate that LDL oxidation is a significant component of early atherogenesis in this model but suggest that hyperlipidemia can outweigh the therapeutic effectiveness of antioxidants. The high sensitivity of aortic lesion initiation to α-tocopherol in hamsters maintained on moderately hypercholesterolemic diets depleted of endogenous antioxidants demonstrates that vascular oxidative stress can be isolated from other causative factors in an in vivo model of atherosclerosis.
Immunologic Detection and Measurement of Hypochlorite-Modified LDL With Specific Monoclonal Antibodies
Authors: Malle Ernst , Hazell Linda , Stocker Roland , Sattler Wolfgang , Esterbauer Hermann , Waeg Georg
Published: June 2011

Abstract
Oxidation of LDL is thought to contribute to the early stages of atherogenesis. Because myeloperoxidase is present in atherosclerotic lesions and can produce the strong oxidant hypochlorous acid (HOCl), which converts LDL into its high-uptake atherogenic form in vitro, we raised polyclonal and monoclonal antibodies (MoAbs) against HOCl-modified LDL (HOCl-LDL). Characterization of the polyclonal anti-human HOCl-LDL Abs showed that they cross-reacted strongly with 4-hydroxynonenal–, malondialdehyde-, and Cu
2+
-oxidized LDL. Similarly, polyclonal and some monoclonal Abs against aldehyde- and Cu
2+
-modified LDL cross-reacted with HOCl-LDL. In contrast to the polyclonal Abs, two selected hybridoma cell line supernatants containing MoAbs raised against HOCl-LDL (MoAb-A and MoAb-B) did not cross-react with either native LDL or aldehyde- or Cu
2+
-modified LDL. MoAb-A (clone 1B10A11, subtype IgG1κ) recognized an epitope that appeared to be specific for HOCl-LDL and depended on the tertiary structure of the (lipo)protein, as judged by a lack of cross-reactivity with HOCl-modified human and bovine serum albumin and a loss of reactivity associated with lipoprotein denaturation. MoAb-B (clone 2D10G9, subtype IgG2bκ), on the other hand, gave identical titration curves with HOCl-LDL and HOCl-modified albumins, suggesting that this antibody recognized epitopes that are commonly generated on proteins that have been oxidized with HOCl. Thus, MoAb-A and MoAb-B may be useful tools for the investigation of a possible role for HOCl-mediated damage to (lipo)proteins in atherosclerosis and other inflammatory diseases.
Immunosuppressive Agents and Endothelial Repair
Authors: Fyfe Alistair I. , Rosenthal Alan , Gotlieb Avrum I.
Published: June 2011

Abstract
Endothelial denudation at areas of predilection to atherosclerosis is balanced by an active repair process that may be inhibited under conditions of accelerated atherosclerosis. After cardiac transplantation, the accelerated atherosclerotic process that develops may be enhanced by immunosuppressive agents that have nonspecific effects on cell signaling, proliferation, and response to injury. To study subtle effects of cyclosporine A, azathioprine, and 6α-methylprednisolone on normal endothelial repair processes, confluent porcine endothelial monolayers were denuded in the presence of clinically relevant concentrations of these agents. The rate of endothelial wound repair was compared and the effects on cell spreading, proliferation, and the cytoskeleton assessed. 6α-Methylprednisolone at concentrations of 1.25 to 50 μmol/L was associated with a transient 30% to 60% inhibition of endothelial wound repair. This was associated with increased cell size at the wound edge and a delay in centrosomal reorientation toward the wound, without any effect on cell proliferation. Cyclosporine and azathioprine in clinically relevant concentrations did not affect endothelial repair. Thus, corticosteroids transiently inhibit endothelial cytoskeletal alterations that are important in endothelial repair after a denuding injury.
Platelet-Derived Growth Factor Enhances Sp1 Binding to the LDL Receptor Gene
Authors: Basheeruddin Khaja , Li Xiaoli , Rechtoris Carol , Mazzone Theodore
Published: June 2011

