bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Variants of Angiotensin II Receptors and Cardiovascular Risk in Hypertension
Authors: Jones Alun , Dhamrait Sukhbir S. , Payne John R. , Hawe Emma , Li Ping , Toor Iqbal S. , Luong Le , Wootton Peter T.E. , Miller George J. , Humphries Steve E. , Montgomery Hugh E.
Published: August 2003

Renin-angiotensin systems may mediate cardiovascular disease pathogenesis through a balance of actions of angiotensin II on (potentially proatherogenic) constitutive type 1 (AT
1
R) and (potentially antiatherogenic) inducible type 2 (AT
2
R) receptors. We explored such potential roles in a prospective candidate gene association study. Cardiovascular end points (fatal, nonfatal, and silent myocardial infarction and coronary artery bypass surgery/angioplasty) were documented among 2579 healthy UK men (mean age, 56.1±3.5 years; median follow-up, 10.1 years) genotyped for the AT
1
R1166A>C and the X chromosome located AT
2
R1675A>G and 3123C>A polymorphisms. Baseline characteristics, including blood pressure, were independent of genotype. The AT
1
R1166CC genotype was associated with relative cardiovascular risk (hazard ratio, 1.65 [1.05 to 2.59];
P
=0.03) independent of blood pressure. Systolic blood pressure was associated with risk (
P
=0.0005), but this association was restricted to AT
2
R1675A allele carriers (
P
<0.00001), with G allele carriers protected from the risk associated with blood pressure (
P
=0.18). Hypertensive carriers with the AT
2
R1675A/3123A haplotype were at most risk, with 37.5% having an event. This is the first study to demonstrate an association of AT
2
R genotype with coronary risk, an effect that was confined to hypertensive subjects and supports the concept that the inducible AT
2
R is protective. Conversely, the AT
1
R1166CC genotype was associated with cardiovascular risk irrespective of blood pressure. These data are important to our understanding of the divergent role of angiotensin II acting at its receptor subtypes and coronary disease pathogenesis and for the development of future cardiovascular therapies.
Interactions Between Vasoconstrictors and Vasodilators in Regulating Hemodynamics of Distinct Vascular Beds
Authors: Gomez-Alamillo Carlos , Juncos Luis A. , Cases Aleix , Haas John A. , Romero J. Carlos
Published: August 2003

We examined whether interactions between angiotensin II (Ang II), endothelin (ET), nitric oxide (NO), and prostaglandins (PGs) differentially regulate perfusion to distinct vascular beds. For this, we blocked either angiotensin AT
1
or ET receptors or both and then sequentially inhibited NO and PG synthesis in anesthetized dogs. Blocking Ang II or ET had similar effects on systemic hemodynamics: Mean arterial pressure fell slightly without altering cardiac output. Blocking both caused a synergistic fall in mean arterial pressure and increased cardiac output. Pulmonary vascular resistance was not altered by blocking Ang II, ET, or both but progressively increased during NO and PG blockade in group 2 (which had unblocked ET receptors), suggesting that endogenous ET exerts pulmonary vasoconstriction that is tempered by NO and PGs. In the kidney, blocking Ang II increased regional blood flow (RBF), glomerular filtration rate (GFR), and fractional excretion of sodium (FENa). In contrast, blocking ET did not alter RBF, and it decreased GFR and FENa. Combined Ang II and ET blockade markedly increased RBF without altering GFR, and FENa was maintained at the levels as when only ET was blocked. Sequentially inhibiting NO and PGs decreased RBF when Ang II or ET were blocked but had little effect when both were blocked. Finally, Ang II or ET blockade did not alter iliac blood flow. Inhibiting NO and PGs decreased iliac blood flow when Ang II or ET but not both were blocked. These results suggest that regional differences in the interactions between endogenous Ang II, ET, NO, and PGs are important determinants in systemic, pulmonary, and regional hemodynamics.
11β-Hydroxysteroid Dehydrogenase Type 2 in Mouse Aorta
Authors: Christy Clare , Hadoke Patrick W.F. , Paterson Janice M. , Mullins John J. , Seckl Jonathan R. , Walker Brian R.
Published: August 2003

