bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Induction of a Releasable Pool of Factor VIII in Human Endothelial Cells
Authors: Rosenberg Jonathan B. , Greengard Judith S. , Montgomery Robert R.
Published: June 2011

Abstract
—Although it is known that factor VIII (FVIII) plasma levels increase rapidly in response to a number of stimuli, the biological stimuli behind this release is less clear. Previously, we showed that FVIII can traffic together with von Willebrand factor (vWF) into storage granules in a pituitary tumor cell line, AtT-20; however, AtT-20 cells could not be used to address the release or functional activity of released FVIII. To investigate the regulated secretion of stored FVIII, endothelial cells with intact agonist-stimulated release pathways were used. Human umbilical vein endothelial cells (HUVECs) were transduced with retroviral FVIII construct [hFVIII(V)] to create a FVIII/vWF storage pool. Immunofluorescent staining of transduced cells demonstrated FVIII in Weibel-Palade bodies. In contrast, the transduction of hFVIII(V) into HT-1080 and HepG2 cells displayed FVIII only in the cytoplasm. We studied the regulated release of both FVIII and vWF from endothelial cells after agonist-induced stimulation and demonstrated a parallel release of FVIII and vWF proteins. This released FVIII was functionally active. Hence, endothelial cells transduced with hFVIII(V) store FVIII together with vWF in Weibel-Palade bodies , creating a releasable storage pool of both proteins. Because FVIII secretion can be physiologically regulated by agonists in culture, this may explain the pharmacological agonist-induced release of FVIII by drugs such as desmopressin in vivo and suggests vascular endothelium as a reasonable target of gene therapy of hemophilia A.
Identification and Localization of a Fatty Acid Response Region in the Human Plasminogen Activator Inhibitor-1 Gene
Authors: Chen YaBing , Billadello Joseph J. , Schneider David J.
Published: June 2011

Abstract
—The increased expression of plasminogen activator inhibitor type-1 (PAI-1) is associated with increased concentrations of fatty acids in blood and may accelerate atherogenesis in diabetes. The present study was designed to define mechanisms by which nonesterified (free) fatty acids (FFAs) augment the expression of PAI-1. FFAs increased PAI-1 protein and mRNA expression by HepG2 cells. To identify potential regulatory elements, we constructed chimeric genes by fusing 1313, 853, 610, or 328 bp of human PAI-1 5′-flanking DNA to a luciferase reporter (PAI-LUC). A 2-fold increase in luciferase activity was seen when cells were transfected with PAI-LUC 1313, 863, or 610 and exposed to FFAs. No response to FFAs was seen with PAI-LUC 328 and after deletion of a 72-bp (−599 to −528) fragment from PAI-LUC 1313. This 72-bp fragment conferred FFA responsiveness to a different (simian virus 40) promoter. Two footprinted regions were demonstrated by DNase I analysis. Gel mobility shift assays indicated specific binding of extracted proteins to an FFA response element: 5′-TG(G/C)
1–2
CTG-3′. This sequence is repeated 4 times and is similar to an Sp1-binding site. Sp1 consensus oligonucleotides inhibited binding of extracted proteins to the regulatory elements. Accordingly, FFA-induced increased expression of PAI-1 in HepG2 cells is mediated by the binding of a transcription factor or factors to the repeated fatty acid response element, 5′-TG(G/C)
1–2
CTG-3′, that is highly homologous to an Sp1-binding site.
Role of RhoA and Rho Kinase in Lysophosphatidic Acid–Induced Endothelial Barrier Dysfunction
Authors: Amerongen Geerten P. van Nieuw , Vermeer Mario A. , van Hinsbergh Victor W. M.
Published: June 2011

Abstract
—In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca
2+
concentration ([Ca
2+
]
i
), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca
2+
]
i
was not elevated by LPA. Chelation of Ca
2+
i
ions by 1,2-bis(2-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from
Clostridium botulinum
inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca
2+
]
i
and requires activation of RhoA and Rho kinase.
Vascular Endothelial Cells and Smooth Muscle Cells Differ in Expression of Fas and Fas Ligand and in Sensitivity to Fas Ligand–Induced Cell Death
Authors: Sata Masataka , Suhara Toshimitsu , Walsh Kenneth
Published: June 2011

Abstract
—Fas ligand (FasL) is a death factor that induces apoptosis in cells bearing its receptor, Fas. Fas and FasL have been detected in the vessel wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the vasculature. Here, we evaluated the expression of Fas in the presence and absence of cytokines on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We also examined the sensitivity of ECs and VSMCs to Fas-mediated apoptosis induced by exposure to multiple Fas agonists: soluble FasL, anti-Fas antibody, and membrane-bound FasL resulting from transduction with a replication-defective adenovirus expressing FasL (Adeno-FasL). Cell-surface FasL expression was detected on human ECs with the use of 4 anti-FasL antibodies, whereas cell-surface FasL expression was not detected on VSMCs. Unstimulated ECs expressed relatively low levels of Fas, but expression was upregulated after treatment with tumor necrosis factor-α (TNF-α) or interferon gamma (IFN-γ). In contrast, VSMCs expressed relatively high levels of Fas, and treatment with TNF-α or IFN-γ induced little or no upregulation under the conditions of these assays. ECs were resistant to death after exposure to soluble FasL or agonist anti-Fas antibody and also after infection with Adeno-FasL in the presence or absence of cytokine treatment. In contrast, VSMCs remained viable in the presence of soluble FasL or agonist anti-Fas antibody, but they underwent apoptosis after infection with Adeno-FasL. IFN-γ enhanced Adeno-FasL-induced death of VSMCs, but TNF-α did not. These findings provide insights about the potential role of Fas-mediated apoptosis in the vessel wall and suggest strategies to treat proliferative vascular diseases by exploiting the differential sensitivity of ECs and VSMCs to FasL-induced cell death.
Zinc Finger Transcription Factor Egr-1 Activates Flt-1 Gene Expression in THP-1 Cells on Induction for Macrophage Differentiation
Authors: Akuzawa Nobuhiro , Kurabayashi Masahiko , Ohyama Yoshio , Arai Masashi , Nagai Ryozo
Published: June 2011

