bluuebunny/crossref_metadata_2025 huggingface dataset:
Association Between Polymorphism in the Chemokine Receptor CX3CR1 and Coronary Vascular Endothelial Dysfunction and Atherosclerosis
Authors: McDermott David H. , Halcox Julian P.J. , Schenke William H. , Waclawiw Myron A. , Merrell Maya N. , Epstein Neal , Quyyumi Arshed A. , Murphy Philip M.
Published: September 2007

Fractalkine, a chemokine expressed by inflamed endothelium, induces leukocyte adhesion and migration via the receptor CX3CR1, and the CX3CR1 polymorphism V249I affects receptor expression and function. Here we show that this polymorphism is an independent risk factor for atherosclerotic coronary artery disease (CAD). Genotyping of the CX3CR1-V249I polymorphism was performed in a cohort of 339 white individuals who underwent cardiac catheterization (n=197 with and n=142 without CAD, respectively). In 203 patients, intracoronary acetylcholine 15 μg/min) and sodium nitroprusside (20 μg/min) were administered to test endothelium-dependent and -independent coronary vascular function, respectively. Change in coronary vascular resistance (ΔCVR) was measured as an index of microvascular dilation. An association was observed between presence of the CX3CR1 I249 allele and reduced prevalence of CAD, independent of established CAD risk factors (odds ratio=0.54 [95% confidence interval, 0.30 to 0.96],P=0.03). Angiographic severity of CAD was also lower in these subjects (P=0.01). Furthermore, endothelium-dependent vasodilation was greater in these individuals compared with individuals homozygous for the CX3CR1-V249 allele (ΔCVR during acetylcholine = −46±3% versus −36±3%, respectively,P=0.02), whereas ΔCVR with sodium nitroprusside was similar in both groups (−55±2% versus −53±2%,P=0.45). The association between CX3CR1 genotype and endothelial function was independent of established risk factors and presence of CAD by multivariate analysis (P=0.02). Thus, the CX3CR1 I249 allele is associated with decreased risk of CAD and improved endothelium-dependent vasodilation. This suggests that CX3CR1 may be involved in the pathogenesis of CAD.
Phosphorylation of Mitochondrial Elongation Factor Tu in Ischemic Myocardium
Authors: He Huaping , Chen Min , Scheffler N. Karoline , Gibson Bradford W. , Spremulli Linda L. , Gottlieb Roberta A.
Published: September 2007

The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart. We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation. We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts. We found that phosphorylation of a 46-kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts. Using 2D gel electrophoresis and mass spectrometry, we have identified the 46-kDa protein as mitochondrial translational elongation factor Tu (EF-Tu
mt
). These data reveal that ischemia and preconditioning modulate the phosphorylation of EF-Tu
mt
and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation. Phosphorylation of mitochondrial EF-Tu has not been previously described; however, in prokaryotes, EF-Tu phosphorylation inhibits protein translation. We hypothesized that phosphorylation of mitochondrial EF-Tu would inhibit mitochondrial protein translation and attempted to reproduce the effect with inhibition of mitochondrial protein synthesis by chloramphenicol. We found that chloramphenicol pretreatment significantly reduced infarct size, suggesting that mitochondrial protein synthesis is one determinant of myocardial injury during ischemia and reperfusion.
Fc Receptor–Mediated Inhibitory Effect of Immunoglobulin Therapy on Autoimmune Giant Cell Myocarditis
Authors: Shioji Keisuke , Kishimoto Chiharu , Sasayama Shigetake
Published: September 2007

Abstract—

In the present study, the mechanisms and importance of the Fc portion of immunoglobulin in experimental giant cell myocarditis were examined. Giant cell myocarditis was induced in rats by immunization of porcine cardiac myosin. Human intact immunoglobulin (1 g · kg
−1
· d
−1
) or F(ab′)
2
fragments of human immunoglobulin (1 g · kg
−1
· d
−1
) were administered intraperitoneally daily on days 1 to 21. Intact immunoglobulin administration significantly ameliorated myocarditis, but F(ab′)
2
fragments did not. The ribonuclease protection assay revealed that therapy with intact immunoglobulin, but not F(ab′)
2
fragments, suppressed the mRNA expressions of inflammatory and proinflammatory cytokines. Immunohistochemical analysis showed that therapy with intact immunoglobulin, but not F(ab′)
2
fragments, suppressed dendritic cell (DC) expression during both the early and the subsequent fulminant phases. Moreover, the early treatment of intact immunoglobulin until the 11th day or 14th day, when the expression of DCs was completely suppressed, ameliorated myocarditis. However, the late treatment of intact immunoglobulin beginning on day 15, when the expression of DCs had already been completed, failed to ameliorate the condition. An in vitro study showed that intact immunoglobulin, but not F(ab′)
2
fragments, suppressed the lipopolysaccharide-induced interleukin-1β production associated with the downregulation of CD32 antigen (Fcγ receptor II) expression. Thus, intact immunoglobulin therapy markedly suppressed myocarditis as a result of Fc receptor–mediated anti-inflammatory action, and the suppression of the disease was associated with the suppression of DCs, ie, the suppression of the initial antigen-priming process in experimental giant cell myocarditis.
Constitutive and Inducible Expression ofCyp1a1andCyp1b1in Vascular Smooth Muscle Cells
Authors: Kerzee J. Kevin , Ramos Kenneth S.
Published: September 2007

