bluuebunny/crossref_metadata_2025 huggingface dataset:
A Quantitative Trait Locus on Chromosome 16q Influences Variation in Plasma HDL-C Levels in Mexican Americans
Authors: Mahaney M.C. , Almasy L. , Rainwater D.L. , VandeBerg J.L. , Cole S.A. , Hixson J.E. , Blangero J. , MacCluer J.W.
Published: February 2003

Objective—

We conducted a whole-genome, multipoint linkage screen to localize a previously reported major locus accounting for 56% to 67% of the additive genetic effects on covariate-adjusted plasma HDL cholesterol (HDL-C) levels in Mexican Americans from the San Antonio Family Heart Study (SAFHS).

Methods and Results—

After using complex segregation analysis to recover the major locus in 472 SAFHS participants from 10 genotyped families, we incorporated covariates required to detect that major locus, including plasma levels of triglycerides and apolipoprotein A-I, in a maximum-likelihood-based variance-components linkage screen. Only chromosome 16 exhibited convincing evidence for a quantitative trait locus (QTL), with a peak multipoint log of the odds (LOD)=3.73 (
P
=0.000034). Subsequent penetrance model-based linkage analysis, incorporating genotypes at the marker locus nearest the multipoint peak (D16S518) into the segregation model, detected linkage with the previously detected major locus (LOD=2.73,
P
=0.000642). Initial estimates place this QTL within a 15-cM region of chromosome 16q near the structural loci for lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP).

Conclusions—

A QTL influencing plasma levels of HDL-C in Mexican Americans from San Antonio maps to a region of human chromosome 16q near
LCAT
and
CETP
.
Phospholipid Hydroxyalkenals
Authors: Hoff Henry F. , O’Neil June , Wu Zhiping , Hoppe George , Salomon Robert L.
Published: February 2003

Objective—

Phosphatidylcholine hydroxyalkenals (PC-HAs) are a class of oxidized PCs derived from lipid peroxidation of arachidonate or linoleate at the
sn
-2 position to form terminal γ-hydroxy, α-, and β-unsaturated aldehydes. The aim of this study was to characterize some of their biological properties, ascertain the mechanism of their action, and assess whether they have in vivo relevance.

Methods and Results—

Combinations of cell biological approaches with radiolabels, mass spectroscopy, and immunochemical as well as immunohistochemical techniques were used to show that PC-HAs reduce the proteolytic degradation by mouse peritoneal macrophages (MPMs) of internalized macromolecules, such as maleylated bovine serum albumin, and that the activity of the lysosomal protease, cathepsin B, in MPMs form Michael adducts with MPM proteins and with
N
-acetylated cysteine in vitro form pyrrole adducts with MPM proteins and reduce the maturation of Rab5a, thereby impairing phagosome-lysosome fusion (maturation) in phagocytes; they are present unbound and as pyrrole adducts in human atherosclerotic lesions.

Conclusions—

PC-HAs are present in vivo and possess multiple functions characteristic of oxidized LDL and 4-hydroxynonenal.
Macrophage-Expressed Group IIA Secretory Phospholipase A
2
Increases Atherosclerotic Lesion Formation in LDL Receptor–Deficient Mice
Authors: Webb Nancy R. , Bostrom Meredith A. , Szilvassy Stephen J. , van der Westhuyzen Deneys R. , Daugherty Alan , de Beer Frederick C.
Published: February 2003

Objective—

Transgenic mice expressing human group IIA secretory phospholipase A
2
(group IIA sPLA
2
) spontaneously develop atherosclerotic lesions. The mechanism for this proatherogenic effect is likely multifactorial, because HDL-cholesterol is significantly lower and LDL/VLDL cholesterol is slightly higher in transgenic mice compared with nontransgenic littermates. In the present study, we show for the first time that elicited peritoneal macrophages from transgenic mice express human group IIA sPLA
2
. This study tested whether macrophage-expressed sPLA
2
contributes to atherogenesis.

Methods and Results—

Bone marrow cells from either sPLA
2
transgenic mice or control C57BL/6 mice were transplanted into LDL receptor–deficient mice. After hematopoietic engraftment, animals were fed a diet enriched with saturated fat and cholesterol for 12 weeks. Despite a lack of effect on serum lipoprotein concentrations, the presence of bone marrow–derived cells expressing human group IIA sPLA
2
resulted in a significant increase in the extent of atherosclerosis in the aortic arch (12.8±1.4% versus 7.4±0.9%;
P
<0.005) and aortic sinus (0.3±0.03 mm
2
versus 0.2±0.04 mm
2
;
P
<0.05).

Conclusions—

Group IIA sPLA
2
can contribute to atherosclerotic lesion development through a mechanism that is independent of systemic lipoprotein metabolism.
Extensive Association Analysis Between Polymorphisms of PON Gene Cluster With Coronary Heart Disease in Chinese Han Population
Authors: Wang Xiaoling , Fan Zhongjie , Huang Jianfeng , Su Shaoyong , Yu Quanjun , Zhao Jiangong , Hui Rutai , Yao Zhijian , Shen Yan , Qiang Boqin , Gu Dongfeng
Published: February 2003

Objective—An extensive association analysis of PON gene cluster (PONs) with coronary heart disease (CHD) was performed in Chinese Han population.

Methods and Results—Thirty polymorphisms of PON1, PON2, and PON3 gene were identified by direct sequencing of genomic DNA derived from 48 randomly selected patients. Twelve polymorphisms were additionally investigated for association with CHD in 474 male patients and 475 controls. Univariate analyses showed the cases had significantly higher frequencies of PON1 192Q allele, 160R allele, −162A allele, and PON2 311C allele than were seen in the controls. Logistic regression analyses revealed only the PON1 R160G and −162G/A polymorphisms remained significantly associated with CHD (P=0.0054 andP=0.0002). Haplotype analyses for various polymorphism combinations additionally confirmed the results of individual polymorphism analyses. Only the frequencies of haplotypes containing −162A allele were significantly higher, whereas only the frequencies of haplotypes containing 160G allele were significantly lower in cases than in controls in various polymorphism combinations.