Abstract
—Activation of macrophages is a hallmark of atherosclerosis. Stimulation of human monocytic leukemia THP-1 cells with phorbol 12-myristate 13-acetate (PMA) is known to induce a variety of genes whose function is relevant to activated macrophages. Flt-1, a receptor tyrosine kinase for vascular endothelial growth factor, is expressed in macrophages as well as in endothelial cells and mediates the biological response to vascular endothelial growth factor. In this study, we investigated the molecular mechanisms underlying the inducible expression of the flt-1 gene during the activation of THP-1 cells. Reverse transcription—polymerase chain reaction analysis showed that exposure of THP-1 cells to PMA increases flt-1 mRNA and protein levels. A transfected reporter gene, consisting of the human flt-1 promoter region coupled to the luciferase gene, indicated a direct effect of PMA on transcriptional activity. Transfection analysis of a series of 5′-deletion constructs and site-directed mutants localized the PMA-responsive region to a DNA stretch from −174 to −166, which represents overlapping Egr-1/Sp1 transcription factor–binding sites. Competitive gel mobility shift assays and supershift assays showed that PMA induces the binding of Egr-1 to this site. Consistent with these findings, the Egr-1 expression plasmid strongly induced flt-1 promoter activity in a sequence-specific manner. Taken together, our data demonstrate that PMA induces flt-1 gene transcription through an induction of Egr-1 in THP-1 cells, thus providing new evidence that the flt-1 gene is a direct target of Egr-1, the transcription factor primarily induced on macrophage differentiation.
Leptin, Peroxisome Proliferator-Activated Receptor-γ, and CCAAT/Enhancer Binding Protein-α mRNA Expression in Adipose Tissue of Humans and Their Relation to Cardiovascular Risk Factors
Authors: Krempler Franz , Breban David , Oberkofler Hannes , Esterbauer Harald , Hell Emanuel , Paulweber Bernhard , Patsch Wolfgang
Published: June 2011

Abstract
—Obesity is a prevalent disorder that increases the risk for premature cardiovascular disease. The adipose tissue itself plays an active role in the regulation of fuel metabolism and energy homeostasis by expressing a number of regulatory genes, such as leptin, peroxisome proliferator–activated receptor-γ (PPARγ), and CCAAT/enhancer binding protein-α (C/EBPα). To study the in vivo relationships among these genes and their associations with cardiovascular risk factors, plasma levels of leptin, lipids, apolipoproteins (apo), insulin, and glucose were measured in 216 obese, 165 nonobese, and 36 weight-losing postobese subjects. mRNA expression of leptin, PPARγ, and C/EBPα in the extraperitoneal and intraperitoneal adipose tissue was quantified in subsets of subjects. In obese individuals, plasma leptin was associated with apoA-I (
r
=0.2346,
P
<0.001) and insulin (
r
=0.2125,
P
<0.002). Leptin and C/EBPα mRNA expression in extraperitoneal and intraperitoneal adipose tissue of obese patients was higher than in the respective tissues of nonobese or postobese subjects. No significant differences among the study groups were found for PPARγ mRNA expression. Leptin, PPARγ, and C/EBPα mRNA levels correlated with each other in the intraperitoneal and extraperitoneal fat of obese subjects, but multivariate analysis revealed that only C/EBPα was a predictor of leptin expression in extraperitoneal tissue (partial
r
=0.6096,
P
<0.001). Intraperitoneal PPARγ expression was inversely related to fasting insulin (
r
=−0.2888,
P
<0.017) and a fasting insulin resistance index (
r
=−0.2814,
P
<0.021) in obese subjects. In postobese patients, intraperitoneal PPARγ expression was associated with plasma HDL cholesterol (
r
=0.5695,
P
<0.018) and apoA-I (
r
=0.6216,
P
<0.008) but was inversely related to LDL cholesterol (
r
=−0.5101,
P
<0.03) and apoB (
r
=−0.6331,
P
<0.007). These findings suggest a relationship between plasma leptin and HDL metabolism as well as adipose-tissue site–dependent associations among leptin, C/EBP-α, and PPAR-γ mRNA expression. Furthermore, our results suggest that C/EBP-α enhances leptin expression in vivo and that PPARγ mRNA expression is inversely associated with cardiovascular risk factors.
New Functional Promoter Polymorphism, CETP/−629, in Cholesteryl Ester Transfer Protein (CETP) Gene Related to CETP Mass and High Density Lipoprotein Cholesterol Levels
Authors: Dachet Christiane , Poirier Odette , Cambien François , Chapman John , Rouis Mustapha
Published: June 2011