Both isozymes of 11β-hydroxysteroid dehydrogenase, which interconvert active and inactive glucocorticoids, are expressed in the mouse aortic wall. Mice deficient in 11HSD type 2 (which converts active corticosterone into inert 11-dehydrocorticosterone) have hypertension and impaired endothelial nitric oxide activity. It has been suggested that 11HSD2 influences vascular function directly by limiting glucocorticoid-mediated inhibition of endothelium-derived nitric oxide. This study sought to determine (1) the cellular distribution of the 11HSD isozymes within the mouse aortic wall and (2) the influence of 11HSD2 on direct glucocorticoid-mediated changes in aortic function. Mouse aortas were separated into their component layers and RNA extracted for RT-PCR. Both types of corticosteroid (mineralocorticoid and glucocorticoid) receptors and both 11HSD isozymes were expressed in the aortic wall. 11HSD1 expression colocalized with α-smooth muscle actin (a marker for smooth muscle cells), whereas 11HSD2 colocalized with TIE-2 (a marker for endothelial cells). Functional relaxation responses of mouse aortic rings were unaltered after exposure to glucocorticoids for 24 hours. In the presence of
l
-arginine, glucocorticoids produced an endothelium-independent reduction of contraction; similar results were obtained with aortas from mice with genetic inactivation of 11HSD2. Incubation in medium containing
l
-arginine reversed the endothelial cell dysfunction associated with 11HSD2 inactivation. Thus, 11HSD2 is appropriately sited to modulate endothelial cell function, but endothelial dysfunction in 11HSD2 knockout mice cannot be explained simply by increased access of corticosterone to endothelial cell corticosteroid receptors. Therefore, additional mechanisms, possibly involving indirect effects of enhanced corticosterone action in the kidney and the resultant hypertension, must be involved.
Beneficial Effects of Antioxidant Vitamins on the Stenotic Kidney
Authors: Chade Alejandro R. , Rodriguez-Porcel Martin , Herrmann Joerg , Krier James D. , Zhu Xiangyang , Lerman Amir , Lerman Lilach O.
Published: August 2003

Renal artery stenosis (RAS) may lead to renal injury, partly mediated through increased oxidative stress. However, the potential effects of chronic oral antioxidant intervention on the stenotic kidney remain unknown. This study was designed to test the hypothesis that chronic antioxidant vitamin supplementation in RAS would preserve renal function and structure. Single-kidney hemodynamics and function were quantified in vivo in pigs using electron-beam CT after 12 weeks of unilateral RAS (n=7), a similar degree of RAS orally supplemented with vitamins C (1 g) and E (100 IU/kg) (RAS+Vitamins, n=7), or controls (normal, n=7). Renal tissue was studied ex vivo using Western blotting and immunohistochemistry. Mean arterial pressure was similarly elevated in both RAS groups, while ischemic renal volume and glomerular filtration rate were similarly reduced. Renal blood flow was decreased in RAS compared with normal (326.5±99.9 versus 553.4±48.7 mL/min, respectively,
P
=0.01), but preserved in RAS+Vitamins (485.2±104.1 mL/min,
P
=0.3 versus normal). The marked increase in the expression of the NADPH-oxidase subunits p47phox and p67phox, nitrotyrosine, endothelial and inducible nitric oxide synthase, and nuclear factor-κB observed in RAS (
P
<0.05 versus normal) was normalized in RAS+Vitamins (
P
>0.1). Furthermore, trichrome staining and the expression of transforming growth factor-β and tissue inhibitor of matrix-metalloproteinase-1 were also decreased in RAS+Vitamins. In conclusion, chronic blockade of the oxidative stress pathway in RAS using antioxidant vitamins improved renal hemodynamics and decreased oxidative stress, inflammation, and fibrosis in the ischemic kidney. These observations underscore the involvement of oxidative stress in renal injury in RAS and support a role for antioxidant vitamins in preserving the ischemic kidney.
Role of the Mitochondrial Permeability Transition in Myocardial Disease
Authors: Weiss James N. , Korge Paavo , Honda Henry M. , Ping Peipei
Published: August 2003

Mitochondria play a key role in determining cell fate during exposure to stress. Their role during ischemia/reperfusion is particularly critical because of the conditions that promote both apoptosis by the mitochondrial pathway and necrosis by irreversible damage to mitochondria in association with mitochondrial permeability transition (MPT). MPT is caused by the opening of permeability transition pores in the inner mitochondrial membrane, leading to matrix swelling, outer membrane rupture, release of apoptotic signaling molecules such as cytochrome
c
from the intermembrane space, and irreversible injury to the mitochondria. During ischemia (the MPT priming phase), factors such as intracellular Ca
2+
accumulation, long-chain fatty acid accumulation, and reactive oxygen species progressively increase mitochondrial susceptibility to MPT, increasing the likelihood that MPT will occur on reperfusion (the MPT trigger phase). Because functional cardiac recovery ultimately depends on mitochondrial recovery, cardioprotection by ischemic and pharmacological preconditioning must ultimately involve the prevention of MPT. Investigations into this area are beginning to unravel some of the mechanistic links between cardioprotective signaling and mitochondria.
Myoendothelial Differentiation of Human Umbilical Cord Blood–Derived Stem Cells in Ischemic Limb Tissues
Authors: Pesce Maurizio , Orlandi Alessia , Iachininoto Maria Grazia , Straino Stefania , Torella Anna Rita , Rizzuti Vania , Pompilio Giulio , Bonanno Giuseppina , Scambia Giovanni , Capogrossi Maurizio C.
Published: August 2003