Conclusions—This extensive association study has identified the PON1 −162G/A and R160G polymorphisms to be independently associated with CHD in Chinese Han population and warrants additional study to elucidate the biological mechanism.
Systematic Family Screening for Familial Hypercholesterolemia in Iceland
Authors: Thorsson Bolli , Sigurdsson Gunnar , Gudnason Vilmundur
Published: February 2003

Objective—

This study compares a novel approach using systematic family screening for patients in Iceland who have familial hypercholesterolemia (FH) with conventional proband screening and assesses the sensitivity and specificity of diagnosing FH by cholesterol measurements compared with mutational testing of family members.

Methods and Results—

Probands with the I4T+2C mutation were traced to common ancestors. A downtracing of each family lineage was performed back to the oldest living offspring (key individuals); these individuals were recruited for cholesterol measurement and mutation testing. The sensitivity and specificity of cholesterol measurements was assessed against mutational analysis. Eleven probands clustered into 4 families. There were 364 key individuals identified among their descendants. Eighty-four percent responded, and 11% were positive for the mutation. There were 78 offspring of the positive key individuals, and 40 of those were carriers. Compared with use of the conventional first-degree relative approach, an additional 19% of FH individuals, including key individuals and their descendants, were identified. As diagnostic criteria, cholesterol measurements in the families had 95% specificity and 94% sensitivity.

Conclusions—

Tracing FH probands to common ancestors and screening the oldest offspring in each family lineage adds considerably to the conventional method of FH screening (testing first-degree relatives). This may have relevance in other founder populations.
ACE- Versus Chymase-Dependent Angiotensin II Generation in Human Coronary Arteries
Authors: Tom Beril , Garrelds Ingrid M. , Scalbert Elizabeth , Stegmann Alexander P.A. , Boomsma Frans , Saxena Pramod R. , Danser A.H. Jan
Published: February 2003

Objective—

The objective of this study was to investigate ACE- and chymase-dependent angiotensin I-to-II conversion in human coronary arteries (HCAs).

Methods and Results—

HCA rings were mounted in organ baths, and concentration-response curves to angiotensin II, angiotensin I, and the chymase-specific substrate Pro
11
-
d
-Ala
12
–angiotensin I (PA–angiotensin I) were constructed. All angiotensins displayed similar efficacy. For a given vasoconstriction, bath (but not interstitial) angiotensin II during angiotensin I and PA–angiotensin I was lower than during angiotensin II, indicating that interstitial (and not bath) angiotensin II determines vasoconstriction. PA–angiotensin I increased interstitial angiotensin II less efficiently than angiotensin I. Separate inhibition of ACE (with captopril) and chymase (with C41 or chymostatin) shifted the angiotensin I concentration-response curve ≈5-fold to the right, whereas a 10-fold shift occurred during combined ACE and chymase inhibition. Chymostatin, but not captopril and/or C41, reduced bath angiotensin II and abolished PA–Ang I–induced vasoconstriction. Perfused HCA segments, exposed luminally or adventitially to angiotensin I, released angiotensin II into the luminal and adventitial fluid, respectively, and this release was blocked by chymostatin.

Conclusions—

Both ACE and chymase contribute to the generation of functionally active angiotensin II in HCAs. However, because angiotensin II loss in the organ bath is chymase-dependent, ACE-mediated conversion occurs more efficiently (ie, closer to AT
1
receptors) than chymase-mediated conversion.
Disparity of MCP-1 mRNA and Protein Expressions Between the Carotid Artery and the Aorta in WHHL Rabbits
Authors: Tanaka Eriko , Shimokawa Hiroaki , Kamiuneten Hitoshi , Eto Yasuhiro , Matsumoto Yasuharu , Morishige Kunio , Koike George , Yoshinaga Masaru , Egashira Kensuke , Tokunaga Osamu , Shiomi Masashi , Takeshita Akira
Published: February 2003

Objective—This study was designed to examine why in WHHL rabbits, muscular arteries, such as the carotid artery, are relatively resistant to atherosclerosis compared with the aorta, with a special reference to monocyte chemoattractant protein (MCP)-1.

Methods and Results—MCP-1 mRNA expression was quantitated by Northern blot analysis, and its protein expression was quantitated by immunostaining and ELISA at the age of 1, 3, 6, and 12 months (n=5 to 6 each). In the aorta, atherosclerotic lesions were progressively developed with aging, and MCP-1 was highly expressed in endothelial cells and infiltrating macrophages. By contrast, in the carotid artery, atherosclerotic lesions and MCP-1 immunoreactivity were not evident throughout the experimental period. Unexpectedly, however, the extent of MCP-1 mRNA expression was comparable between the aorta and the carotid artery throughout the experimental period. Endothelial cells in primary culture from the aorta and the carotid artery expressed the same extent of MCP-1 mRNA on stimulation by oxidized LDL. There was no abnormality in primary structure of MCP-1 cDNA in WHHL.