Abstract
—A new polymorphism located at position −629 (CETP/−629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The −629A allele was associated with lower CETP mass (
P
<0.0001) and higher high density lipoprotein cholesterol (
P
<0.001) than the C allele in a sample of 536 control subjects from the ECTIM study. Transfection studies in HepG2 cells with a luciferase expression vector incorporating a 777-bp fragment of the CETP promoter and containing either A or C at position −629 showed significantly lower luciferase activity with the promoter fragment of the A allele (−25%,
P
<0.05). By gel-shift assay, DNA-protein interactions were evaluated in nuclear extracts of HepG2 cells with the use of 2 probes (A or C probe) composed of 20 bp of the promoter sequence surrounding the polymorphic site. Two specific complexes of distinct migration rate were identified with the A and the C probe. Competition with an excess of oligonucleotide containing the Sp1 consensus binding site showed that a protein(s) of the Sp transcription factor family was implicated in complex formation with the A probe but not with the C probe. Incubation with specific antibodies indicated that Sp1 and Sp3 bound specifically to the A probe. We introduced mutations in the −629-Sp1 binding site to test its functionality and to define the characteristics of transcription factor binding. We showed, by gel-shift assay, that no nuclear proteins bound to the mutated sequence. Transient transfection of HepG2 cells revealed that the expression of the mutated fragment was significantly increased compared with that of the A promoter fragment (25%,
P
<0.05). The mutated fragment displayed the same activity as that of the C promoter. These results indicate that Sp1 and/or Sp3 repress CETP promoter activity, whereas nuclear factors binding the C allele are without effect on promoter expression.
Lipoprotein(a) in Homozygous Familial Hypercholesterolemia
Authors: Kraft H. G. , Lingenhel A. , Raal F. J. , Hohenegger M. , Utermann G.
Published: June 2011

Abstract
—Lipoprotein(a) [Lp(a)] is a quantitative genetic trait that in the general population is largely controlled by 1 major locus—the locus for the apolipoprotein(a) [apo(a)] gene. Sibpair studies in families including familial defective apolipoprotein B or familial hypercholesterolemia (FH) heterozygotes have demonstrated that, in addition, mutations in apolipoprotein B and in the LDL receptor (LDL-R) gene may affect Lp(a) plasma concentrations, but this issue is controversial. Here, we have further investigated the influence of mutations in the LDL-R gene on Lp(a) levels by inclusion of FH homozygotes. Sixty-nine members of 22 families with FH were analyzed for mutations in the LDL-R as well as for apo(a) genotypes, apo(a) isoforms, and Lp(a) plasma levels. Twenty-six individuals were found to be homozygous for FH, and 43 were heterozygous for FH. As in our previous analysis, FH heterozygotes had significantly higher Lp(a) than did non-FH individuals from the same population. FH homozygotes with 2 nonfunctional LDL-R alleles had almost 2-fold higher Lp(a) levels than did FH heterozygotes. This increase was not explained by differences in apo(a) allele frequencies. Phenotyping of apo(a) and quantitative analysis of isoforms in family members allowed the assignment of Lp(a) levels to both isoforms in apo(a) heterozygous individuals. Thus, Lp(a) levels associated with apo(a) alleles that were identical by descent could be compared. In the resulting 40 allele pairs, significantly higher Lp(a) levels were detected in association with apo(a) alleles from individuals with 2 defective LDL-R alleles compared with those with only 1 defective allele. This difference of Lp(a) levels between allele pairs was present across the whole size range of apo(a) alleles. Hence, mutations in the LDL-R demonstrate a clear gene-dosage effect on Lp(a) plasma concentrations.
Physical Training Improves Flow-Mediated Dilation in Patients With the Polymetabolic Syndrome
Authors: Lavrenc̆ic̆ Ales̆a , Salobir Barbara Guz̆ic̆ , Keber Irena
Published: June 2011

Abstract
—Endothelial dysfunction that can be detected as impaired flow-mediated dilation by ultrasonography is an early event in atherogenesis and has been demonstrated in healthy subjects with risk factors for atherosclerosis many years before the appearance of atheromatous plaques. We examined the influence of physical training on flow-mediated dilation in patients with the polymetabolic syndrome. Twenty-nine asymptomatic men aged 40 to 60 years with the polymetabolic syndrome were randomly divided between the control group and the training group, which trained 3 times a week for 12 weeks. On high-resolution ultrasound images, the diameter of the brachial artery was measured at rest, after reactive hyperemia (causing flow-mediated, endothelium-dependent dilation), and after sublingual glyceryltrinitrate (causing endothelium-independent vasodilation) in all subjects before and after the training period. The training program induced an increase of 18% in physical fitness. Flow-mediated dilation increased from 5.3±2.8% to 7.3±2.7% (
P
<0.05). There was no change in body mass index, blood pressure, insulin resistance, lipids, and big endothelin-1 in either group. Flow-mediated dilation measured before training was negatively correlated with resting heart rate, waist-to-hip ratio, and insulin resistance. Resting heart rate emerged as the only independent determinant, which explained 22% of the variation in flow-mediated dilation. In conclusion, our findings suggest that a 3-month physical training program, which improved maximal exercise capacity, enhances flow-mediated dilation in patients with the polymetabolic syndrome.
Regulation of cdk2 Activity in Endothelial Cells That Are Inhibited From Growth by Cell Contact
Authors: Chen Donghui , Walsh Kenneth , Wang Jian
Published: June 2011