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34
+
/CD133
+
EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34
+
cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34
−
cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34
+
cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34
+
cells after ex vivo expansion. Myogenic conversion of human cord blood CD34
+
cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34
+
cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.
Serine Protease Inhibitor Serp-1 Strongly Impairs Atherosclerotic Lesion Formation and Induces a Stable Plaque Phenotype in ApoE
−/−
Mice
Authors: Bot Ilze , von der Thüsen Jan H. , Donners Marjo M.P.C. , Lucas Alexandra , Fekkes Madelon L. , de Jager Saskia C.A. , Kuiper Johan , Daemen Mat J.A.P. , van Berkel Theo J.C. , Heeneman Sylvia , Biessen Erik A.L.
Published: August 2003

The myxoma virus protein Serp-1 is a member of the serine protease inhibitor superfamily. Serp-1 potently inhibits human serum proteases including plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA). Serp-1 also displays a high antiinflammatory activity, rendering it a promising candidate for antiatherosclerotic therapy. In this study, we have thus examined the effect of Serp-1 on de novo atherosclerotic plaque formation and on advanced lesions. Perivascular collars were placed around carotid arteries of ApoE
−/−
mice to induce atherosclerotic plaques and Serp-1 treatment started at week 1 and week 5 after collar placement. Effects of Serp-1 on de novo atherogenesis were characterized by a significantly lower plaque size than that of control mice (18±5×10
3
versus 57±12×10
3
μm
2
, respectively;
P
=0.007). Immunostaining showed a 50% (
P
=0.004) decrease in the MOMA-2–stained lesion area of Serp-1–treated mice. Treatment of advanced lesions with Serp-1 resulted in a decrease in plaque size and lumen stenosis (
P
=0.028). α-Actin staining of these lesions was significantly increased compared with the control (
P
=0.017). In both studies, a higher cellularity of the plaque and increased collagen content was observed in Serp-1–treated mice. In vitro studies showed that Serp-1 induces proliferation and migration of vascular smooth muscle cells. In conclusion, Serp-1 inhibits carotid artery plaque growth and progression in ApoE
−/−
mice. Equally relevant, it enhances cellularity of the plaque core potentially leading to improved plaque stability. The above results indicate that Serp-1 constitutes a promising lead in antiatherosclerotic therapy.
Endothelin B Receptor Deficiency Predisposes to Pulmonary Edema Formation via Increased Lung Vascular Endothelial Cell Growth Factor Expression
Authors: Carpenter Todd , Schomberg Stacey , Steudel Wolfgang , Ozimek John , Colvin Kelley , Stenmark Kurt , Ivy D. Dunbar
Published: August 2003

Endothelin (ET) may contribute to pulmonary edema formation, particularly under hypoxic conditions, and decreases in ET-B receptor expression can lead to reduced ET clearance. ET increases vascular endothelial cell growth factor (VEGF) production in vitro, and VEGF overexpression in the lung causes pulmonary edema in vivo. We hypothesized that pulmonary vascular ET-B receptor deficiency leads to increased lung ET, that excess ET increases lung VEGF levels, promoting pulmonary edema formation, and that hypoxia exaggerates these effects. We studied these hypotheses in ET-B receptor–deficient rats. In normoxia, homozygous ET-B–deficient animals had significantly more lung vascular leak than heterozygous or control animals. Hypoxia increased vascular leak regardless of genotype, and hypoxic ET-B–deficient animals leaked more than hypoxic control animals. ET-B–deficient animals had higher lung ET levels in both normoxia and hypoxia. Lung HIF-1α and VEGF content was greater in the ET-B–deficient animals in both normoxia and hypoxia, and both HIF-1α and VEGF levels were reduced by ET-A receptor antagonism. Both ET-A receptor blockade and VEGF antagonism reduced vascular leak in hypoxic ET-B–deficient animals. We conclude that ET-B receptor–deficient animals display an exaggerated lung vascular protein leak in normoxia, that hypoxia exacerbates that leak, and that this effect is in part attributable to an ET-mediated increase in lung VEGF content.
Ca
2+
-Dependent Activation of Rho and Rho Kinase in Membrane Depolarization–Induced and Receptor Stimulation–Induced Vascular Smooth Muscle Contraction
Authors: Sakurada Sotaro , Takuwa Noriko , Sugimoto Naotoshi , Wang Yu , Seto Minoru , Sasaki Yasuharu , Takuwa Yoh
Published: August 2003

Ca
2+
sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase–dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca
2+
-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca
2+
or the addition of a dihydropyridine Ca
2+
channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N
19
RhoA suppressed Ca
2+
-induced Thr
695
phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca
2+
removal and depletion of the intracellular Ca
2+
store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A
2
analogue–induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca
2+
-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.
Role of Phosphodiesterase 3 in NO/cGMP-Mediated Antiinflammatory Effects in Vascular Smooth Muscle Cells
Authors: Aizawa Toru , Wei Heng , Miano Joseph M. , Abe Jun-ichi , Berk Bradford C. , Yan Chen
Published: August 2003