Conclusions—These results suggest that in WHHL, the atherosclerosis process, including MCP-1 protein expression, may be reduced in the carotid artery (and possibly in other muscular arteries), accounting in part for the regional resistance to atherosclerosis.
Blockade of the Platelet P2Y
12
Receptor by AR-C69931MX Sustains Coronary Artery Recanalization and Improves the Myocardial Tissue Perfusion in a Canine Thrombosis Model
Authors: Wang Kai , Zhou Xiaorong , Zhou Zhongmin , Tarakji Khaldoun , Carneiro Marcelo , Penn Marc S. , Murray Daniel , Klein Allan , Humphries Robert G. , Turner Jonathan , Thomas James D. , Topol Eric J. , Lincoff A. Michael
Published: February 2003

Objective—

Reperfusion therapy for myocardial infarction is limited by a significant reocclusion rate and less optimal myocardial tissue perfusion due to excessive platelet accumulation and recruitment at the sites of vascular injury. We assessed the influence of a selective P2Y
12
-receptor antagonist (AR-C69931MX), in conjunction with thrombolytic therapy, on the prevention of platelet aggregation and thrombus formation.

Methods and Results—

A canine coronary electrolytic injury thrombosis model was used. Tissue-type plasminogen activator (t-PA; 1 mg/kg in phase I, 0.5 mg/kg in phase II in the AR-C69931MX group, and 1 mg/kg in the placebo group in phase I and II) was administered 30 minutes after thrombus formation; either saline or AR-C69931MX (4 μg · kg
−1
· min
−1
) was given to all animals intravenously 10 minutes before t-PA administration for a total of 2 hours. All animals received heparin (80 U/kg) as an intravenous bolus followed by a continuous infusion of 17 U · kg
−1
· h
−1
. Myocardial tissue perfusion was evaluated by use of the colored microsphere technique and real-time myocardial contrast echocardiography. The incidences of reocclusion and cyclic flow variation were significantly decreased in the AR-C69931MX group (
P
<0.05). Myocardial tissue flow with AR-C69931MX treatment improved significantly at 20 and 120 minutes after reflow, whereas tissue flow with placebo remained at a level similar to that during occlusion (
P
<0.05).

Conclusions—

The adjunctive administration of AR-C69931MX blocked ADP-mediated platelet aggregation and recruitment and prevented platelet-mediated thrombosis, resulting in prolongation of reperfusion time and a decrease in reocclusion and cyclic flow variations. Importantly, myocardial tissue perfusion was significantly improved in the P2Y
12
antagonist group.
HEX Acts as a Negative Regulator of Angiogenesis by Modulating the Expression of Angiogenesis-Related Gene in Endothelial Cells In Vitro
Authors: Nakagawa Tomowaki , Abe Mayumi , Yamazaki Tohru , Miyashita Hiroki , Niwa Hitoshi , Kokubun Shoichi , Sato Yasufumi
Published: February 2003

Objective—

The hematopoietically expressed homeobox (HEX) is transiently expressed in endothelial cells (ECs) during vascular formation in embryo. Here, we investigated whether HEX played any role in angiogenesis-related properties of ECs in vitro.

Methods and Results—

We transiently overexpressed HEX in human umbilical vein ECs (HUVECs). To our surprise, HEX completely abrogated the response of HUVECs to vascular endothelial growth factor (VEGF) with regard to proliferation, migration, and invasion and abolished network formation by HUVECs on Matrigel. cDNA microarray analysis and quantitative real-time reverse transcription–polymerase chain reaction combined with Western blotting revealed that HEX significantly repressed the expression of VEGF receptor-1, VEGF receptor-2, neuropilin-1, tyrosine kinase with Ig and EGF homology domains (TIE)-1, TIE-2, and the integrin αv subunit, whereas it augmented the expression of endoglin in HUVECs. We established murine embryonic stem cells that were stably transfected with HEX sense cDNA or antisense cDNA, and we examined the in vitro differentiation to ECs. Although the expression of VEGF receptor-2 was decreased in sense transfectants, the number of cells expressing VE-cadherin, a specific marker of ECs, was not altered.

Conclusions—

Our present results suggest that HEX may not affect the differentiation of ECs but acts as a negative regulator of angiogenesis.
Lysophosphatidylcholine Inhibits Endothelial Cell Migration by Increasing Intracellular Calcium and Activating Calpain
Authors: Chaudhuri Pinaki , Colles Scott M. , Damron Derek S. , Graham Linda M.
Published: February 2003

Objective—

Endothelial cell (EC) migration, essential for reestablishing arterial integrity after vascular injury, is inhibited by oxidized LDL (oxLDL) and lysophosphatidylcholine (lysoPC) that are present in the arterial wall. We tested the hypothesis that a mechanism responsible for lysoPC-induced inhibition is increased intracellular free calcium concentration ([Ca
2+
]
i
).

Methods and Results—

LysoPC, at concentrations that inhibit in vitro EC migration to 35% of control, increased [Ca
2+
]
i
levels 3-fold. These effects of lysoPC were concentration dependent and reversible. LysoPC induced Ca
2+
influx within 10 minutes, and [Ca
2+
]
i
remained elevated for 2 hours. The calcium ionophore A23187 also increased [Ca
2+
]
i
and inhibited EC migration. Chelators of intracellular Ca
2+
(BAPTA/AM and EGTA/AM) and nonvoltage-sensitive channel blockers (lanthanum chloride and gadolinium chloride) blunted the lysoPC-induced [Ca
2+
]
i
rise and partially preserved EC migration. After lysoPC treatment, calpain, a calcium-dependent cysteine protease, was activated, and cytoskeletal changes occurred. Calpain inhibitors (calpastatin, MDL28170, and calpeptin) added before lysoPC prevented cytoskeletal protein cleavage and preserved EC migration at 60% of control levels.

Conclusions—

LysoPC increases [Ca
2+
]
i
. In turn, activating calpains that can alter the cytoskeleton are activated and EC migration is inhibited.
Role of p44/p42 MAP Kinase in the Age-Dependent Increase in Vascular Smooth Muscle Cell Proliferation and Neointimal Formation
Authors: Gennaro Giuseppa , Ménard Catherine , Giasson Edith , Michaud Sophie-Élise , Palasis Maria , Meloche Sylvain , Rivard Alain
Published: February 2003

Objective—

Age-dependent increase in vascular smooth muscle cell (VSMC) proliferation is thought to contribute to the pathology of atherosclerotic diseases. In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) on VSMC proliferation and neointimal formation in the context of aging.