Abstract—Ahr is a ligand-activated bHLH/PAS transcription factor involved in cytochrome P450 (CYP) gene regulation and murine susceptibility to atherogenic stimuli. The present studies were conducted to examine constitutive and inducible expression ofCyp1a1andCyp1b1in vascular smooth muscle cells (VSMCs) from Ahr+/+and Ahr−/−mice.Cyp1a1mRNA was not expressed constitutively in VSMCs irrespective of Ahr phenotype. AlthoughCyp1a1was inducible in Ahr+/+by 3 μmol/L benzo(a)pyrene, a known hydrocarbon inducer, the protein was uninducible. In contrast,Cyp1b1mRNA and protein were expressed under constitutive and inducible conditions irrespective of Ahr phenotype or growth status. CYP-encoded aryl hydrocarbon hydroxylase activity was higher in Ahr−/−VSMCs under constitutive conditions and induced by benzo(a)pyrene in Ahr+/+and Ahr−/−VSMCs. CYP expression was influenced by mitogenic status, because randomly cycling cells consistently exhibited higher levels than growth-arrested counterparts. Actinomycin D (2 μg/mL) or cycloheximide (10 μmol/L) did not inhibit constitutive or hydrocarbon-inducible aryl hydrocarbon hydroxylase activity in VSMCs. These data indicate that in murine VSMCs, expression ofCyp1alandCyp1b1is differentially influenced by Ahr phenotype and mitogenic status, with patterns that may dictate inherent susceptibility to atherogenic stimuli.
Integrins in Vascular Development
Authors: Rupp Paul A. , Little Charles D.
Published: September 2007

In recent years, there has been a sustained interest in vascularization processes. Much, if not all, of the work has included the concept of new vessel morphogenesis. Surprisingly, most of the work has not addressed developmental mechanisms directly, but rather as an offshoot of a disease process, wound healing process, or from the perspective of inducing vessels in an ischemic site. One theme has dominated the various studies on capillary or endothelial tube morphogenesis—integrin-mediated cell behavior. Integrin biology impacts virtually every known step of nascent vessel formation. In this review article, we attempted to summarize key findings from the viewpoint of developmental biologists/morphologists. We also attempted to summarize and contrast data obtained using integrin gene ablation approaches in mice with other experimental systems. It is hoped this review will provide a distinct cell biological perspective to vascular scientists from the clinical, molecular, and tissue engineering communities.
Proangiogenic Effect of Angiotensin-Converting Enzyme Inhibition Is Mediated by the Bradykinin B
2
Receptor Pathway
Authors: Silvestre Jean-Sébastien , Bergaya Sonia , Tamarat Radia , Duriez Micheline , Boulanger Chantal M. , Levy Bernard I.
Published: September 2007

Recent studies have suggested a proangiogenic effect of angiotensin-converting enzyme (ACE) inhibition. We hypothesized that such a proangiogenic effect of ACE inhibition may be mediated, in part, by bradykinin (BK) B
2
-receptor pathway. This study therefore examined the neovascularization induced by ACE inhibitor treatment in B
2
receptor–deficient mice (B
2
−/−
) in a model of surgically induced hindlimb ischemia. After artery femoral occlusion, wild-type and B
2
−/−
mice were treated with or without ACE inhibitor (perindopril, 3 mg/kg/d) for 28 days. Angiogenesis was then quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. The protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were determined by Western blot. In wild-type animals, vessel density and capillary number in the ischemic leg were raised by 1.8- and 1.4-fold, respectively, in mice treated with ACE inhibitor when compared with the nontreated animals (
P
<0.01). This corresponded to an improved ischemic/nonischemic leg perfusion ratio by 1.5-fold in ACE inhibitor–treated animals when compared with the untreated ones (0.87±0.07 versus 0.59±0.05, respectively,
P
<0.01). Activation of the angiogenic process was also associated with a 1.7-fold increase in tissue eNOS protein level in mice treated with ACE inhibitor (
P
<0.05 versus control) but not with changes in VEGF protein level. Conversely, ACE inhibition did not affect vessel density, blood flow, and eNOS protein level in ischemic hindlimb of B
2
−/−
mice. Therefore, proangiogenic effect of ACE inhibition is mediated by B
2
-receptor signaling and was associated with upregulation of eNOS content, independently of VEGF expression.
Rapid Reversal of the Diabetic Endothelial Dysfunction by Pharmacological Inhibition of Poly(ADP-Ribose) Polymerase
Authors: Soriano Francisco Garcia , Pacher Pál , Mabley Jon , Liaudet Lucas , Szabó Csaba
Published: September 2007