Abstract
We have previously demonstrated that growth activation of quiescent cells enhances LDL receptor gene transcription and that the proximal 5′ flanking region of the LDL receptor gene could transduce a platelet-derived growth factor (PDGF) response. This portion of the LDL receptor gene encompasses a previously characterized sterol response element and an adjacent Sp1 binding site. By use of mobility shift analyses we show that PDGF activation of quiescent cells enhances binding of Sp1 to the LDL receptor gene. Transfection analyses indicated that the Sp1 site, but not the sterol response element binding protein site, could confer PDGF responsiveness to a heterologous promoter in quiescent cells. Furthermore, cotransfection of an LDL receptor reporter gene (containing −141 to +35 bp of the LDL receptor gene promoter) along with an expression construct coding for high-level constitutive expression of an Sp1 cDNA led to marked enhancement in expression of the LDL receptor reporter gene in quiescent cells. Increased Sp1 binding due to PDGF could be due to enhanced production of Sp1; alternatively, posttranslational activation of binding could be involved. Western blot analysis showed no difference in Sp1 abundance in quiescent cells versus PDGF-stimulated cells, suggesting a posttranslational mechanism for activation of Sp1 binding by growth induction. Our data demonstrate that PDGF stimulation of quiescent cells leads to enhanced Sp1 binding to the LDL receptor gene. This enhanced binding could participate in PDGF induction of LDL receptor gene transcription.
Alterations of Bone Matrix Protein mRNA Expression in Rat Aorta In Vitro
Authors: Hao Hiroyuki , Hirota Seiichi , Tsukamoto Yoshitane , Imakita Masami , Ishibashi-Ueda Hatsue , Yutani Chikao
Published: June 2011

AbstractWe examined the expression of matrix Gla protein (MGP), osteopontin (OPN), and osteonectin (ON) mRNAs in aortic rings excised from 3-month-, 10-month-, and 2-week-old rats during 72-hour incubations in serum-free media. In the aortic rings from 3-month-old rats, the expression of MGP mRNA was strong before incubation and increased during the 72-hour incubation. The expression of OPN mRNA was first detected after a 5-hour incubation and increased thereafter, and that of ON mRNA was strong before the incubation and decreased during the incubation. The expression of MGP and OPN mRNAs in 10-month- and 2-week-old rats was similar to that in 3-month-old rats. In contrast, expression of ON mRNA in 10-month-old rats and the expression of ON mRNA in 2-week-old rats was stronger than that in 3-month-old rats at every incubation period. In situ hybridization and immunohistochemistry identified the MGP, OPN, and ON mRNA–expressing cells as vascular smooth muscle cells. These results suggest that the expression of these mRNAs was regulated in incubation time–dependent and age-specific ways. We believe that this organ culture model is useful for further studies of the function of these bone matrix proteins and regulation of their expression in the vessel wall.
Peripheral Vascular Alterations During Experimental Heart Failure in the Rat
Authors: Heeneman Sylvia , Leenders Peter J. A. , Aarts Petra J. J. W. , Smits Jos F. M. , Arends Jan Willem , Daemen Mat J. A. P.
Published: June 2011

Abstract
Structural changes of the peripheral vascular component as seen during hypertension and atherosclerosis have been suggested during heart failure but have never been reported. Therefore, we studied possible structural alterations in the peripheral vasculature in an experimental model of heart failure, induced by ligation of the left coronary artery in rats. Large conduit and resistance-type arteries were excised at 1, 3, 5, and 12 weeks after myocardial infarct induction (MI) or sham surgery. Vessel dimensions (medial cross-sectional area [CSA], internal and external diameters, and media-to-lumen ratios) as well as medial collagen and elastin volume fractions were measured by computerized morphometry. The hydroxyproline assay was used to determine collagen and elastin content biochemically. In separate groups of animals, peripheral tissue flows were measured by using radioactive microspheres 5 and 12 weeks after MI. To evaluate the effects of the degree of heart failure, the animals of the 12-week group (n=10) were subdivided into groups of moderate (<45% infarct size) and large (>45% infarct size) infarction. At all time points, body weights of sham-operated and MI rats were comparable. Lung weights of infarcted animals were increased proportionally to infarct size. No major changes in vessel dimensions were seen at the earlier time points. Twelve weeks after coronary artery ligation, significantly smaller CSAs were observed in several large conduit arteries such as the thoracic aorta, carotid artery, and superior mesenteric artery. These changes coincided with reductions in both internal and external diameters. In contrast, internal and external diameters of mesenteric and pulmonary resistance arteries were increased after 12 weeks of coronary artery ligation. The medial CSAs of these resistance arteries showed a tendency to increase. Collagen and elastin volume fractions of the large conduit arteries were comparable in both sham-operated and MI animals during the entire experimental period. This latter observation was confirmed by the results of the hydroxyproline assay, which showed no differences in collagen and elastin content. In general, no major changes could be observed in the absolute blood flow to peripheral tissues measured in resting conditions at 5 and 12 weeks after coronary artery ligation. The data indicate that MI induces diverse adaptive changes in the vascular system. Medial CSAs and internal and external diameters of large conduit arteries of MI-operated animals are smaller 12 weeks after infarction. These parameters show a tendency to increase in resistance-type arteries.
Genotype Distribution of Angiotensin-Converting Enzyme Polymorphism in Australian Healthy and Coronary Populations and Relevance to Myocardial Infarction and Coronary Artery Disease
Authors: Wang X.L. , McCredie R.M. , Wilcken D.E.L.
Published: June 2011