Methods and Results—

VSMCs were isolated from the aorta of young and old rabbits. The proliferative index after serum stimulation was significantly increased in old versus young VSMCs. This was associated with a significant and specific age-dependent increase in p44/p42 MAPK activation. Treatment with MEK inhibitor PD98059 successfully inhibited p44/p42 MAPK activities and VSMC proliferation. These results were confirmed in vivo using a model of balloon injury in rabbit iliac arteries. p44/p42 MAPK activities were rapidly induced by angioplasty in young and old animals. However, the levels of p44/p42 MAPK activities achieved in arteries of old rabbits were significantly higher than those of young rabbits. This was associated with a higher cellular proliferative index and a significant increase in neointimal formation in old animals. Local delivery of PD98059 in old rabbits successfully inhibited p44/p42 MAPK activities after angioplasty, which led to a significant reduction in cellular proliferation and neointimal formation in treated animals.

Conclusions—

Our study suggests for the first time that increased p44/p42 MAPK activation contributes to augmented VSMC proliferation and neointimal formation with aging. p44/p42 MAPK inhibition could represent a novel therapeutic avenue against atherosclerotic diseases.
A Novel Nonobstructive Intravascular MRI Coil
Authors: Worthley Stephen G. , Helft Gérard , Fuster Valentin , Fayad Zahi A. , Shinnar Meir , Minkoff Lawrence A. , Schechter Clyde , Fallon John T. , Badimon Juan J.
Published: February 2003

Objective—

MRI is being used to characterize the composition of atherosclerotic plaques. However, the resolution achievable using surface radiofrequency coils is limited by the signal to noise ratio. We studied the utility of a new intravascular (IV) MRI probe for high-resolution in vivo imaging of atherosclerotic lesions.

Methods and Results—

Balloon-injured Watanabe heritable hyperlipidemic rabbits served as the experimental model of atherosclerosis. The newly developed IV MRI probe is 1.3 mm in diameter and can be positioned over a guidewire. MRI was performed with both an external phased-array coil and the IV MR coil. MR observations were correlated with histopathology. After MRI, the animals were killed and analysis of agreement between MR and histopathology was performed. The IV MR coil allows aortic images to be obtained with 156×156 μm
2
in-plane resolution versus 352×352 μm
2
when used with the external phased-array coil. No significant motion artifacts were noted, despite the continuation of arterial blood flow during image acquisition around the IV probe. The different components of the atherosclerotic lesions (lipid core and fibrous cap) were easily identified. There was an excellent agreement between MRI with the IV coil and histopathology by simple linear regression for both the mean wall thickness (
r
=0.88, slope 0.82,
P
<0.0001) and vessel wall area (
r
=0.86, slope 1.08,
P
<0.0001).

Conclusions—

The new nonobstructive design for this intravascular coil provides great promise for additional work in high-resolution MRI characterization of atherosclerotic plaques in vivo. The ability to position the probe with a guidewire allows its placement under fluoroscopic or MRI guidance, whereas its size is compatible with human coronary arteries.
Smallest LDL Particles Are Most Strongly Related to Coronary Disease Progression in Men
Authors: Williams Paul T. , Superko H. Robert , Haskell William L. , Alderman Edwin L. , Blanche Patricia J. , Holl Laura Glines , Krauss Ronald M.
Published: February 2003

Objective—

LDLs include particle subclasses that have different mobilities on polyacrylamide gradient gels: LDL-I (27.2 to 28.5 nm), LDL-IIa (26.5 to 27.2 nm), LDL-IIb (25.6 to 26.5 nm), LDL-IIIa (24.7 to 25.6 nm), LDL-IIIb (24.2 to 24.7 nm), LDL-IVa (23.3 to 24.2 nm), and LDL-IVb (22.0 to 23.3 nm in diameter). We hypothesized that the association between smaller LDL particles and coronary artery disease (CAD) risk might involve specific LDL subclasses.

Methods and Results—

Average 4-year onstudy lipoprotein measurements were compared with annualized rates of stenosis change from baseline to 4 years in 117 men with CAD. The percentages of total LDL and HDL occurring within individual subclasses were measured by gradient gel electrophoresis. Annual rate of stenosis change was related concordantly to onstudy averages of total cholesterol (
P
=0.04), triglycerides (
P
=0.05), VLDL mass (
P
=0.03), total/HDL cholesterol ratio (
P
=0.04), LDL-IVb (
P
=0.01), and HDL
3a
(
P
=0.02) and inversely to HDL
2
-mass (
P
=0.02) and HDL
2b
(
P
=0.03). The average annual rate in stenosis change was 6-fold more rapid in the fourth quartile of LDL-IVb (≥5.2%) than in the first quartile (<2.5%,
P
=0.03). Stepwise multiple regression analysis showed that LDL-IVb was the single best predictor of stenosis change.

Conclusions—

LDL-IVb was the single best lipoprotein predictor of increased stenosis, an unexpected result, given that LDL-IVb represents only a minor fraction of total LDL.
Molecular Basis of Cell Membrane Estrogen Receptor Interaction With Phosphatidylinositol 3-Kinase in Endothelial Cells
Authors: Simoncini Tommaso , Rabkin Elena , Liao James K.
Published: February 2003

Objective—

Nontranscriptional signaling mechanisms mediate some of the biological effects of estrogen, such as the rapid actions on the blood vessels. By interacting with phosphatidylinositol 3-kinase (PI3K), estrogen receptor (ER) α leads to activation of protein kinase Akt and to subsequent increase in endothelial nitric oxide synthase activity. Because PI3K is mainly a cytoplasmic complex, we studied the cellular site of interaction between this enzyme and ERα, and we dissected the molecular mechanisms that mediate this interaction.