Atherosclerosis involves cellular immune responses and altered vascular smooth muscle cell (VSMC) function. Nitric oxide (NO)/cGMP is uniquely capable of inhibiting key processes in atherosclerosis. In this study, we determined the effects of NO/cGMP and their molecular mechanisms in the regulation of NF-κB–dependent gene expression in VSMCs. We found that cGMP-elevating agents such as the NO donor
S
-nitroso-
N
-acetylpenicillamine (SNAP) and C-type natriuretic peptide (CNP), reduced TNF-α–induced NF-κB–dependent reporter gene expression in rat aortic VSMCs in a cGMP-dependent manner. The effects of SNAP and CNP on NF-κB are mediated by cAMP-dependent protein kinase (PKA) but not cGMP-dependent protein kinase (PKG) based on the findings that the selective PKA inhibitor, PKI, abolished the effects of SNAP and CNP on NF-κB, whereas the PKG inhibitor Rp-8-Br-PET-cGMP had no effect. Inhibition of cGMP-inhibited cAMP-hydrolyzing phosphodiesterase 3 (PDE3) blocked SNAP- and CNP-elicited effects on NF-κB–dependent transcription. Furthermore, cGMP analogues such as 8-pCPT-cGMP, which selectively activates PKG but does not inhibit PDE3, had no effect on NF-κB–mediated transcription. Activation of PKA by SNAP or cAMP-elevating agents not only inhibited TNF-α–induced NF-κB–dependent reporter gene expression but also reduced endogenous NF-κB–dependent adhesion molecule and chemokine expression. These results suggest that SNAP and CNP exert inhibitory effects on NF-κB–dependent transcription by activation of PKA via cGMP-dependent inhibition of PDE3 activity. Therefore, PDE3 is a novel mediator of inflammation in VSMCs.
Gap Junction Communication Mediates Transforming Growth Factor-β Activation and Endothelial-Induced Mural Cell Differentiation
Authors: Hirschi Karen K. , Burt Janis M. , Hirschi Kendal D. , Dai Cuiping
Published: August 2003

During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-β (TGF-β) activation. We investigated whether gap junction channels are formed between endothelial cells and recruited mesenchymal progenitors and whether intercellular communication is necessary for endothelial-induced mural cell differentiation. Mesenchymal progenitors from Cx43
−/−
murine embryos and Cx43
+/+
littermates were cocultured with prelabeled endothelial cells. Intracellular dye injection and dual whole-cell voltage clamp revealed that endothelial cells formed gap junction channels with Cx43
+/+
but not Cx43
−/−
progenitors. In coculture with endothelial cells, Cx43
−/−
progenitors did not undergo mural cell differentiation as did Cx43
+/+
cells. Stable reexpression of Cx43 in Cx43
−/−
cells (reCx43) restored their ability to form gap junctions with endothelial cells and undergo endothelial-induced mural cell differentiation. Cocultures of endothelial cells and either Cx43
+/+
or reCx43 mesenchymal cells produced activated TGF-β; endothelial-Cx43
−/−
cocultures did not. However, Cx43
−/−
cells did produce latent TGF-β and undergo mural cell differentiation in response to exogenous TGF-β1. These studies indicate that gap junction communication between endothelial and mesenchymal cells mediates TGF-β activation and subsequent mural cell differentiation.
Reduction in Preretinal Neovascularization by Ribozymes That Cleave the A
2B
Adenosine Receptor mRNA
Authors: Afzal A. , Shaw L.C. , Caballero S. , Spoerri P.E. , Lewin A.S. , Zeng D. , Belardinelli L. , Grant M.B.
Published: August 2003

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein–coupled receptors (A
1
, A
2A
, A
2B
, and A
3
) and is a potential mediator of angiogenesis through the A
2B
receptor. The lack of a potent, selective A
2B
receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A
2B
receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A
2B
receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A
2B
receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a V
max
of 515±125 pmol/min and a K
cat
of 36.1±8.3 min
−1
(
P
≤1×10
−5
). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A
2B
receptor mRNA levels by 45±4.8% (
P
=5.1×10
−5
). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3±1.2% (
P
=7×10
−4
). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5±8.2% (
P
=4.5×10
−5
). Our results suggest that the A
2B
receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A
2B
receptor in retinal angiogenesis.
Mitochondrial Sources of H
2
O
2
Generation Play a Key Role in Flow-Mediated Dilation in Human Coronary Resistance Arteries
Authors: Liu Yanping , Zhao Hongtao , Li Hongwei , Kalyanaraman B. , Nicolosi Alfred C. , Gutterman David D.
Published: August 2003