Abstract
—Endothelial cells (ECs) are quiescent in normal blood vessels but undergo rapid bursts of proliferation after vascular injury and during angiogenesis. Here we show that the activity of cyclin-dependent kinase-2 (cdk2), a key regulator of the G1 and S phases of the cell cycle, is expressed at high levels in proliferating ECs but at low levels in ECs that are contact-inhibited for growth. Despite these differences in kinase activity, the protein levels of cdk2 and 1 of its activating subunits, cyclin E, are not modulated by these different growth conditions. The cdk inhibitor p27 is highly expressed in contact-inhibited but not proliferating ECs, whereas the level of cyclin A protein is preferentially expressed in proliferating ECs. p27 protein was detected in immunoprecipitable complexes with cdk2 or cyclin E in cultures that were contact-inhibited for growth. The functional significance of the p27 induction was indicated by the detection of a heat-stable cdk2 inhibitory activity that was induced by endothelial cell-cell contact and could be immunodepleted with anti-p27 antibodies. In a confluent EC monolayer, cdk2 kinase activity was activated by a scraping injury that led to cell migration and proliferation. The injury-induced activation of cdk2 coincided with the downregulation of p27 and the induction of cyclin A. These data demonstrate that p27 is induced in confluent cultures of ECs. They also indicate that both p27 induction and cyclin A downregulation contribute to the inhibition of cdk2 and cell proliferation by cell-cell contact in ECs.
Gene Transfer and Hepatic Overexpression of the HDL Receptor SR-BI Reduces Atherosclerosis in the Cholesterol-Fed LDL Receptor–Deficient Mouse
Authors: Kozarsky Karen F. , Donahee Mary H. , Glick Jane M. , Krieger Monty , Rader Daniel J.
Published: June 2011

Abstract
—HDL cholesterol levels in humans are inversely correlated with the risk of atherosclerosis. The class B scavenger receptor type I (SR-BI) is the first molecularly well-defined HDL receptor, and hepatic overexpression of SR-BI in normal mice has been shown to result in decreased plasma HDL cholesterol levels. To determine whether SR-BI overexpression is proatherogenic or is protective against atherosclerosis, LDL receptor–deficient mice were placed on a high-fat/high-cholesterol diet for 2 or 12 weeks to induce atherosclerotic lesions of different stages and then were injected with a recombinant adenovirus encoding murine SR-BI. Transient hepatic overexpression of SR-BI in mice with both early and advanced lesions significantly decreased atherosclerosis. SR-BI expression was associated with markedly decreased HDL cholesterol and either unchanged or only modestly reduced non-HDL cholesterol levels; in all experiments, the mean HDL cholesterol levels were significantly correlated with atherosclerotic lesion size. These data suggest that interventions that promote HDL cholesterol transport and lower plasma HDL cholesterol levels can suppress atherosclerosis, even when initiated after significant lesion development. Thus, stimulation of hepatic SR-BI activity may provide a novel target for therapeutic intervention in atherosclerotic cardiovascular disease.
In Vivo Evidence for Both Lipolytic and Nonlipolytic Function of Hepatic Lipase in the Metabolism of HDL
Authors: Dugi K. A. , Amar M. J. A. , Haudenschild C. C. , Shamburek R. D. , Bensadoun A. , Hoyt R. F. , Fruchart-Najib J. , Madj Z. , Brewer H. B. , Santamarina-Fojo S.
Published: June 2011

Abstract
—To investigate the in vivo role that hepatic lipase (HL) plays in HDL metabolism independently of its lipolytic function, recombinant adenovirus (rAdV) expressing native HL, catalytically inactive HL (HL-145G), and luciferase control was injected in HL-deficient mice. At day 4 after infusion of 2×10
8
plaque-forming units of rHL-AdV and rHL-145G-AdV, similar plasma concentrations were detected in postheparin plasma (HL=8.4±0.8 μg/mL and HL-145G=8.3±0.8 μg/mL). Mice expressing HL had significant reductions of cholesterol (−76%), phospholipids (PL; −68%), HDL cholesterol (−79%), apolipoprotein (apo) A-I (−45%), and apoA-II (−59%;
P
<0.05 for all), whereas mice expressing HL-145G decreased their cholesterol (−49%), PL (−40%), HDL cholesterol (−42%), and apoA-II (−89%;
P
<0.005 for all) but had no changes in apoA-I. The plasma kinetics of
125
I-labeled apoA-I HDL,
131
I-labeled apoA-II HDL, and [
3
H]cholesteryl ester (CE) HDL revealed that compared with mice expressing luciferase control (fractional catabolic rate [FCR] in d
−1
: apoA-I HDL=1.3±0.1; apoA-II HDL=2.1±0; CE HDL=4.1±0.7), both HL and HL-145G enhanced the plasma clearance of CEs and apoA-II present in HDL (apoA-II HDL=5.6±0.5 and 4.4±0.2; CE HDL=9.3±0.0 and 8.3±1.1, respectively), whereas the clearance of apoA-I HDL was enhanced in mice expressing HL (FCR=4.6±0.3) but not HL-145G (FCR=1.4±0.4). These combined findings demonstrate that both lipolytic and nonlipolytic functions of HL are important for HDL metabolism in vivo. Our study provides, for the first time, in vivo evidence for a role of HL in HDL metabolism independent of lipolysis and provides new insights into the role of HL in facilitating distinct metabolic pathways involved in the catabolism of apoA-I– versus apoA-II–containing HDL.
Reversible Regulation of Tissue Factor–Induced Coagulation by Glycosyl Phosphatidylinositol–Anchored Tissue Factor Pathway Inhibitor
Authors: Ott Ilka , Miyagi Yohei , Miyazaki Kaoru , Heeb Mary Jo , Mueller Barbara M. , Rao L. Vijay M. , Ruf Wolfram
Published: June 2011