Methods and Results—

By using cultured human saphenous vain endothelial cells, we found that cell membrane–bound ERα colocalizes with PI3K and may be responsible for PI3K activation. Furthermore, we characterized the subsequent steps in the activation of the PI3K/Akt signaling cascade, comparing the molecular events that follow insulin or estradiol activation of PI3K.

Conclusions—

We provide novel evidence for an important role of nonnuclear estrogen receptor in rapid, nontranscriptional responses of human endothelial cells to estrogen.
Antidiabetic PPARγ-Activator Rosiglitazone Reduces MMP-9 Serum Levels in Type 2 Diabetic Patients With Coronary Artery Disease
Authors: Marx Nikolaus , Froehlich Johannes , Siam Laila , Ittner Jochen , Wierse Gerhard , Schmidt Arnold , Scharnagl Hubert , Hombach Vinzenz , Koenig Wolfgang
Published: February 2003

Background—

Matrix metalloproteinases (MMPs) are critically involved in the development of unstable plaques. Although arteriosclerotic lesions in patients with diabetes mellitus are more unstable than those of nondiabetic subjects, nothing is known about serum levels of MMPs in these patients or about mechanisms to modulate MMP levels. We investigated MMP levels in diabetic and nondiabetic coronary artery disease (CAD) patients and performed a clinical trial to assess the effect of the PPARγ-activating, antidiabetic thiazolidinedione rosiglitazone on MMP levels in diabetic CAD patients.

Methods and Results—

In CAD patients, MMP-2, -8, and -9 serum levels were significantly higher in type 2 diabetic subjects compared with age-, sex-, and body mass index–matched nondiabetics. Thirty-nine diabetic patients with CAD were randomized to receive rosiglitazone 4 mg (twice daily) or placebo for 12 weeks. Rosiglitazone treatment, but not placebo, significantly reduced MMP-9 levels already after 2 weeks by −19.6% (−38.3% to 8.6%,
P
<0.05), and levels remained suppressed until the end of the study. In addition, rosiglitazone significantly decreased serum amyloid A (SAA) and tumor necrosis factor-α levels.

Conclusion—

MMP-9 levels are increased in type 2 diabetic patients with CAD, and treatment of these patients with the antidiabetic PPARγ-activator rosiglitazone significantly reduces MMP-9, tumor necrosis factor-α, and SAA serum levels. These data support anti-inflammatory and potential antiatherogenic effects of thiazolidinediones.
Involvement of RhoA/Rho Kinase Signaling in VEGF-Induced Endothelial Cell Migration and Angiogenesis In Vitro
Authors: van Nieuw Amerongen Geerten P. , Koolwijk Pieter , Versteilen Amanda , van Hinsbergh Victor W.M.
Published: February 2003

Objective—

Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced signaling pathways, but its role has not been clarified.

Methods and Results—

VEGF induced the activation of RhoA and recruited RhoA to the cell membrane of human ECs. This increase in RhoA activity is necessary for the VEGF-induced reorganization of the F-actin cytoskeleton, as demonstrated by adenoviral transfection of dominant-negative RhoA. Rho kinase mediated this effect of RhoA, as was demonstrated by the use of Y-27632, a specific inhibitor of Rho kinase. Inhibition of Rho kinase prevented the VEGF-enhanced EC migration in response to mechanical wounding but had no effect on basal EC migration. Furthermore, in an in vitro model for angiogenesis, inhibition of either RhoA or Rho kinase attenuated the VEGF-mediated ingrowth of ECs in a 3-dimensional fibrin matrix.

Conclusions—

VEGF-induced cytoskeletal changes in ECs require RhoA and Rho kinase, and activation of RhoA/Rho kinase signaling is involved in the VEGF-induced in vitro EC migration and angiogenesis.
Isoform-Dependent Cholesterol Efflux From Macrophages by Apolipoprotein E Is Modulated by Cell Surface Proteoglycans
Authors: Hara Masumi , Matsushima Teruhiko , Satoh Hiroaki , Iso-o Naoyuki , Noto Hiroshi , Togo Masako , Kimura Satoshi , Hashimoto Yoshiaki , Tsukamoto Kazuhisa
Published: February 2003

Objective—

Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression.

Methods and Results—

When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-β-
d
-xyloside (β-DX) resulted in all 3 isoforms’ reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with β-DX resulted in all 3 exogenously derived apoE isoforms’ reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4.

Conclusions—

The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.
Cholesteryl Ester Transfer Protein
Authors: Barter Philip J. , Brewer H. Bryan , Chapman M. John , Hennekens Charles H. , Rader Daniel J. , Tall Alan R.
Published: February 2003

Cholesteryl ester transfer protein (CETP) promotes the transfer of cholesteryl esters from antiatherogenic HDLs to proatherogenic apolipoprotein B (apoB)–containing lipoproteins, including VLDLs, VLDL remnants, IDLs, and LDLs. A deficiency of CETP is associated with increased HDL levels and decreased LDL levels, a profile that is typically antiatherogenic. Studies in rabbits, a species with naturally high levels of CETP, support the therapeutic potential of CETP inhibition as an approach to retarding atherogenesis. Studies in mice, a species that lacks CETP activity, have provided mixed results. Human subjects with heterozygous CETP deficiency and an HDL cholesterol level >60 mg/dL have a reduced risk of coronary heart disease. Evidence that atherosclerosis may be increased in CETP-deficient subjects whose HDL levels are not increased is difficult to interpret and may reflect confounding or bias. Small-molecule inhibitors of CETP have now been tested in human subjects and shown to increase the concentration of HDL cholesterol while decreasing that of LDL cholesterol and apoB. Thus, it seems important and timely to test the hypothesis in randomized trials of humans that pharmacological inhibition of CETP retards the development of atherosclerosis.
HMG-CoA Reductase Inhibitor Increases GTP Cyclohydrolase I mRNA and Tetrahydrobiopterin in Vascular Endothelial Cells
Authors: Hattori Yoshiyuki , Nakanishi Nobuo , Akimoto Kazumi , Yoshida Mika , Kasai Kikuo
Published: February 2003

Objective—

Endothelial nitric oxide synthase (eNOS) activity is supported by tetrahydrobiopterin (BH4), which appears to be important for generating protective NO but decreases uncoupling formation of superoxide. We investigated the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, in terms of BH4 metabolism in human umbilical vein endothelial cells (HUVECs).