AbstractAngiotensin-converting enzyme is a key component of the renin-angiotensin system that plays an important role in cardiovascular regulation. An association between the angiotensin-converting enzyme insertion/deletion (I/D) polymorphism and increased coronary risk has been found in some studies but not in others. To explore this further in an Australian white population, we compared the ACE genotype distribution in 550 patients aged 37 to 65 years with coronary artery disease documented by angiography with the genotype distribution in 404 healthy school children aged 6 to 13 years. We also explored associations in the patients between the angiotensin-converting enzyme I/D polymorphism and a history of myocardial infarction and coronary artery disease severity assessed by the number of major coronary arteries with more than 50% luminal obstructions and by the Green Lane coronary score. The frequencies of the angiotensin-converting enzyme genotype in the coronary artery disease patients were 0.236 for I/I, 0.395 for I/D, and 0.369 for D/D genotypes. This distribution with an excess of the D/D genotype was significantly different (χ2=23.69,P<.0001) from that in the school children, in whom the genotype distribution was in Hardy-Weinberg equilibrium (I/I, 0.21; I/D, 0.54; D/D, 0.25). There was also a significant excess of D/D genotype among patients with a history of myocardial infarction (χ2=9.42,P=.009), and there was the same D/D excess in the subgroup of children (n=60) with two or more grandparents who had had coronary artery disease. We found no associations between the angiotensin-converting enzyme polymorphism and the number of significantly stenosed coronary arteries (χ2=2.069,P=.91). We conclude that the D/D genotype is a significant predictor for coronary artery disease events in the Australian white population but is not a marker for angiographically assessed coronary artery disease severity. The angiotensin-converting enzyme genotype–associated increased risk for coronary events may be mediated more by angiotensin II–induced coronary vasoconstriction than by an increase in injury-related smooth muscle cell proliferation in the coronary vasculature.
Regulation of Vascular Smooth Muscle Cell Migration and Proliferation In Vitro and in Injured Rat Arteries by a Synthetic Matrix Metalloproteinase Inhibitor
Authors: Zempo Nobuya , Koyama Noriyuki , Kenagy Richard D. , Lea Holly J. , Clowes Alexander W.
Published: June 2011

AbstractSmooth muscle cell (SMC) migration and proliferation and extracellular matrix remodeling are essential aspects of the arterial response to injury, vessel development, and atherogenesis. Matrix metalloproteinase (MMP) expression is associated with SMC proliferation and migration after arterial injury. To assess the role of MMPs in SMC proliferation and migration and intimal thickening, we measured the effect of the synthetic MMP inhibitor BB94 (Batimastat) on DNA synthesis and migration of SMCs in vitro as well as the formation of a neointima after balloon injury to the rat carotid artery. BB94 dose-dependently inhibited SMC migration induced by platelet-derived growth factor (PDGF)–BB through a filter coated with a thick basement membrane matrix (Matrigel) layer but did not show any inhibitory effect on SMC migration through a lightly coated filter. At concentrations up to 1 μmol/L, BB94 did not alter DNA synthesis induced by PDGF-AA or PDGF-BB. Treatment with 30 mg BB94·kg−1·d−1IP for 7 or 14 days after balloon injury to the rat carotid artery decreased the total number of intimal SMC nuclei and suppressed intimal thickening. SMC proliferation (5-bromo-2′-deoxyuridine labeling) was decreased in the media at 2 days, whereas it was increased in the intima at 7 but not 14 days. These results suggest that BB94 inhibits intimal thickening after arterial injury by decreasing SMC migration and proliferation and support the conclusion that MMPs play a significant role in regulating intimal thickening in injured arteries.
Regression or Progression
Authors: Candipan Robert C. , Wang Bing-yin , Buitrago Ricardo , Tsao Philip S. , Cooke John P.
Published: June 2011