Abstract
—Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-1), which regulates tissue factor (TF) function by TF · VIIa · Xa · TFPI-1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF · VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF · VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.
Apolipoprotein E Inhibition of Vascular Smooth Muscle Cell Proliferation but Not the Inhibition of Migration Is Mediated Through Activation of Inducible Nitric Oxide Synthase
Authors: Ishigami Masato , Swertfeger Debi K. , Hui Michele S. , Granholm Norman A. , Hui David Y.
Published: June 2011

Abstract
—Initial experiments revealed that low concentrations of apolipoprotein (apo) E (0.1 to 5 μg/mL) were effective in inhibiting platelet-derived growth factor (PDGF)–directed smooth muscle cell (SMC) migration by 60% to 80%. In contrast, higher concentrations of apoE, at 25 and 50 μg/mL, were necessary to achieve similar inhibition of PDGF-induced SMC proliferation. The potential role of nitric oxide (NO) in mediating the inhibitory effects of apoE was explored. Results showed that, although 0.1 to 5 μg/mL of apoE had no effect on NO production by SMCs, physiological concentrations of apoE (25 to 50 μg/mL) enhanced NO synthesis by 2-fold in a dose-dependent manner (
P
<0.001). Reverse transcription–polymerase chain reaction amplification of RNA obtained from control and apoE-treated SMCs demonstrated a direct role of apoE in activating inducible nitric oxide synthase (iNOS) gene expression. The apoE-induced nitric oxide production was significantly reduced by coincubation of the cells with aminoguanidine or
N
G
-monomethyl-
l
-arginine (
P
<0.05) or with antisense iNOS oligodeoxynucleotides (
P
<0.01). Moreover, the inhibition of iNOS was shown to overcome apoE suppression of PDGF-induced vascular SMC proliferation. However, apoE suppression of PDGF-directed SMC migration was not affected by these treatments. Taken together, these results document that apoE exerts its inhibitory effects on cell proliferation via activation of iNOS. However, apoE inhibition of cell migration is mediated by a mechanism independent of iNOS activation.
Upregulation of Endothelial Receptor for Oxidized LDL (LOX-1) by Oxidized LDL and Implications in Apoptosis of Human Coronary Artery Endothelial Cells
Authors: Li Dayuan , Mehta Jawahar L.
Published: June 2011

Abstract
—A specific lectinlike endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL–induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 μg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL–mediated upregulation of LOX-1 was suppressed (
P
<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL–mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL–mediated apoptosis of HCAECs. Nuclear factor (NF)-κB was markedly activated in ox-LDL–treated HCAECs. The critical role of NF-κB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL–mediated NF-κB activation. In this process, an NF-κB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL–mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL–induced apoptosis is mediated by the action of LOX-1. In this process, NF-κB activation may play an important role as a signal transduction mechanism.
Influence of PAI-1 on Adipose Tissue Growth and Metabolic Parameters in a Murine Model of Diet-Induced Obesity
Authors: Morange P. E. , Lijnen H. R. , Alessi M. C. , Kopp F. , Collen D. , Juhan-Vague I.
Published: June 2011

Abstract
—An increased plasma plasminogen activator inhibitor-1 (PAI-1) level is a risk factor for myocardial infarction, particularly when associated with visceral obesity. Although the link between PAI-1 and obesity is well documented, little is known about the physiological relevance of PAI-1 production by adipose tissue. Therefore, we have compared adipose tissue development and insulin resistance plasma parameters in PAI-1–deficient mice (PAI-1
−/−
) and wild-type littermates (PAI-1
+/+
) in a model of nutritionally induced obesity. After 17 weeks of consuming a high-fat diet (HFD), PAI-1
+/+
mice showed marked obesity, with a 52% increase in body weight compared with mice that were kept on a standard fat diet (
P
<0.0001). This weight gain was accompanied by adipocyte hypertrophy and an increase in the number of stroma cells in the gonadal fat pad, expressed as stroma cells/adipocytes (0.67±0.05 versus 0.43±0.02;
P
<0.001). In plasma, the HFD induced a marked increase in PAI-1 antigen (5.1±0.56 versus 2±0.22 ng/mL;
P
<0.001), fasting insulinemia (1.1±0.21 versus 0.21±0.04 ng/mL;
P
<0.001), and glycemia (7.4±0.5 versus 5±0.3 mmol/L;
P
<0.001), whereas plasma triglyceride levels were not affected. When we compared PAI-1
−/−
and PAI-1
+/+
mice on the HFD, PAI-1
−/−
mice gained weight faster than did PAI-1
+/+
mice, with a significant difference in body weight between 3 and 8 weeks of the diet (32±1.7 versus 26±1.6 g at 6 weeks;
P
<0.05). After 17 weeks of the HFD, its effect on weight gain and the number and size of adipocytes was similar in PAI-1
+/+
and PAI-1
−/−
mice. By contrast, the increase in the number of stroma cells presented by PAI-1
+/+
mice was not observed in PAI-1
−/−
mice. In obese PAI-1
−/−
mice, tissue-type PA activity and antigen levels in the gonadal fat pad were significantly higher than in obese PAI-1
+/+
mice (230±50 versus 47±20 arbitrary units/g,
P
<0.01; 40±13 versus 17±13 ng/g,
P
<0.05, respectively), whereas urokinase-type PA activity and antigen levels were similar in both groups. In plasma, nonobese PAI-1
−/−
mice displayed 62% higher insulin levels (
P
<0.05) than did PAI-1
+/+
mice. Obese PAI-1
−/−
mice displayed 68% higher triglyceride levels (
P
<0.01) and 21% lower glucose levels (
P
<0.05) than did PAI-1
+/+
mice. These data support an effect of PAI-1 on weight gain and adipose tissue cellularity in the induction of obesity in mice. Moreover, PAI-1 influences glucidolipidic metabolism. The elevated expression of PAI-1 observed in human obesity could be involved in mechanisms that control adipose tissue development.
Reduced Thrombus Formation by Hyaluronic Acid Coating of Endovascular Devices
Authors: Verheye Stefan , Markou Christos P. , Salame Mahomed Y. , Wan Barbara , King Spencer B. , Robinson Keith A. , Chronos Nicolas A. F. , Hanson Stephen R.
Published: June 2011