Methods and Results—

We measured the mRNA levels of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the first step of de novo BH4 synthesis, by real-time polymerase chain reaction. The mRNA of GTPCH, as well as of eNOS, was upregulated in HUVECs treated with cerivastatin. This increase was time and dose dependent. Fluvastatin was also observed to enhance GTPCH and eNOS mRNA levels. In parallel with this observation, cerivastatin increased intracellular BH4. Incubating HUVECs with tumor necrosis factor (TNF-α) was observed to increase GTPCH mRNA while decreasing eNOS mRNA. In the presence of cerivastatin, the TNF-α–mediated increase in GTPCH mRNA was enhanced, and the TNF-α–mediated decrease in eNOS mRNA was attenuated. Cerivastatin increased the stability of eNOS mRNA. However, it did not alter the stability of GTPCH mRNA but increased GTPCH gene transcription, as shown by nuclear run-on assays. Preteatment of HUVECs with the selective GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, caused a decrease in intracellular BH4 and decreased citrulline formation after stimulation with ionomycin. Furthermore, the potentiating effect of cerivastatin was decreased by limiting the cellular availability of BH4.

Conclusions—

Our data demonstrate that statins elevate GTPCH mRNA, thereby increasing BH4 levels in vascular endothelial cells. In addition to augmenting eNOS expression, statins potentiate GTPCH gene expression and BH4 synthesis, thereby increasing NO production and preventing relative shortages of BH4.
Lysophosphatidic Acid Induction of Tissue Factor Expression in Aortic Smooth Muscle Cells
Authors: Cui Mei-Zhen , Zhao Guojun , Winokur Allison L. , Laag Essam , Bydash Jason R. , Penn Marc S. , Chisolm Guy M. , Xu Xuemin
Published: February 2003

Objective—Tissue factor (TF), the initiator of the coagulation cascade, is expressed by cells in atherosclerotic lesions. Lysophosphatidic acid (LPA) is a component of oxidized lipoproteins and an agent released by activated platelets. The present study investigated whether and how TF expression is regulated by LPA.

Methods and Results—Northern blotting, Western blotting, and TF activity assays demonstrated that LPA markedly induced TF mRNA, protein, and activity in vascular smooth muscle cells. LPA-induced TF expression is primarily controlled at the transcriptional level. Phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signaling–regulated kinases (ERK1/2) was rapidly and markedly induced by LPA. MEK inhibitors U0126 and PD98059 blocked both ERK activation and the increase in TF mRNA. In contrast, the specific p38 MAP kinase inhibitor SB203580 had no effect on LPA-induced TF mRNA increase. The Gαi protein inhibitor, pertussis toxin, abolished LPA-induced phosphorylation of MEKs and ERKs, as well as the induction of TF mRNA.

Conclusions—Our data demonstrate that a Gαi protein and activation of MEKs and ERKs mediate LPA-induced TF expression. Our data suggest that elevated LPA could be a thrombogenic risk factor by upregulating TF expression. These results may have important implications in vascular remodeling and vascular diseases.
Distinct Roles of Ephrin-B2 Forward and EphB4 Reverse Signaling in Endothelial Cells
Authors: Hamada Koichi , Oike Yuichi , Ito Yasuhiro , Maekawa Hiromitsu , Miyata Keishi , Shimomura Taizo , Suda Toshio
Published: February 2003

Objective—

The transmembrane ligand ephrin-B2 and its receptor tyrosine kinase EphB4 are specifically expressed on arterial and venous endothelial cells, respectively, and bidirectional signals mediated by both proteins play an important role in vascular development. However, how such bidirectional signals are required for cell-cell adhesion or repulsion remains unclear.

Methods and Results—

Using a cell line and sorted primary endothelial cells, we show that ephrin-B2 forward signaling through the EphB4 receptor inhibits cell adhesion, whereas EphB4 reverse signaling by the transmembrane ephrin-B2 ligand does not. Cell migration is also inhibited on immobilized ephrin-B2-Fc but not on EphB4-Fc protein.

Conclusions—

Ephrin-B2 forward signaling and EphB4 reverse signaling differentially affect cell adhesion and migration between arterial and venous endothelial cells.
Bicuspid Aortic Valve Associated With Aortic Dilatation
Authors: Nkomo Vuyisile T. , Enriquez-Sarano Maurice , Ammash Naser M. , Melton L. Joseph , Bailey Kent R. , Desjardins Valerie , Horn Robin A. , Tajik A. Jamil
Published: February 2003

Objective—

This study was undertaken to examine the association between bicuspid aortic valve (BAV) and aortic dilatation in the community. The association between BAV and aortic dilatation has been reported predominantly in retrospective studies in patients mostly with valvular dysfunction or selected surgical patients from tertiary referral centers. An independent association of BAV and aortic dilatation in a community-based study has not been demonstrated.