Oxygen- and nitrogen-derived free radicals and oxidants play an important role in the pathogenesis of diabetic endothelial dysfunction. Recently we proposed the importance of oxidant-induced DNA strand breakage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in the pathogenesis of diabetic endothelial dysfunction. In this study, we tested whether established diabetic endothelial dysfunction is reversible by PARP inhibition. The novel PARP inhibitor PJ34 (10 mg/kg per day PO) was given at various lengths (4 weeks or 3 days) for established streptozotocin-diabetic animals. In addition, we also tested whether incubation of the aortic rings with PJ34 (3 μmol/L) or a variety of other PARP inhibitors for 1 hour affects the diabetic vascular changes. Both 4-week and 3-day PARP-inhibitor treatment of streptozotocin-diabetic mice with established endothelial dysfunction fully reversed the acetylcholine-induced endothelium-dependent relaxations in vitro. Furthermore, 1-hour in vitro incubation of aortae from streptozotocin-diabetic mice with various PARP inhibitors was able to reverse the endothelial dysfunction. ATP, NAD
+
, and NADPH levels were markedly reduced in diabetic animals, and PARP-inhibitor treatment was able to restore these alterations. Unexpectedly, pharmacological inhibition of PARP not only prevents the development of the endothelial dysfunction but is also able to rapidly reverse it. Thus, PARP activation and the associated metabolic compromise represent an ongoing process in diabetic blood vessels. Pharmacological inhibition of this process is able to reverse diabetic endothelial dysfunction.
Reciprocal Antagonism Between Estrogen Receptor and NF-κB Activity In Vivo
Authors: Evans Mark J. , Eckert Amy , Lai KehDih , Adelman Steven J. , Harnish Douglas C.
Published: September 2007

The functional interaction, or “cross-talk,” between estrogen receptor (ER) and the proinflammatory transcription factor nuclear factor (NF)-κB demonstrated in vitro has been suggested to play a role in estrogen prevention of cardiovascular disease. Here, we demonstrate that this reciprocal cross-talk occurs in vivo. Ovariectomized C57BL/6 mice fed an atherogenic diet had increased hepatic levels of active NF-κB and numerous inflammatory genes, including MHC invariant chain (Ii), vascular cell adhesion molecule-1, tumor necrosis factor-α, and RANTES. Treatment with 17α-ethinylestradiol (EE) strongly blocked induction of these genes but had no effect on their basal expression levels. ER was required for this activity, because the antagonist ICI 182,780 completely blocked the inhibitory activity of EE. Gene activation by EE was not required for inhibition of inflammatory gene expression, because both the phytoestrogen genistein and low doses of EE were effective in blocking inflammatory gene induction without inducing marker genes such as intestinal trefoil factor (ITF) or myo-inositol-1-phosphate synthase (IPS). The in vivo transcriptional interference was reciprocal, with EE induction of ITF and IPS greatly reduced in animals fed the atherogenic diet versus chow-fed controls. This interference was specific to the liver, because diet had no effect on uterine weight increases produced by EE. Transfection experiments confirmed that the extent of inhibition of ER-mediated transcription by inflammatory stimuli correlated with the extent of NF-κB activation. These results suggest that the cross-talk between ER and NF-κB does occur in vivo and may indeed contribute significantly to the cardioprotective effects of estrogen.
Extracellular Nucleotides Induce Arterial Smooth Muscle Cell Migration Via Osteopontin
Authors: Chaulet Hervé , Desgranges Claude , Renault Marie-Ange , Dupuch Françoise , Ezan Gérome , Peiretti Franck , Loirand Gervaise , Pacaud Pierre , Gadeau Alain-Pierre
Published: September 2007

Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y
2
ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors α
v
β
3
/β
5
by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.
Cytochrome P450 and Vascular Homeostasis
Authors: Fleming Ingrid
Published: September 2007

Since the initial reports that renal cytochrome P450 (CYP) enzymes can metabolize arachidonic acid to substances which affect arterial tone, it has become increasingly clear that CYP enzymes expressed within the cardiovascular system play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP epoxygenase in endothelial cells is an essential step in nitric oxide and prostacyclin-independent vasodilatation of several vascular beds, particularly in the heart and kidney. A smooth muscle CYP ω-hydroxylase, on the other hand, generates a vasoconstrictor eicosanoid that is central to the myogenic response. Moreover, CYP epoxygenase and ω-hydroxylase products, as well as CYP-derived reactive oxygen species, are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. This review summarizes the vascular effects of epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid, both of which are CYP-derived metabolites of arachidonic acid, endogenously generated within endothelial and vascular smooth muscle cells. Although the link between CYP expression/activity and cardiovascular disease is currently tentative, the evidence being accumulated to suggest that CYP pathways are altered in animal models of hypertension and atherosclerosis can no longer be ignored. The development of selective pharmacological tools is, however, a prerequisite for the analysis of the involvement of specific CYP isoforms in the regulation of vascular homeostasis in human subjects.
Mitochondrial ATP-Sensitive Potassium Channels Attenuate Matrix Ca
2+
Overload During Simulated Ischemia and Reperfusion
Authors: Murata Mitsushige , Akao Masaharu , O’Rourke Brian , Marbán Eduardo
Published: September 2007

Mitochondrial ATP-sensitive potassium (mitoK
ATP
) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca
2+
concentration ([Ca
2+
]
m
), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoK
ATP
channel opener diazoxide attenuated the accumulation of [Ca
2+
]
m
during simulated I/R (EC
50
=18 μmol/L). These effects of diazoxide were blocked by the mitoK
ATP
channel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca
2+
]
m
accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, ΔΨ
m
, in permeabilized myocytes revealed that diazoxide depolarized ΔΨ
m
(by 12% at 10 μmol/L,
P
<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca
2+
overload, as a consequence of partial mitochondrial membrane depolarization by mitoK
ATP
channels, underlies cardioprotection. Furthermore, mitoK
ATP
channels and the MPT differentially affect mitochondrial calcium homeostasis: mitoK
ATP
channels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion.
Maternal Hypercholesterolemia and Treatment During Pregnancy Influence the Long-Term Progression of Atherosclerosis in Offspring of Rabbits
Authors: Palinski Wulf , D’Armiento Francesco P. , Witztum Joseph L. , de Nigris Filomena , Casanada Florencia , Condorelli Mario , Silvestre Mercedes , Napoli Claudio
Published: September 2007