Abstract
—Biocompatible stent coatings may alleviate problems of increased (sub)acute thrombosis after stent implantation. Hyaluronic acid (HA), a ubiquitous, nonsulfated glycosaminoglycan, inhibits platelet adhesion and aggregation and prolongs bleeding when administered systemically. However, the effects of immobilized HA for reducing stent platelet deposition in vivo are unknown. We therefore quantified the antithrombotic effects of coating stainless steel stents and tubes with HA using an established baboon thrombosis model under physiologically relevant blood flow conditions. HA-coated and uncoated (control) stents (3.5 mm in diameter, n=32) and stainless steel tubes (4.0 mm in diameter, n=18) were deployed into exteriorized arteriovenous shunts of conscious, nonanticoagulated baboons. Accumulation of
111
In-radiolabeled platelets was quantified by continuous gamma-camera imaging during a 2-hour blood exposure period. HA coating resulted in a significant reduction in platelet deposition in long (4 cm) tubes (0.24±0.15×10
9
versus 6.12±0.49×10
9
platelets;
P
<0.03), short (2 cm) stainless steel tubes (0.18±0.06×10
9
versus 3.03±0.56×10
9
platelets;
P
<0.008), and stents (0.82±0.20×10
9
versus 1.83±0.23×10
9
platelets;
P
<0.02) compared with uncoated control devices. Thus, HA coating reduces platelet thrombus formation on stainless steel stents and tubes in primate thrombosis models. These results indicate that immobilized HA may represent an attractive strategy for improving the thromboresistance of endovascular devices.
Regulation of Lysyl Oxidase by Interferon-γ in Rat Aortic Smooth Muscle Cells
Authors: Song Yun Ling , Ford John W. , Gordon David , Shanley Charles J.
Published: June 2011

Abstract
—Lysyl oxidase is an essential catalyst for the cross-linking of extracellular collagen and elastin. Abnormalities in lysyl oxidase activity may contribute to the pathogenesis of arterial diseases characterized by abnormal matrix remodeling. This study tested the hypothesis that interferon (IFN)-γ, a proinflammatory cytokine present in aortic aneurysm and arteriosclerotic plaque rupture, downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells. Steady-state lysyl oxidase mRNA levels decreased in a concentration- and time-dependent manner to 30% of control levels after 24 hours of treatment with IFN-γ. Cell layer lysyl oxidase activity decreased in parallel with the observed changes in steady-state mRNA. Nuclear runoff studies suggested that transcriptional regulation was responsible for at least 40% of the observed downregulation. mRNA decay studies suggested that IFN-γ also decreased lysyl oxidase mRNA half-life from 9 to 6 hours. Downregulation of lysyl oxidase by IFN-γ did not appear to require new protein synthesis. This study documents that IFN-γ downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms. If similar regulation occurs in vivo, it is possible that IFN-γ–mediated changes in lysyl oxidase may contribute to arterial diseases characterized by abnormal extracellular matrix.
Matrix Metalloproteinase and αvβ3 Integrin–Dependent Vascular Smooth Muscle Cell Invasion Through a Type I Collagen Lattice
Authors: Kanda Shigeru , Kuzuya Masafumi , Ramos Miguel A. , Koike Teruhiko , Yoshino Kohichiro , Ikeda Shoji , Iguchi Akihisa
Published: June 2011

Abstract
—Smooth muscle cell (SMC) migration from the tunica media to the intima is a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. SMCs require not only migratory but also degradative abilities that enable them to migrate through extracellular matrix proteins, which surround and embed these cells. We used a collagen type I lattice as a coating on top of a porous filter as a matrix barrier in a chamber to test the invasive behavior of SMCs in response to a chemoattractant (invasion assay) and compared that behavior with simple SMC migration through collagen type I–coated filters (migration assay). Inhibitors of matrix metalloproteinase, KB-R8301, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TIMP-2, and peptide 74, attenuated platelet-derived growth factor-BB (PDGF-BB)–directed SMC invasion across the collagen lattice, whereas no effect was seen with these inhibitors on simple SMC migration through collagen-coated filters. RGD peptide inhibited SMC invasion but did not affect SMC migration. Anti-αvβ3 integrin antibody attenuated PDGF-BB–directed SMC invasion, whereas other antibodies against RGD-recognizing integrins, namely αvβ5 and α5, had no effect. None of these antibodies had any effect on simple SMC migration. RGD peptide and anti-αvβ3 antibody inhibited the attachment and spreading of SMCs on denatured collagen but not on native collagen. These findings indicate that there is a difference in the mechanisms between simple SMC migration across a collagen-coated filter and SMC invasion through a fibrillar collagen barrier. A proteolytic process is required for SMC invasion, and the degradation of matrix proteins alters the relationship between matrix protein molecules and SMC surface integrins.
Relationship Between Homocysteine and Superoxide Dismutase in Homocystinuria
Authors: Wilcken David E. L. , Wang Xing Li , Adachi Tetsuo , Hara Hirokazu , Duarte Natalia , Green Kathryn , Wilcken Bridget
Published: June 2011