We previously showed that hydrogen peroxide (H
2
O
2
) contributes to flow-induced dilation in human coronary resistance arteries (HCRAs); however, the source of this H
2
O
2
is not known. We hypothesized that the H
2
O
2
is derived from superoxide (O
2
·−
) generated by mitochondrial respiration. HCRAs were dissected from right atrial appendages obtained from patients during cardiac surgery and cannulated with micropipettes. H
2
O
2
-derived radicals and O
2
·−
were detected by electron spin resonance (ESR) using BMPO as the spin trap and by histofluorescence using hydroethidine (HE, 5 μmol/L) and dichlorodihydrofluorescein (DCFH, 5 μmol/L). Diameter changes to increases in pressure gradients (20 and 100 cm H
2
O) were examined in the absence and the presence of rotenone (1 μmol/L), myxothiazol (100 nmol/L), cyanide (1 μmol/L), mitochondrial complex I, III, and IV inhibitors, respectively, and apocynin (3 mmol/L), a NADPH oxidase inhibitor. At a pressure gradient of 100 cm H
2
O, ubisemiquinone and hydroxyl radicals were detected from effluents of vessels. Including superoxide dismutase and catalase in the perfusate reduced the ESR signals. Relative ethidium and DCFH fluorescence intensities in HCRAs exposed to flow were enhanced (1.45±0.15 and 1.57±0.12, respectively compared with no-flow) and were inhibited by rotenone (0.87±0.17 and 0.95±0.07). Videomicroscopic studies showed that rotenone and myxothiazol blocked flow-induced dilation (% max. dilation at 100 cm H
2
O: rotenone, 74±3% versus 3±13%; myxothiazol, 67±3% versus 28±4%;
P
<0.05). Neither cyanide nor apocynin altered flow-induced dilation. These results suggest that shear stress induced H
2
O
2
formation, and flow-induced dilation is derived from O
2
·−
originating from mitochondrial respiration.
Ryanodine Receptor Mutations Associated With Stress-Induced Ventricular Tachycardia Mediate Increased Calcium Release in Stimulated Cardiomyocytes
Authors: George Christopher H. , Higgs Gemma V. , Lai F. Anthony
Published: August 2003

Ca
2+
release from the sarcoplasmic reticulum mediated by the cardiac ryanodine receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2 mutations suggested to cause defective Ca
2+
channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S
2246
L, N
4104
K, and R
4497
C) in HL-1 cardiomyocytes displaying correct targeting to the endoplasmic reticulum. N
4104
K also localized to the Golgi apparatus. Phenotypic characteristics including intracellular Ca
2+
handling, proliferation, viability, RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca
2+
release was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine and 4-chloro-m-cresol) or β-adrenergic stimulation (isoproterenol). RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation, but was dramatically disrupted by isoproterenol or forskolin, an activator of adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar magnitudes in all cells and were associated with equivalent hyperphosphorylation of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting cardiomyocyte phenotype but mediated augmented Ca
2+
release on RyR-agonist or β-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated.
Transcriptional Stimulation of the eNOS Gene by the Stable Prostacyclin Analogue Beraprost Is Mediated Through cAMP-Responsive Element in Vascular Endothelial Cells
Authors: Niwano Kazuo , Arai Masashi , Tomaru Koichi , Uchiyama Tsuyoshi , Ohyama Yoshio , Kurabayashi Masahiko
Published: August 2003

Beraprost sodium (BPS), an orally active prostacyclin analogue, has been reported to be beneficial in the treatment of primary pulmonary hypertension and obstructive peripheral arterial disease. Although BPS was originally described for its effects on platelet aggregation and vasodilatory response, the effect on endothelial cells has been poorly understood. In this study, we examined the effects of BPS on the eNOS gene expression in mouse aorta and cultured human and bovine aortic endothelial cells. Treatment of these cells with BPS increased the eNOS expression as assessed by Northern blots, Western blots, and NO production by NO-specific fluorescence (DAF2-DA) and by the Griess method. Standard mRNA decay assays showed that BPS increases the stability of eNOS mRNA. In addition, BPS increased the promoter activity of the human eNOS gene, as determined by luciferase assays of the eNOS promoter gene. Progressive 5′-deletion and site-specific mutation analyses defined the BPS-responsive sequences as cAMP-responsive elements (CRE) located at −733 and −603. By using the oligonucleotide probe containing this CRE sequence in electrophoretic mobility shift assays, we showed that the phosphorylated form of CRE-binding protein is a major constituent of the complex in BPS-treated cells. Western blot analyses indicate that BPS but not endogenous prostacyclin phosphorylates CRE-binding protein. The presence of functional CRE sites within human eNOS promoter may represent a novel mechanism for regulating eNOS gene expression.
Randomized Clinical Trial of Therapeutic Exercise in Subacute Stroke
Authors: Duncan Pamela , Studenski Stephanie , Richards Lorie , Gollub Steven , Lai Sue Min , Reker Dean , Perera Subashan , Yates Joni , Koch Victoria , Rigler Sally , Johnson Dallas
Published: August 2003

Background and Purpose—Rehabilitation care after stroke is highly variable and increasingly shorter in duration. The effect of therapeutic exercise on impairments and functional limitations after stroke is not clear. The objective of this study was to determine whether a structured, progressive, physiologically based exercise program for subacute stroke produces gains greater than those attributable to spontaneous recovery and usual care.