Maternal hypercholesterolemia during pregnancy is associated with enhanced fatty streak formation in human fetuses and faster progression of atherosclerosis during childhood even under normocholesterolemic conditions. A causal role of maternal hypercholesterolemia in lesion formation during fetal development has previously been established in rabbits. The same experimental model is now used to establish that maternal hypercholesterolemia or ensuing pathogenic events in fetal arteries enhance atherogenesis later in life. Five groups of rabbit mothers were fed chow, cholesterol-enriched chow, or cholesterol-enriched chow plus 1000 IU vitamin E, 3% cholestyramine, or both during pregnancy. Offspring of all groups (n=136) were fed a mildly hypercholesterolemic diet for up to a year and had similar cholesterol levels. Aortic lesion sizes and lipid peroxidation products in plasma and lesions in offspring were determined at birth, 6 months, or 12 months. Lesion progression in offspring of hypercholesterolemic mothers was greater than in all other groups. At each time point, offspring of hypercholesterolemic mothers had 1.5- to 3-fold larger lesions than offspring of normocholesterolemic mothers (
P
<0.01), with the greatest absolute differences at 12 months. Maternal treatment reduced lesions by 19% to 53%, compared with offspring of untreated hypercholesterolemic mothers (
P
<0.01), with the greatest effect in the vitamin E groups. At 12 months, lesions in offspring of all vitamin E and cholestyramine-treated mothers were similar to those of normocholesterolemic mothers. Lipid peroxidation end-products in lesions and plasma showed analogous differences between groups as lesions (
P
<0.01). Thus, pathogenic programming in utero increases the susceptibility to atherogenic risk factors later in life and maternal intervention with cholesterol-lowering drugs or antioxidants reduce postnatal lipid peroxidation and atherosclerosis in their offspring.
Shear Stress Regulates Endothelial Nitric Oxide Synthase Expression Through c-Src by Divergent Signaling Pathways
Authors: Davis Michael E. , Cai Hua , Drummond Grant R. , Harrison David G.
Published: September 2007

In this study, we defined the signaling cascade responsible for increased eNOS mRNA expression in response to laminar shear stress. This pathway depends on the tyrosine kinase c-Src because shear induction of eNOS mRNA is blocked by the c-Src inhibitors PP1 and PP2, as well as an adenovirus encoding kinase inactive c-Src. After activation of c-Src, this pathway diverges. One arm is responsible for the short-term (6 hour) increase in eNOS mRNA. This involves a transient, 1-hour increase in eNOS transcription, as detected by nuclear run-on, that is dependent on activation of Ras and is blocked by adenoviral infection with dominant negative Ras. Downstream of Ras, MEK1/2 and ERK1/2 are important in this pathway, as 2 inhibitors of MEK1/2, PD98059 and UO126, completely prevented this early increase in eNOS mRNA. ERK1/2 was rapidly phosphorylated in response to shear, and this was prevented by c-Src and Ras inhibition. Further, Raf is phosphorylated in response to shear stress, and this is prevented by c-Src inhibition, suggesting that Raf may transduce the signal between Ras and ERK1/2. The second arm of the pathway linking activation of c-Src to eNOS expression involves stabilization of eNOS mRNA by shear stress. This response to shear is completely abrogated by the c-Src inhibitor PP1 but not altered by Ras or MEK1/2 inhibition. Thus, c-Src plays a central role in modulation of eNOS expression in response to shear stress via divergent pathways involving a short-term increase in eNOS transcription and a longer-term stabilization of eNOS mRNA.
The Complete Gene Sequence of Titin, Expression of an Unusual ≈700-kDa Titin Isoform, and Its Interaction With Obscurin Identify a Novel Z-Line to I-Band Linking System
Authors: Bang Marie-Louise , Centner Thomas , Fornoff Friderike , Geach Adam J. , Gotthardt Michael , McNabb Mark , Witt Christian C. , Labeit Dietmar , Gregorio Carol C. , Granzier Henk , Labeit Siegfried
Published: September 2007

Titin is a giant vertebrate striated muscle protein with critical importance for myofibril elasticity and structural integrity. We show here that the complete sequence of the human titin gene contains 363 exons, which together code for 38 138 residues (4200 kDa). In its central I-band region, 47 novel PEVK exons were found, which contribute to titin’s extensible spring properties. Additionally, 3 unique I-band titin exons were identified (named novex-1 to -3). Novex-3 functions as an alternative titin C-terminus. The novex-3 titin isoform is ≈700 kDa in size and spans from Z1-Z2 (titin’s N-terminus) to novex-3 (C-terminal exon). Novex-3 titin specifically interacts with obscurin, a 721-kDa myofibrillar protein composed of 57 Ig/FN3 domains, followed by one IQ, SH3, DH, and a PH domain at its C-terminus. The obscurin domains Ig48/Ig49 bind to novex-3 titin and target to the Z-line region when expressed as a GFP fusion protein in live cardiac myocytes. Immunoelectron microscopy detected the C-terminal Ig48/Ig49 obscurin epitope near the Z-line edge. The distance from the Z-line varied with sarcomere length, suggesting that the novex-3 titin/obscurin complex forms an elastic Z-disc to I-band linking system. This system could link together calcium-dependent, SH3-, and GTPase-regulated signaling pathways in close proximity to the Z-disc, a structure increasingly implicated in the restructuring of sarcomeres during cardiomyopathies.
Contribution of the 5-HT
1B
Receptor to Hypoxia-Induced Pulmonary Hypertension
Authors: Keegan Alan , Morecroft Ian , Smillie Diane , Hicks Martin N. , MacLean Margaret R.
Published: September 2007