Abstract
—A modest homocysteine elevation is associated with an increased cardiovascular risk. Marked circulating homocysteine elevations occur in homocystinuria due to cystathionine β-synthase (CβS) deficiency, a disorder associated with a greatly enhanced cardiovascular risk. Lowering homocysteine levels reduces this risk significantly. Because homocysteine-induced oxidative damage may contribute to vascular changes and extracellular superoxide dismutase (EC-SOD) is an important antioxidant in vascular tissue, we assessed EC-SOD and homocysteine in patients with homocystinuria. We measured circulating EC-SOD, total homocysteine (free plus bound), and methionine levels during the treatment of 21 patients with homocystinuria, 18 due to CβS deficiency, aged 8 to 59 years, and 3 with remethylating defects. We measured total homocysteine by immunoassay, EC-SOD by ELISA, and methionine by amino acid analysis and assessed interindividual and intraindividual relationships. There was a significant, positive relationship between EC-SOD and total homocysteine. For the interindividual assessment, levels were highly correlated,
r
=0.746, N=21,
P
<0.0001. This relationship was maintained after taking into account intraindividual patient variation (
r
=0.607, N=62,
P
<0.0001). In 2 newly diagnosed CβS-deficient patients, treatment that lowered the markedly elevated pretreatment homocysteine level (from 337 to 72 and from 298 to 50 μmol/L) reduced the associated elevated EC-SOD in each by 50%. EC-SOD and methionine levels were unrelated (
r
=0.148, n=39,
P
=0.368). The positive relationship between circulating EC-SOD and homocysteine could represent a protective antioxidant response to homocysteine-induced oxidative damage and contribute to reducing cardiovascular risk in homocystinuric patients. EC-SOD levels may be relevant to the pathogenesis of vascular disease in other patient groups.
Doxazosin Inhibits Retinoblastoma Protein Phosphorylation and G
1
→S Transition in Human Coronary Smooth Muscle Cells
Authors: Kintscher Ulrich , Wakino Shu , Kim Sarah , Jackson Simon M. , Fleck Eckart , Hsueh Willa A. , Law Ronald E.
Published: June 2011

Abstract
—Previous studies have demonstrated that the α
1
-adrenergic receptor antagonist doxazosin (Dox) inhibits multiple mitogenic signaling pathways in human vascular smooth muscle cells. This broad antiproliferative activity of Dox occurs through a novel mechanism unrelated to its blocking the α
1
-adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitogen-induced G
1
→S progression of human coronary artery smooth muscle cells (CASMCs) in a dose-dependent manner, with a maximal reduction of S-phase transition by 88±10.5% in 20 ng/mL platelet-derived growth factor and 1 μmol/L insulin (P+I)–stimulated cells (
P
<0.01 for 10 μmol/L Dox versus P+I alone) and 52±18.7% for 10% FBS-induced mitogenesis (
P
<0.05 for 10 μmol/L Dox versus 10% FBS alone). Inhibition of G
1
exit by Dox was accompanied by a significant blockade of retinoblastoma protein (Rb) phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G
1
arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CASMCs to progress through G
1
and enter the S phase. E2F-mediated G
1
exit was not affected by Dox, suggesting that it targets events upstream from Rb hyperphosphorylation. Downregulation of the cyclin-dependent kinase inhibitory protein p27 is important for maximal activation of G
1
cyclin/cyclin-dependent kinase holoenzymes to overcome the cell cycle inhibitory activity of Rb. In Western blot analysis, p27 levels decreased after mitogenic stimulation (after P+I, 43±1.8% of quiescent cells [
P
<0.01 versus quiescent cells]; after 10% FBS, 55±7.7% of quiescent cells [
P
<0.05 versus quiescent cells]), whereas the addition of Dox (10 μmol/L) markedly attenuated its downregulation (after P+I, 90±8.3% of quiescent cells [
P
<0.05 versus P+I alone]; after 10% FBS, 78±8.3% of quiescent cells [
P
<0.05 versus 10% FBS alone]). Furthermore, Dox inhibited cyclin A expression, an E2F regulated gene that is essential for cell cycle progression into the S phase. The present study demonstrates that Dox inhibits CASMC proliferation by blocking cell cycle progression from the G
0
/G
1
phase to the S phase. This G
1
→S blockade likely results from an inhibition of mitogen-induced Rb hyperphosphorylation through prevention of p27 downregulation.
In Vivo Metabolism of ApoB, ApoA-I, and VLDL Triglycerides in a Form of Hypobetalipoproteinemia Not Linked to the ApoB Gene
Authors: Elias Nizar , Patterson Bruce W. , Schonfeld Gustav
Published: June 2011