Methods and Results—

In a geographically defined population of Olmsted County, Minnesota, residents with BAV (n=44, age 35±13 years) without hemodynamically significant obstruction or regurgitation and matched controls with normal tricuspid aortic valves were identified by transthoracic echocardiography. The two groups were compared with respect to measurements of the aorta. The BAV and control groups differed with respect to size of the aortic anulus (23.2±2.4 versus 21.6±2.4 mm;
P
=0.002), aortic sinus (33.5±4.6 versus 30.3±4.1 mm;
P
=0.0001), and proximal ascending aorta (33.3±6.5 versus 27.9±3.6 mm;
P
=0.0001). There was no difference in the size of the aortic arch (24.2±3.6 versus 25.3±3.4 mm;
P
=0.16). These differences were maintained when the groups were stratified by sex and blood pressure. The relationship between bicuspid aortic valve and aortic dilatation was maintained when adjusting for factors related to fluid mechanics and hemodynamics such as systolic blood pressure, diastolic blood pressure, left ventricular ejection time, and peak aortic valve velocity.

Conclusions—

In a community-based study, BAV is associated with an alteration of aortic dimensions even in the absence of hemodynamically significant aortic valve stenosis or regurgitation.
Wall Tension Is a Potent Negative Regulator of In Vivo Thrombomodulin Expression
Authors: Sperry Jason L. , Deming Clayton B. , Bian Ce , Walinsky Peter L. , Kass David A. , Kolodgie Frank D. , Virmani Renu , Kim Antony Y. , Rade Jeffrey J.
Published: February 2003

Thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is a major contributor to vascular thromboresistance. We previously found that TM protein expression is dramatically reduced in autologous vein grafts during the first two weeks after implantation, coincident to a local inflammatory response, and remains suppressed for at least 6 weeks. To determine the proximate cause of TM loss, in vivo gene expression was quantified by real-time PCR. TM gene expression in vein grafts declined >85% during the first postoperative week and remained suppressed >55% at 6 weeks, accounting for the observed changes in protein expression. The effects of vein graft inflammation were evaluated in animals rendered leukopenic with vinblastine before graft implantation. Abrogating the local inflammatory response affected neither TM protein nor gene expression. To determine how hemodynamic forces might modulate TM expression, the surgical protocol was modified to alter blood flow and pressure-induced vessel distension. TM protein and gene expression did not correlate to changes in shear stress but highly correlated to changes in wall tension, both acutely and over time. We conclude that the primary stimulus for altered TM expression in vein grafts is the exposure to arterial pressure. Furthermore, these data identify strain as a novel and important pathway for in vivo TM gene regulation.
DNA Microarray Profiling to Identify Angiotensin-Responsive Genes in Vascular Smooth Muscle Cells
Authors: Campos Alexandre H. , Zhao Ying , Pollman Matthew J. , Gibbons Gary H.
Published: February 2003

Angiotensin II (Ang II) induces changes in vessel structure by its capacity to activate genes that are coupled to signaling pathways such as extracellular signal–regulated kinase (ERK), p38, and phosphatidylinositol 3-kinase (PI3K). Using a DNA microarray containing 5088 genes and expressed sequence tags, we initially established a database of replicated experiments (n=4) to define the variances in mRNA expression in response to Ang II versus vehicle treatment. We observed a wide range of values for the coefficients of variation in a gene-specific manner. Guided by power calculations, we used statistical inference on a sufficient number of experimental replicates to minimize the number of false-negatives and define a subset of Ang II–responsive genes (
P
<0.05). To further characterize the molecular circuitry that couples Ang II stimulation with mRNA expression, we assessed expression profiles in the presence and absence of inhibitors of ERK, p38, and PI3K. Using two different methods of computational cluster analysis, we identified a subset of six matricellular proteins (eg, osteopontin and plasminogen activator inhibitor-1) that are coordinately upregulated by Ang II via an ERK/p38-dependent pathway. In addition, these cluster analyses identified calpactins I and II as novel Ang II–responsive genes. Given that Ang II promotes vascular lesion formation, we examined whether this matricellular gene cluster was also coordinately regulated in vivo. Indeed, we demonstrate that both calpactin I and osteopontin are upregulated in response to vascular injury. Taken together, the combined use of DNA microarrays, statistical inference, and cluster analysis identified novel, coordinately regulated Ang II–responsive genes that may mediate vascular lesion formation.
Glucocorticoid Excess Induces Superoxide Production in Vascular Endothelial Cells and Elicits Vascular Endothelial Dysfunction
Authors: Iuchi Takahiko , Akaike Masashi , Mitsui Takao , Ohshima Yasushi , Shintani Yasumi , Azuma Hiroyuki , Matsumoto Toshio
Published: February 2003

Glucocorticoid (GC) excess often elicits serious adverse effects on the vascular system, such as hypertension and atherosclerosis, and effective prophylaxis for these complications is limited. We sought to reveal the mechanism underlying GC-induced vascular complications. Responses in forearm blood flow to reactive hyperemia in 20 GC-treated patients were significantly decreased to 43±8.9% (mean±SEM) from the values obtained before GC therapy (130±14%). An administration of vitamin C almost normalized blood flow responses. In human umbilical vein endothelial cells (HUVECs), production of hydrogen peroxide was increased up to 166.5±3.3% of control values by 10
−7
mol/L dexamethasone (DEX) treatment (
P
<0.01). Concomitant with DEX-induced hydrogen peroxide production, intracellular amounts of peroxynitrite significantly increased and those of nitric oxide (NO) decreased, respectively (
P
<0.01). Immunoblotting analysis using anti-nitrotyrosine antibody showed that peroxynitrite formation was increased in DEX-treated HUVECs. Using inhibitors against metabolic pathways for generation of reactive oxygen species (ROS), we identified that the major production sources of ROS by DEX treatment were mitochondrial electron transport chain, NAD(P)H oxidase, and xanthine oxidase. These findings suggest that GC excess causes overproduction of ROS and thereby perturbs NO availability in the vascular endothelium, leading to vascular complications in patients with GC excess.
Relaxin, a Pregnancy Hormone, Is a Functional Endothelin-1 Antagonist
Authors: Dschietzig Thomas , Bartsch Cornelia , Richter Christoph , Laule Michael , Baumann Gert , Stangl Karl
Published: February 2003