5-Hydroxytryptamine (5-HT)
1B
receptors mediate contraction in human pulmonary arteries, and 5-HT
1B
receptor-mediated contraction is enhanced in pulmonary arteries from hypoxic rats. Here we further examine the role of this receptor in the development of pulmonary hypertension (PHT) by examining (1) the effects of a 5-HT
1B/1D
-receptor antagonist (GR127935) on hypoxia-induced PHT (CHPHT) in rats and (2) CHPHT in 5-HT
1B
-receptor knockout mice. In rats, hypoxia increased right ventricular pressure and right ventricular hypertrophy and induced pulmonary vascular remodeling associated with an increase in pulmonary arterial wall thickness. GR127935 (3 mg · kg
−1
· d
−1
) reduced all of these indices. 5-HT
1
-mediated contraction was enhanced in pulmonary arteries of the CHPHT rats. The effects of GR127935 on PHT indices were associated with an attenuation of the enhanced contractile responses to 5-HT and the 5-HT
1
-receptor agonist, 5-carboxamidotryptamine (5-CT), in isolated pulmonary arteries. In wild-type mice, hypoxia increased right ventricular hypertrophy, which was absent in 5-HT
1B
-receptor knockout mice. Hypoxia increased pulmonary vascular remodeling in wild-type mice, and this was reduced in the 5-HT
1B
-receptor knockout mice. Hypoxia increased 5-HT
1
-mediated contraction in pulmonary arteries from the wild-type mice and this was attenuated in the 5-HT
1B
-receptor knockout mice. In conclusion, the 5-HT
1B
receptor plays a role in the development of CHPHT. One possible mechanism may be via enhanced 5-HT
1
receptor-mediated contraction of the pulmonary arterial circulation.
Transforming Growth Factor-β
1
Modulates Oxidatively Modified LDL–Induced Expression of Adhesion Molecules
Authors: Chen Hongjiang , Li Dayuan , Saldeen Tom , Mehta Jawahar L.
Published: September 2007

Oxidatively modified LDL (ox-LDL) activates a lectin-like receptor, LOX-1, which results in the expression of adhesion molecules on endothelial surface. We investigated the regulation of the expression of transforming growth factor-β
1
(TGF-β
1
) and its receptors by ox-LDL and the functional significance of this interaction with regard to adhesion molecule expression in human coronary artery endothelial cells (HCAECs). Ox-LDL, in a time- and concentration-dependent manner, upregulated the expression of all 3 subtypes (1, 2, and 3 [including endoglin]) of TGF-β
1
receptors and decreased active TGF-β
1
synthesis (all
P
<0.05 versus control and native-LDL–treated cells). Treatment of HCAECs with a monoclonal antibody to LOX-1 attenuated ox-LDL–mediated upregulation of TGF-β
1
receptors and decrease in TGF-β
1
synthesis (
P
<0.05 versus ox-LDL alone). Ox-LDL also enhanced the expression of P-selectin and ICAM-1 as well as monocyte adhesion to HCAECs (
P
<0.05 versus control untreated cells). Pretreatment with recombinant TGF-β
1
attenuated the enhanced expression of adhesion molecules and monocyte adhesion to HCAECs (
P
<0.05 versus ox-LDL alone). Effects of recombinant TGF-β
1
were blocked by antibody to TGF-β
1
receptor type 2, but not by antibody to endoglin. Thus ox-LDL, via activation of LOX-1, increases the expression of TGF-β
1
receptors and decreases TGF-β
1
synthesis in HCAECs. Recombinant TGF-β
1
, by binding to TGF-β
1
type 2 receptors, modulates ox-LDL–mediated expression of adhesion molecules and monocyte adhesion to HCAECs.
Intrinsic Diurnal Variations in Cardiac Metabolism and Contractile Function
Authors: Young Martin E. , Razeghi Peter , Cedars Ari M. , Guthrie Patrick H. , Taegtmeyer Heinrich
Published: September 2007

Diurnal variation of cardiac function in vivo has been attributed primarily to changes in factors such as sympathetic activity. No study has investigated previously the intrinsic properties of the heart throughout the day. We therefore investigated diurnal variations in metabolic flux and contractile function of the isolated working rat heart and how this related to circadian expression of metabolic genes. Contractile performance, carbohydrate oxidation, and oxygen consumption were greatest in the middle of the night, with little variation in fatty acid oxidation. The expression of all metabolic genes investigated (including regulators of carbohydrate utilization, fatty acid oxidation, and mitochondrial function) showed diurnal variation, with a general peak in the night. In contrast, pressure overload–induced cardiac hypertrophy completely abolished this diurnal variation of metabolic gene expression. Thus, over the course of the day, the normal heart anticipates, responds, and adapts to physiological alterations within its environment, a trait that is lost by the hypertrophied heart. We speculate that loss of plasticity of the hypertrophied heart may play a role in the subsequent development of contractile dysfunction.
Recombinant Annexin II Modulates Impaired Fibrinolytic Activity In Vitro and in Rat Carotid Artery
Authors: Ishii Hideto , Yoshida Masayuki , Hiraoka Megumi , Hajjar Katherine A. , Tanaka Akira , Yasukochi Yukio , Numano Fujio
Published: September 2007