Methods—This randomized, controlled, single-blind clinical trial was conducted in a metropolitan area and 17 participating healthcare institutions. We included persons with stroke who were living in the community. One hundred patients (mean age, 70 years; mean Orpington score, 3.4) consented and were randomized from a screened sample of 582. Ninety-two subjects completed the trial. Intervention was a structured, progressive, physiologically based, therapist-supervised, in-home program of thirty-six 90-minute sessions over 12 weeks targeting flexibility, strength, balance, endurance, and upper-extremity function. Main outcome measures were postintervention strength (ankle and knee isometric peak torque, grip strength), upper- and lower-extremity motor control (Fugl Meyer), balance (Berg and functional reach), endurance (peak aerobic capacity and exercise duration), upper-extremity function (Wolf Motor Function Test), and mobility (timed 10-m walk and 6-minute walk distance).

Results—In the intention-to-treat multivariate analysis of variance testing the overall effect, the intervention produced greater gains than usual care (Wilk’s λ=0.64,P=0.0056). Both intervention and usual care groups improved in strength, balance, upper- and lower-extremity motor control, upper-extremity function, and gait velocity. Gains for the intervention group exceeded those in the usual care group in balance, endurance, peak aerobic capacity, and mobility. Upper-extremity gains exceeded those in the usual care group only in patients with higher baseline function.

Conclusions—This structured, progressive program of therapeutic exercise in persons who had completed acute rehabilitation services produced gains in endurance, balance, and mobility beyond those attributable to spontaneous recovery and usual care.
Race-Ethnic Differences in Patent Foramen Ovale, Atrial Septal Aneurysm, and Right Atrial Anatomy Among Ischemic Stroke Patients
Authors: Rodriguez Carlos J. , Homma Shunichi , Sacco Ralph L. , Di Tullio Marco R. , Sciacca Robert R. , Mohr J.P.
Published: August 2003

Background and Purpose—

Stroke remains a substantial cause of mortality and morbidity in the United States. Racial differences in stroke incidence and mortality persist with well-known excesses among blacks. Information on stroke among Hispanics is limited. In particular, little is known about whether patent foramen ovale (PFO), atrial septal aneurysm (ASA), and other atrial anomalies associated with cryptogenic stroke differ among minority populations.

Methods—

As a part of the PFO in Cryptogenic Stroke Study, transesophageal echocardiography was performed in a cohort of 630 ischemic stroke patients (mean age, 59±12 years; 44% women; 45% whites, 35% blacks, 17% Hispanics, 3% other). The prevalences of PFO, ASA, and right atrial (RA) anatomy favoring paradoxical embolization were compared among race-ethnic groups. Statistical analyses used analysis of variance for continuous variables and logistic regression for dichotomous variables with adjustments for age and sex.

Results—

Age- and sex-adjusted prevalences of PFO and ASA were similar across race-ethnic subgroups. However, large PFO was significantly less prevalent among blacks than among whites (odds ratio, 0.47; 95% confidence interval, 0.24 to 0.91;
P
=0.02). RA anatomy favoring paradoxical embolization was also significantly less prevalent among blacks compared with whites (odds ratio, 0.62; 95% confidence interval, 0.43 to 0.91;
P
=0.01). There were no significant differences in prevalence between whites and Hispanics.

Conclusions—

Although the frequency of PFO did not vary among race-ethnic groups, a large PFO and RA anatomy favoring paradoxical embolization were significantly more prevalent among whites and Hispanics compared with blacks. These may be relatively more important risk factors for stroke among whites and Hispanics than among blacks.
Impact of Viral and Bacterial Burden on Cognitive Impairment in Elderly Persons With Cardiovascular Diseases
Authors: Strandberg Timo E. , Pitkala Kaisu H. , Linnavuori Kimmo H. , Tilvis Reijo S.
Published: August 2003

Background and Purpose—

Inflammation and infectious etiology have been implicated in the pathogenesis of dementia. We sought to investigate whether the seropositivity of common infections was associated with cognitive function.

Methods—

Viral burden (seropositivity for herpes simplex virus type 1 [HSV-1], herpes simplex virus type 2 [HSV-2], or cytomegalovirus [CMV]) and bacterial burden (
Chlamydia pneumoniae
and
Mycoplasma pneumoniae
) were related to cognitive status and its impairment among 383 home-dwelling elderly with cardiovascular diseases (mean age, 80 years). The Mini-Mental State Examination (MMSE) and its changes and the Clinical Dementia Rating (CDR) were used to define cognitive impairment.