We have recently demonstrated that relaxin (RLX) acts as compensatory mediator in human heart failure. RLX inhibits the stimulation of endothelin-1, the most potent vasoconstrictor in heart failure. Upregulation of the endothelin type-B receptor (ET
B
), which mediates endothelin-1 clearance and endothelial release of NO, represents a pivotal mode of RLX action. However, signal transduction and abundance of this phenomenon are unknown. Therefore, we investigated RLX-induced regulation of ET
B
in human umbilical vein endothelial, epithelial (HeLa), and vascular smooth muscle cells. In human umbilical vein endothelial cells and HeLa cells, but not in human vascular smooth muscle cells, RLX upregulated ET
B
expression and activated extracellular signal–regulated kinase-1/2 (ERK-1/2) and nuclear factor-κB (NF-κB), a transcription factor. PD-98059, a selective inhibitor of the mitogen-activated protein kinase kinase-1 (MEK-1)–ERK-1/2 pathway, abolished ERK-1/2 and NF-κB activation and ET
B
upregulation. NF-κB inhibition also prevented RLX-mediated ET
B
stimulation. In NF-κB-luciferase reporter assays, we demonstrated complete inhibition of RLX-induced NF-κB activation in cells transfected with dominant-negative Raf-1, MEK-1, or ERK-1/2 constructs, whereas dominant-negative Ras had no effect. In rat aorta and mesenteric artery, RLX pretreatment, in an ET
B
-dependent fashion, mitigated the maximum contractile response to endothelin-1, by 38±4% and 43±6%, and the endothelin-1 sensitivity (−log[EC
50
]: aorta, 8.2±0.2 for vehicle versus 7.2±0.2 for RLX; mesenteric artery, 8.0±0.2 for vehicle versus 7.1±0.1 for RLX). RLX pretreatment augmented the dilator effect of the ET
B
agonist endothelin-3 by 100±8% and 133±13%. In conclusion, RLX stimulates endothelial and epithelial ET
B
via a Ras-independent Raf-1–MEK-1–ERK-1/2 pathway that activates NF-κB. On vascular smooth muscle cells, ET
B
, a contributor to endothelin-mediated vasoconstriction, remains unaffected. This renders RLX a functional endothelin-1 antagonist.
Focal Adhesion Kinase Is Required for Blood Vessel Morphogenesis
Authors: Ilic Dusko , Kovacic Branka , McDonagh Susan , Jin Fang , Baumbusch Clark , Gardner David G. , Damsky Caroline H.
Published: February 2003

The nonreceptor tyrosine kinase focal adhesion kinase (FAK) is a point of convergence for signals from extracellular matrix, soluble factors, and mechanical stimuli. Targeted disruption of the
fak
gene in mice leads to death at embryonic day 8.5 (E8.5). FAK
−/−
embryos have severely impaired blood vessel development. Gene expression and in vitro differentiation studies revealed that endothelial cell differentiation was comparable in FAK
−/−
and wild-type E8.5 embryos. We examined the role of FAK in blood vessel morphogenesis using an in vitro tubulogenesis assay and three different culture systems: FAK
+/+
and FAK
−/−
embryoid bodies, FAK
+/+
and FAK
−/−
endothelial cells, and human umbilical vein endothelial cells expressing antisense FAK, a dominant-negative fragment of FAK, or wild-type FAK. In all of these systems, endothelial cells deficient in FAK expression or function displayed a severely reduced ability to form tubules in Matrigel. These studies demonstrate clearly that the vascular defects in FAK
−/−
mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular networks, rather than from defects in tissue-specific differentiation.
Nitric Oxide Enhances Angiogenesis via the Synthesis of Vascular Endothelial Growth Factor and cGMP After Stroke in the Rat
Authors: Zhang Ruilan , Wang Lei , Zhang Li , Chen Jieli , Zhu Zhenping , Zhang Zhenggang , Chopp Michael
Published: February 2003

We investigated the effects of NO on angiogenesis and the synthesis of vascular endothelial growth factor (VEGF) in a model of focal embolic cerebral ischemia in the rat. Compared with control rats, systemic administration of an NO donor,
DETANONOate
, to rats 24 hours after stroke significantly enlarged vascular perimeters and increased the number of proliferated cerebral endothelial cells and the numbers of newly generated vessels in the ischemic boundary regions, as evaluated by 3-dimensional laser scanning confocal microscopy. Treatment with
DETANONOate
significantly increased VEGF levels in the ischemic boundary regions as measured by ELISA. A capillary-like tube formation assay was used to investigate whether
DETANONOate
increases angiogenesis in ischemic brain via activation of soluble guanylate cyclase.
DETANONOate
-induced capillary-like tube formation was completely inhibited by a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Blocking VEGF activity by a neutralized antibody against VEGF receptor 2 significantly attenuated
DETANONOate
-induced capillary-like tube formation. Moreover, systemic administration of a phosphodiesterase type 5 inhibitor (Sildenafil) to rats 24 hours after stroke significantly increased angiogenesis in the ischemic boundary regions. Sildenafil and an analog of cyclic guanosine monophosphate (cGMP) also induced capillary-like tube formation. These findings suggest that exogenous NO enhances angiogenesis in ischemic brain, which is mediated by the NO/cGMP pathway. Furthermore, our data suggest that NO, in part via VEGF, may enhance angiogenesis in ischemic brain.