Fibrinolytic activity has been reported to be decreased in atherosclerosis. Recently, annexin II was identified as a coreceptor on endothelial cells for plasminogen and tissue plasminogen activator. In this study, we examined whether recombinant annexin II (rAN II) protein can modulate fibrinolytic activity on vascular endothelium in vitro and in vivo. The effect of rAN II on human umbilical vein endothelial cells (HUVECs) was measured. Addition of a fluorescent plasmin substrate revealed that HUVECs treated with rAN II exhibited significantly more plasmin generation than those treated with BSA. Moreover, rAN II treatment of HUVECs restored plasmin generation impaired by plasminogen activator inhibitor-1 or homocysteine pretreatment. In a rat carotid artery thrombus model, the patency of thrombosed carotid arteries was significantly enhanced by rAN II injection, in contrast to BSA injection, without systemic blood coagulation dysregulation. We found that rAN II enhanced plasmin generation on vascular endothelium in vitro and reduced thrombus formation in vivo, and concluded that enhancement of endothelial fibrinolytic activity by annexin II could modulate the hypercoagulable state of atherosclerosis. Further study of rAN II in vitro and in vivo may lead to the establishment of novel therapeutic approaches to thrombogenic vascular disease.
CD40 Signaling and Plaque Instability
Authors: Schönbeck Uwe , Libby Peter
Published: September 2007

Abstract
—

Today, multiple lines of evidence support the view of atherosclerosis as a chronic inflammatory disease and implicate components of the immune system in atherogenesis. Recent work has documented overexpression of the potent immune mediator CD40 and its counterpart CD40 ligand (CD40L) in experimental and human atherosclerotic lesions. Notably, interruption of CD40/CD40L interactions not only diminished the formation and progression of mouse atheroma, but also fostered changes in lesion biology and structure, which are associated in humans with “plaque stabilization.” In accordance with the hypothesis that CD40 signaling promotes plaque instability, in vitro studies demonstrated that ligation of CD40 on atheroma-associated cell types, namely endothelial cells, smooth muscle cells, and macrophages, mediates functions considered crucial to the process of atherogenesis, such as the expression of cytokines, chemokines, growth factors, matrix metalloproteinases, and procoagulants. The combination of the broad gamut of proatherogenic biological responses triggered by ligation of CD40 on endothelial cells, smooth muscle cells, and macrophages in vitro and the results of in vivo studies of interruption of CD40 signaling suggests a central role for this receptor/ligand dyad during atherogenesis, proposing CD40/CD40L interactions as a novel potential therapeutic target for this prevalent human disease.
Characteristics and Superoxide-Induced Activation of Reconstituted Myocardial Mitochondrial ATP-Sensitive Potassium Channels
Authors: Zhang David X. , Chen Ya-Fei , Campbell William B. , Zou Ai-Ping , Gross Garrett J. , Li Pin-Lan
Published: September 2007

Mitochondrial ATP-sensitive potassium (mitoKATP) channels have been suggested as triggers and end effectors in myocardial ischemic preconditioning. However, the intracellular mechanism regulating mitoKATPchannels remains unclear. In the present study, mitoKATPchannels from bovine ventricular myocardium were reconstituted using planar lipid bilayers, and the effect of superoxide (O2−·) on the activity of these reconstituted channels was examined. After incorporation, a potassium-selective current was recorded. The mean conductance of this current was 56 pS at 150 mmol/L KCl, which was substantially inhibited by 1 mmol/L MgATP. 5-Hydroxydecanoate (5-HD, 10 to 100 μmol/L), a selective mitoKATPantagonist, reduced the open state probability (NPo) of these channels in a concentration-dependent manner, whereas diazoxide (10 μmol/L), a selective mitoKATPagonist, significantly increased channel activity. HMR-1098 (100 μmol/L), a selective sarcolemmal KATPantagonist, had no effect on the activity of reconstituted channels. Addition of xanthine/xanthine oxidase (100 μmol/L per 0.038 U/mL), an O2−·-generating system, resulted in a marked activation of mitoKATPchannels; the NPo of the channels was increased from 0.60±0.10 to 1.94±0.02. This O2−·-induced channel activation was completely abolished by pretreatment with 5-HD (100 μmol/L) or a sulfhydryl alkylating compound,N-ethylmaleimide (2 mmol/L). It is concluded that myocardial mitoKATPchannels can be reconstituted into lipid bilayers and that O2−·activates these channels. The effect of O2−·may be associated with its direct action on the sulfhydryl groups of the channel protein.
Molecular Basis for Angiotensin II-Induced Increase of Chloride/Bicarbonate Exchange in the Myocardium
Authors: Alvarez Bernardo V. , Fujinaga Jocelyne , Casey Joseph R.
Published: September 2007