Results—

At baseline, 0 to 1, 2, and 3 positive titers toward viruses were found in 48 (12.5%), 229 (59.8%), and 106 individuals (27.7%), respectively. MMSE points decreased with increasing viral burden (
P
=0.03). At baseline, 58 individuals (15.1%) had cognitive impairment, which after adjustments was significantly associated with seropositivity for 3 viruses (hazard ratio, 2.5; 95% CI, 1.3 to 4.7). MMSE score decreased in 150 (43% of 348) during 12-month follow-up. After adjustment for MMSE score at baseline and with 0 to 1 seropositivities as reference (1.0), the hazard ratios were 1.8 (95% CI, 0.9 to 3.6) and 2.3 (95% CI, 1.1 to 5.0) for 2 and 3 seropositivities, respectively. The prevalence of possible or definite dementia according to CDR also increased with viral burden. No significant associations were observed between bacterial burden and cognition.

Conclusions—

Viral pathogen burden of HSV and CMV was associated with cognitive impairment in home-dwelling elderly persons with cardiovascular diseases. The results need to be tested in larger databases, but they may offer a preventable cause of cognitive decline.
Neuroprotective Effects of MK-801 in Different Rat Stroke Models for Permanent Middle Cerebral Artery Occlusion
Authors: Gerriets T. , Stolz E. , Walberer M. , Kaps M. , Bachmann G. , Fisher M.
Published: August 2003

Background and Purpose—

Permanent middle cerebral artery occlusion (MCAO) with the use of the suture technique causes hypothalamic damage with subsequent hyperthermia, which can confound neuroprotective drug studies. In the present study the neuroprotective effects of dizocilpine (MK-801) were compared in different permanent MCAO models with and without hypothalamic damage and hyperthermia.

Methods—

Sixty Sprague-Dawley rats were treated with MK-801 or placebo, beginning 15 minutes before MCAO, and assigned to the following groups: suture MCAO (group I), macrosphere MCAO without hypothalamic damage (group II), or macrosphere MCAO with intentionally induced hypothalamic infarction (group III). Body temperature was measured at 3, 6, and 24 hours. Lesion size was determined after 24 hours (2,3,5-triphenyltetrazolium chloride staining).

Results—

Hypothalamic damage was present in animals in group I and was intentionally induced in group III with the use of a modified macrosphere MCAO technique. Body temperature was significantly increased 3, 6, and 24 hours after MCAO in these 2 groups of animals. Hypothalamic damage and subsequent hyperthermia could be avoided effectively by limiting the number of macrospheres (group II). MK-801 provided a highly significant neuroprotective effect in group II but not in groups I and III.

Conclusions—

Hypothalamic damage with subsequent hyperthermia masked the neuroprotective effect of MK-801. This side effect can be avoided by using the macrosphere MCAO technique with a limited number of spheres. This model therefore may be more appropriate to study the effects of neuroprotective drugs in permanent focal cerebral ischemia than the suture method.
Association of Parental History of Stroke With Clinical Parameters in Patients With Ischemic Stroke or Transient Ischemic Attack
Authors: Tentschert S. , Greisenegger S. , Wimmer R. , Lang W. , Lalouschek W.
Published: August 2003

Background and Purpose—

Data regarding hereditary influences on stroke remain controversial. We investigated possible associations of a family history of stroke with clinical parameters in a large cohort of well-documented patients with ischemic cerebrovascular events, with special reference to sex-specific differences.

Methods—

We analyzed the association between a maternal and/or paternal history of stroke and clinical parameters in 1564 patients with known maternal and paternal history of stroke and suffering from ischemic stroke or transient ischemic attack within the Vienna Stroke Registry.

Results—

Patients with a maternal history of stroke were significantly more often female (54%) than those without (44%;
P
=0.003). Hypertension was more prevalent in female patients with than in those without a maternal history of stroke (87% versus 74%;
P
=0.001). These associations remained significant after multivariate adjustment (adjusted odds ratio, 1.9; 95% CI, 1.1 to 3.5;
P
=0.024). Of those female patients with an echocardiogram (n=225), those with a maternal history of stroke more often had left ventricular hypertrophy (48%) than those without (20%) (adjusted odds ratio, 3.6; 95% CI, 1.5 to 8.2;
P
=0.003). In contrast, hypertension was equally prevalent in male patients with or without a maternal history of stroke (75% versus 74%;
P
=0.754). We found no significant associations of clinical parameters with a paternal history of stroke.

Conclusions—

Our results indicate a sex-specific relationship between a maternal history of stroke and the prevalence of hypertension and left ventricular hypertrophy in female patients with ischemic cerebrovascular events.
Systematic Review of the Risks of Carotid Endarterectomy in Relation to the Clinical Indication for and Timing of Surgery
Authors: Bond R. , Rerkasem K. , Rothwell P.M.
Published: August 2003

Background and Purpose—Reliable data on the risk of carotid endarterectomy (CEA) in relation to clinical indication and timing of surgery are necessary to target CEA more effectively, to inform patients, to adjust risks for case mix, and to understand the mechanisms of operative stroke.

Methods—We performed a systematic review of all studies published from 1980 to 2000 inclusive that reported the risk of stroke and death resulting from CEA. Pooled estimates of risk by type of presenting ischemic event and time since the last event were obtained by Mantel-Haenszel meta-analysis.