Abstract
—Familial hypobetalipoproteinemia (FHBL) is an autosomal codominant disorder that may result from different mutations in the apolipoprotein B (apoB) gene or chromosome 2. However, linkage of FHBL to the apoB gene was ruled out in 2 kindreds reported to date, and the genetic and metabolic bases for FHBL remain unknown. One of the reported kindreds is our 40-member F kindred, in which we found linkage of FHBL to a novel susceptibility region on chromosome 3p21.1-2. In addition to having low apoB levels, some, but not all, of the affected subjects in the F kindred also had low levels of high density lipoprotein (HDL) cholesterol and apoA-I. Our aim was to define the metabolic bases of the disorder in the F kindred. Therefore, we studied the in vivo kinetics of apoB and apoA-I and very low density lipoprotein (VLDL) triglycerides in 4 affected subjects and 5 normolipidemic relatives. Deuterated leucine and deuterated glycerol were used to label the apolipoproteins and triglycerides, respectively. Compartmental modeling was used to obtain the kinetic parameters. Affected subjects had (1) normal fractional catabolic rates (FCRs) for VLDL apoB, (2) increased FCRs for low density lipoprotein (LDL) apoB (0.050±0.009 versus 0.030±0.006 pools per hour for normal subjects,
P
=0.005), and (3) decreased production rates of VLDL apoB (11.4±1.7 versus 25.6±4.9 mg · kg
−1
· d
−1
,
P
=0.003), LDL apoB (7.8±1.3 versus 12.7±3.7 mg · kg
−1
· d
−1
,
P
=0.04), and VLDL triglycerides (8.2±4.5 versus 19.6±10.8 58 μmol · kg
−1
· h
−1
,
P
=0.09). These data differ from those obtained in previously studied FHBL heterozygotes bearing apoB-2 and apoB-9, 2 very short truncations of apoB. Low HDL cholesterol and apoA-I levels were caused by higher apoA-I FCRs (0.035±0.005 versus 0.018±0.005 pools per hour in controls,
P
<0.01) without significant decrease in apoA-I production rates (18.7±2.7 versus 22.8±5.6 mg · kg
−1
· d
−1
). In conclusion, decreased secretion of apoB-containing lipoproteins and hypercatabolism of LDL account for low apoB and cholesterol levels in this novel form of FHBL.
Association of Cholesteryl Ester Transfer Protein–
Taq
IB Polymorphism With Variations in Lipoprotein Subclasses and Coronary Heart Disease Risk
Authors: Ordovas Jose M. , Cupples L. Adrienne , Corella Dolores , Otvos James D. , Osgood Doreen , Martinez Antonia , Lahoz Carlos , Coltell Oscar , Wilson Peter W. F. , Schaefer Ernst J.
Published: June 2011

Abstract
—Cholesteryl ester transfer protein (CETP) facilitates the exchange of triglycerides and cholesteryl esters between lipoprotein particles, a key step in reverse cholesterol transport in humans. Variations at the CETP locus have been shown to be determinants of the levels and activity of CETP and high density lipoprotein (HDL) plasma concentration. The associations of the common CETP polymorphism,
Taq
IB in intron 1, with lipoprotein levels and particle size distribution, CETP activity, and coronary heart disease (CHD) risk were examined in a population-based sample of 1411 men and 1505 women from the Framingham Offspring Study. The B2 allele frequency was 0.444 in men and 0.433 in women, and its presence was significantly (
P
<0.05) associated with decreased CETP activity. B1B1 men had lower HDL cholesterol (HDL-C) levels (1.07 mmol/L) compared with B1B2 (1.14 mmol/L) and B2B2 (1.18 mmol/L) men (
P
<0.001). Likewise, B1B1 women had lower HDL-C levels (1.40 mmol/L) compared with B1B2 (1.46 mmol/L) and B2B2 (1.53 mmol/L) women (
P
<0.001). In men, the B2 allele was associated with increased particle size for HDL and low density lipoprotein. In women, a similar effect was demonstrated only for HDL particle size. The odds ratio for prevalent CHD associated with the B2 allele was 0.696 (
P
=0.035) in men. After adjusting for age, body mass index, systolic blood pressure, diabetes, smoking, alcohol consumption, β-blocker use, total cholesterol, and HDL-C, this odds ratio was 0.735 (
P
=0.187), suggesting that the protective effect of the B2 allele was due in part to its association with HDL-C levels. No significant protective effects were observed in women. These data demonstrate that variation at the CETP gene locus is a significant determinant of HDL-C levels, CETP activity, and lipoprotein size in this population. Moreover, these effects appear to translate into a lower CHD risk among those men with the B2 allele.
Viewpoint: Are Studies in Genetically Altered Mice Out of Control?
Authors: Sigmund Curt D.
Published: June 2011

Abstract
—Because the use of transgenic and gene-targeted models has increased in popularity, the number of reports describing unpredictable phenotypic effects caused by variation in the genetic background used to generate or propagate these models has steadily increased. There are now many examples in which animals containing the same exact genetic manipulation exhibit profoundly different phenotypes when present on diverse genetic backgrounds, demonstrating that genes unrelated, per se, to the ones being targeted can play a significant role in the observed phenotype. Herein, I will discuss (1) the source of genetic variability in mutant mouse models, (2) the appropriateness of using inbred mice as controls, and (3) strategies to help minimize genetic variation between experimental and control mice.