Plasma membrane anion exchangers (AEs) regulate myocardial intracellular pH (pHi) by Na+-independent Cl−/HCO3−exchange. Angiotensin II (Ang II) activates protein kinase C (PKC) and increases anion exchange activity in the myocardium. Elevated anion exchange activity has been proposed to contribute to the development of cardiac hypertrophy. Our Northern blots showed that adult rat heart expresses AE1, AE2, AE3fl, and AE3c. Activity of each AE isoform was individually measured by following changes of pHi, associated with bicarbonate transport, in transfected HEK293 cells. Exposure to the PKC activator, PMA (150 nmol/L), increased the transport activity of only the AE3fl isoform by 50±11% (P<0.05, n=6), consistent with the increase observed in intact myocardium. Cotransfection of HEK293 cells with AE3fl and AT1a-Ang II receptors conferred sensitivity of anion transport to Ang II (500 nmol/L), increasing the transport activity by 39±3% (P<0.05, n=4). PKC inhibition by chelerythrine (10 μmol/L) blocked the PMA effect. To identify the PKC-responsive site, 7 consensus PKC phosphorylation sites of AE3fl were individually mutated to alanine. Mutation of serine 67 of AE3 prevented the PMA-induced increase of anion transport activity. Inhibition of MEK1/2 by PD98059 (50 μmol/L) did not affect the response of AE3fl to Ang II, indicating that PKC directly phosphorylates AE3fl. We conclude that following Ang II stimulation of cells, PKCε phosphorylates serine 67 of the AE3 cytoplasmic domain, inducing the Ang II-induced increase in anion transport observed in the hypertrophic myocardium.
Aortic Aneurysm Diameter and Risk of Cardiovascular Mortality
Authors: Brady Anthony R. , Fowkes F. Gerald R. , Thompson Simon G. , Powell Janet T.
Published: September 2007

Abstract
—After successful surgical repair of an abdominal aortic aneurysm, patients have for many years an increased risk of death from cardiovascular causes. We have tested the hypothesis that for patients with abdominal aortic aneurysms, the risk of nonaneurysm cardiovascular mortality before and after surgery increased with aneurysm diameter. Records of aneurysm repair or rupture and mortality were available from 2305 patients entered into the UK Small Aneurysm Trial and Study. Two hundred fifty-nine deaths occurred before aneurysm repair or rupture (mean follow-up 1.7 years), and 325 occurred after surgical repair (mean follow-up 3.6 years). The risk of nonaneurysm-related mortality and cardiovascular death before and after surgery increased with aneurysm diameter at baseline, even after adjustment for other known risk factors. The adjusted hazard ratios for cardiovascular mortality, per standard deviation (0.8-cm) increase in aneurysm diameter, were 1.34 (95% CI 1.01 to 1.79) and 1.31 (95% CI 1.06 to 1.63) in the periods before aneurysm repair or rupture and after aneurysm repair, respectively. The significant association between aortic diameter and cardiovascular mortality, excluding aneurysm-related deaths, suggests that aneurysm diameter is an independent marker of cardiovascular disease risk.
Folate Improves Endothelial Function in Coronary Artery Disease
Authors: Doshi Sagar N. , McDowell Ian F. W. , Moat Stuart J. , Lang Derek , Newcombe Robert G. , Kredan Mahmud B. , Lewis Malcolm J. , Goodfellow Jonathan
Published: September 2007

Abstract
—Homocysteine is a risk factor for coronary artery disease (CAD). Folic acid lowers homocysteine and may improve endothelial function in CAD, although the mechanism is unclear. We investigated the effect of folic acid on endothelial function, homocysteine, and oxidative stress in patients with CAD. We also examined the acute effect of 5-methyltetrahydrofolate (5-MTHF), the principal circulating folate, on endothelial function in vivo and on intracellular superoxide in cultured endothelial cells. A randomized crossover study of folic acid (5 mg daily) for 6 weeks was undertaken in 52 patients with CAD. Ten further patients were given intra-arterial 5-MTHF. Endothelial function was assessed by flow-mediated dilatation (FMD). Folic acid increased plasma folate (
P
<0.001), lowered homocysteine by 19% (
P
<0.001), and improved FMD (
P
<0.001). FMD improvement did not correlate with homocysteine reduction. Malondialdehyde and total plasma antioxidant capacity, markers of oxidative stress, were unchanged. 5-MTHF acutely improved FMD (
P
<0.001) without altering homocysteine (
P
=0.47). In vitro, 5-MTHF abolished homocysteine-induced intracellular superoxide increase (
P
<0.001); this effect was also observed with folic acid and tetrahydrobiopterin. Our data support the beneficial effect of folic acid on endothelial function in CAD but suggest that the mechanism is independent of homocysteine. Reduction of intracellular endothelial superoxide may have contributed to the effect.
Cell Cycle Protein Expression in Vascular Smooth Muscle Cells In Vitro and In Vivo Is Regulated Through Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin
Authors: Braun-Dullaeus Ruediger C. , Mann Michael J. , Seay Ulrike , Zhang Lunan , von der Leyen Heiko E. , Morris Randall E. , Dzau Victor J.
Published: September 2007