AbstractWe have shown that chronic administration of the nitric oxide (NO) precursorl-arginine inhibits atherogenesis in the hypercholesterolemic rabbit. However, the effect of supplemental arginine on preexisting lesions is not known and was the focus of the present study. New Zealand White rabbits received normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently,l-arginine (2.25% in drinking water; ARG group) or vehicle (CHOL group) was administered for an additional 13 weeks, while the high-cholesterol diet was continued. Thoracic aortae were harvested at weeks 10, 14, 18, or 23. Rings of aorta were used to assess NO-dependent vasodilation to acetylcholine. Maximal relaxation to acetylcholine in the CHOL rabbits became progressively attenuated from 53.4% (at week 10) to 17.4% (by week 23). Planimetry of the luminal surface of the aortae from CHOL animals revealed a progressive increase in lesion surface area from 30.3% (at week 10) to 56.5% (by week 23). By contrast, animals in the ARG groups manifested improved endothelium-dependent relaxation associated with a reduction of lesion surface area at 14 and 18 weeks. The arginine-induced improvement in endothelium-dependent relaxation was associated with an increased generation of vascular NO and a reduced generation of vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had persistent improvement in NO-dependent vasodilation and exhibited a further reduction of lesion surface area to 5.4%. We conclude that hypercholesterolemia induces a progressive loss of NO-dependent vasodilation associated with progressive intimal lesion formation. Administration ofl-arginine to animals with preexisting intimal lesions augments vascular NO elaboration, reduces superoxide anion generation, and is associated with a reduction in lesion surface area. This is the first demonstration that restoration of NO activity can induce regression of preexisting intimal lesions and provides evidence thatl-arginine therapy may be of potential clinical benefit.
Plasminogen Activator Inhibitor Type-1 Synthesis and mRNA Expression in HepG2 Cells Are Regulated by VLDL
Authors: Sironi Luigi , Mussoni Luciana , Prati Livia , Baldassarre Damiano , Camera Marina , Banfi Cristina , Tremoli Elena
Published: June 2011

Abstract
The effect of VLDL on plasminogen activator inhibitor type 1 biosynthesis in HepG2 cells was investigated. Exposure of HepG2 cells to VLDL (range, 10 to 100 μg protein per milliliter) for 16 hours resulted in an enhanced release of PAI-1 antigen and PAI activity into conditioned medium, accompanied by the accumulation of intracellular triglycerides. By using a monoclonal antibody (IgG C7) specific to the LDL receptor, we showed that the effect of VLDL is mediated by its interaction with the LDL receptor. Enhanced PAI-1 release was due to increased biosynthesis: PAI-1 mRNA was doubled, mainly because of the effect on the 2.2-kb PAI-1 mRNA rather than the 3.2-kb transcript. Addition of insulin with the VLDL further enhanced PAI-1 antigen release and PAI-1 mRNA accumulation. The effect of VLDL on steady state levels of PAI-1 mRNA was apparently not due to an increase of gene transcription but to stabilization of both PAI-1 mRNA transcripts. The enhancing effect of VLDL on PAI-1 biosynthesis in HepG2 cells may raise PAI-1 antigen levels not only in hypertriglyceridemic states but also in those conditions in which both insulin and VLDL are elevated.
Hypercholesterolemia in the Rabbit Induced by Feeding Graded Amounts of Low-Level Cholesterol
Authors: Kolodgie Frank D. , Katocs Andrew S. , Largis Elwood E. , Wrenn Simeon M. , Cornhill J. Fredrick , Herderick Edward E. , Lee Sue J. , Virmani Renu
Published: June 2011

While a number of studies have presented detailed examinations of lesion development in the cholesterol-fed rabbit, individual variability in response to cholesterol feeding and type of lesion produced relative to the degree of cholesterol exposure is not well defined. This study analyzed such critical parameters in an attempt to further characterize the model and establish a baseline for future testing of treatments targeted at limiting atherosclerosis. For these experiments, male New Zealand White rabbits were fed atherogenic diets consisting of 0.05%, 0.10%, 0.15%, 0.20%, or 0.25% cholesterol dissolved in 6% peanut oil for 31 to 32 weeks. Raising dietary cholesterol from 0.05% to 0.15% resulted in a less than twofold stepwise increase in total plasma cholesterol (TPC) exposure (area under plasma cholesterol versus time curve), whereas further increases in cholesterol intake resulted in an exponential four- to fivefold increase in TPC exposure. Regression analysis of TPC exposure with aortic sudanophilia demonstrated a threshold of ≈5000 cholesterol weeks; below this limit lesions were minimal, and above this value, the degree of plaque correlated with TPC exposure. Furthermore, a wide biological variability occurred among rabbits with respect to individual responsiveness to dietary cholesterol. In the aorta, various types of plaques, from fatty streaks to atheromatous lesions, were observed, depending on the degree of cholesterol intake. Diets consisting of <0.15% cholesterol resulted in the development of fatty streak lesions, while transitional lesions and atheromatous plaques were mostly found with higher cholesterol feeding. Coronary artery atherosclerosis was present in >50% of animals fed diets ≥0.15% cholesterol. Despite the level of TPC exposure, coronary lesions in epicardial vessels were generally the fibrous type, whereas intramyocardial arteries demonstrated predominantly intimal foam cells. In conclusion, by adjusting dietary cholesterol intake and selecting rabbits with a similar responsiveness to cholesterol, the overall cholesterol exposure can be more closely controlled to minimize the inherent individual variability among animals in this model. The nature of the target lesion must also be carefully considered, because the efficacy of some treatments may depend on the type of atherosclerotic plaque.