Abstract—Cell cycle progression represents a key event in vascular proliferative diseases, one that depends on an increased rate of protein synthesis. An increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity is associated with vascular smooth muscle cell proliferation, and rapamycin, which blocks the activity of the mammalian target of rapamycin, inhibits this proliferation in vitro and in vivo. We hypothesized that these 2 molecules converge on a critical pathway of translational regulation that is essential for successful upregulation of cell cycle–regulatory proteins in activated smooth muscle cells. p70S6kinase, a target of PI 3-kinase and the mammalian target of rapamycin, was rapidly activated on growth factor stimulation of quiescent coronary artery smooth muscle cells and after balloon injury of rat carotid arteries. The translational repressor protein 4E-binding protein 1 was similarly hyperphosphorylated under these conditions. These events were associated with increases in the protein levels of cyclin B1, cyclin D1, cyclin E, cyclin-dependent kinase 1, cyclin-dependent kinase 2, proliferating cell nuclear antigen, and p21Cip1in vivo and in vitro, whereas inhibition of the PI 3-kinase signaling pathway with either rapamycin or wortmannin blocked the upregulation of these cell cycle proteins, but not mRNA, and arrested the cells in vitro before S phase. In contrast to findings in other cell types, growth factor– or balloon injury–induced downregulation of the cell cycle inhibitor p27Kip1was not affected by rapamycin treatment. These data suggest that cell cycle progression in vascular cells in vitro and in vivo depends on the integrity of the PI 3-kinase signaling pathway in allowing posttranscriptional accumulation of cell cycle proteins.
Complement Components, but Not Complement Inhibitors, Are Upregulated in Atherosclerotic Plaques
Authors: Yasojima K. , Schwab C. , McGeer E. G. , McGeer P. L.
Published: September 2007

Abstract
—Complement activation occurs in atherosclerotic plaques. The capacity of arterial tissue to inhibit this activation through generation of the complement regulators C1 inhibitor, decay accelerating factor, membrane cofactor protein (CD46), C4 binding protein (C4BP), and protectin (CD59) was evaluated in pairs of aortic atherosclerotic plaques and nearby normal artery from 11 human postmortem specimens. All 22 samples produced mRNAs for each of these proteins. The ratios of plaque versus normal artery pairs was not significantly different from unity for any of these inhibitors. However, in plaques, the mRNAs for C1r and C1s, the substrates for the C1 inhibitor, were increased 2.35- and 4.96-fold, respectively, compared with normal artery; mRNA for C4, the target for C4BP, was elevated l.34-fold; and mRNAs for C7 and C8, the targets for CD59, were elevated 2.61- and 3.25-fold, respectively. By Western blotting and immunohistochemistry, fraction Bb of factor B, a marker of alternative pathway activation, was barely detectable in plaque and normal arterial tissue. These data indicate that it is primarily the classical, not the alternative pathway, that is activated in plaques and that key inhibitors are not upregulated to defend against this activation.
Human-Derived Anti-Oxidized LDL Autoantibody Blocks Uptake of Oxidized LDL by Macrophages and Localizes to Atherosclerotic Lesions In Vivo
Authors: Shaw Peter X. , Hörkkö Sohvi , Tsimikas Sotirios , Chang Mi-Kyung , Palinski Wulf , Silverman Gregg J. , Chen Pojen P. , Witztum Joseph L.
Published: September 2007

Autoantibodies to oxidation-specific epitopes of low density lipoprotein (LDL), such as malondialdehyde-modified LDL (MDA-LDL), occur in plasma and atherosclerotic lesions of humans and animals. Plasma titers of such antibodies are correlated with atherosclerosis in murine models, and several such autoantibodies have been cloned. However, human-derived monoclonal antibodies to epitopes of oxidized LDL (OxLDL) have not yet been reported. We constructed a phage display antibody library from a patient with high plasma anti-MDA-LDL titers and isolated 3 monoclonal IgG Fab antibodies, which specifically bound to MDA-LDL. One of these, IK17, also bound to intact OxLDL as well as to its lipid and protein moieties but not to those of native LDL. IK17 inhibited the uptake of OxLDL by macrophages and also bound to apoptotic cells and inhibited their phagocytosis by macrophages. IK17 strongly immunostained necrotic cores of human and rabbit atherosclerotic lesions. When
125
I-IK17 was injected intravenously into LDL receptor-deficient mice, its specific uptake was greatly enriched in atherosclerotic plaques versus normal aortic tissue. Human autoantibodies to OxLDL have important biological properties that could influence the natural course of atherogenesis.
Mice Expressing Only the Mutant APOE3Leiden Gene Show Impaired VLDL Secretion
Authors: Mensenkamp Arjen R. , Teusink Bas , Baller Julius F.W. , Wolters Henk , Havinga Rick , van Dijk Ko Willems , Havekes Louis M. , Kuipers Folkert
Published: September 2007

Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and show impaired very low density lipoprotein (VLDL)-triglyceride (TG) secretion. These effects are normalized on the introduction of the human APOE3 gene. To assess whether this apoE effect is isoform specific, we studied hepatic lipid metabolism in mice expressing either APOE3 or the mutant APOE3Leiden onapoe−/− orapoe+/− backgrounds. The transgenes were expressed mainly in periportal hepatocytes, as revealed by in situ hybridization. Mice expressing APOE3Leiden, on theapoe−/− andapoe+/− backgrounds, had fatty livers, which were absent in APOE3/apoe−/− mice. APOE3Leiden/apoe−/− mice showed a strongly reduced VLDL-TG secretion compared with APOE3/apoe−/− mice (48±14 versus 82±10 μmol/kg per hour, respectively). The presence of a single mouseapoeallele increased VLDL-TG secretion in APOE3Leiden/apoe+/− mice (121±43 μ mol/kg per hour) compared with APOE3Leiden/apoe−/− mice. These results show that APOE3Leiden does not prevent development of a fatty liver and does not normalize VLDL-TG secretion in mice with an apoE-deficient background. The presence of a single mouseapoeallele is sufficient to normalize the APOE3Leiden-associated reduction of VLDL-TG secretion but does not prevent steatosis. We conclude that apoE-mediated stimulation of VLDL secretion is isoform specific.