Results—Of 383 published studies, only 103 stratified risk by indication. Although the operative risk for symptomatic stenosis overall was higher than for asymptomatic stenosis (odds ratio [OR], 1.62; 95% confidence interval [CI], 1.45 to 1.81;P<0.00001; 59 studies), risk in patients with ocular events only tended to be lower than for asymptomatic stenosis (OR, 0.75, 95% CI, 0.50 to 1.14; 15 studies). Operative risk was the same for stroke and cerebral transient ischemic attack (OR, 1.16; 95% CI, 0.99 to 1.35;P=0.08; 23 studies) but higher for cerebral transient ischemic attack than for ocular events only (OR, 2.31; 95% CI, 1.72 to 3.12;P<0.00001; 19 studies) and for CEA for restenosis than primary surgery (OR, 1.95; 95% CI, 1.21 to 3.16;P=0.018; 6 studies). Urgent CEA for evolving symptoms had a much higher risk (19.2%, 95% CI, 10.7 to 27.8) than CEA for stable symptoms (OR, 3.9; 95% CI, 2.7 to 5.7;P<0.001; 13 studies), but there was no difference between early (<3 to 6 weeks) and late (>3 to 6 weeks) CEA for stroke in stable patients (OR, 1.13; 95% CI, 0.79 to 1.62;P=0.62; 11 studies). All observations were highly consistent across studies.

Conclusions—Risk of stroke and death resulting from CEA is highly dependent on the clinical indication. Audits of risk should be stratified accordingly, and patients should be informed of the risk that relates to their presenting event.
Cerebral Autoregulation in Carotid Artery Occlusive Disease Assessed From Spontaneous Blood Pressure Fluctuations by the Correlation Coefficient Index
Authors: Reinhard M. , Roth M. , Müller T. , Czosnyka M. , Timmer J. , Hetzel A.
Published: August 2003

Background and Purpose—

Estimation of dynamic cerebral autoregulation from spontaneous fluctuations of arterial blood pressure (ABP) and cerebral blood flow velocity (CBFV) is an attractive monitoring option for cerebral hemodynamic impairment. We evaluated the correlation coefficient index method in patients with severe obstructive carotid disease and compared it with transfer function analysis (frequency domain approach to cerebral autoregulation) and CO
2
vasomotor reactivity.

Methods—

In 139 patients with severe unilateral carotid stenosis (≥70%) or occlusion, CBFV (transcranial Doppler) and ABP (Finapres method) were recorded over 10 minutes. Correlations between systolic pressure, diastolic pressure, and mean ABP and CBFV oscillations over 1-minute epochs were averaged over 10 minutes to form the correlation coefficient indexes (Sx, Dx, Mx, respectively). Transfer function parameters (phase shift and gain between ABP and CBFV oscillations) were determined from the entire 10-minute period. CO
2
reactivity was assessed by inhalation of 7% CO
2
.

Results—

The correlation indexes Dx and Mx were significantly higher ipsilateral to stenosis and increased with degree of stenosis, indicating increasing dependence of CBFV on ABP and thus impairment of cerebral autoregulation. Dx and Mx correlated moderately but highly significantly with transfer function parameters and CO
2
reactivity and showed a good level of agreement in detecting pathological values. Patients with a small variance of the 1-minute source correlations of Dx and Mx showed clearly better correlation values. Transfer function parameters and CO
2
reactivity but not Dx and Mx were significantly poorer in patients with symptomatic stenosis or occlusion.

Conclusions—

The potential of the correlation coefficient indexes Dx and Mx in detecting hemodynamic impairment in patients with carotid stenosis is comparable to that of transfer function analysis and CO
2
reactivity testing. In future, a combination of various hemodynamic tests might help to identify patients at risk for ischemic events.
Functional and Histological Evidence for the Protective Effect of NXY-059 in a Primate Model of Stroke When Given 4 Hours After Occlusion
Authors: Marshall Jonathan W.B. , Cummings Rosalyn M. , Bowes Laura J. , Ridley Rosalind M. , Green A. Richard
Published: August 2003

Background and Purpose—

NXY-059 has substantial protective effects when administered immediately after the onset of ischemia in a primate model of stroke. This study examined the efficacy of this drug when administered 4 hours after onset, a more clinically relevant time point.

Methods—

Before surgery, marmosets were trained and tested on a number of neurological tests, which assessed general neurological function, motor ability, and spatial awareness. Four hours after permanent middle cerebral artery occlusion (pMCAO), marmosets received a bolus of saline (n=13) or NXY-059 (n=13), and osmotic minipumps were implanted, providing 48-hour saline or drug (85 μmol/kg per hour) infusion. The monkeys were retested 3 and 10 weeks after surgery. Finally, infarct size was evaluated with histological analysis.

Results—

The unbound plasma NXY-059 concentration was 200±9 μmol/L after 24-hour infusion, a concentration well tolerated in stroke patients. Drug treatment ameliorated the long-term motor impairment produced by pMCAO; the marmosets were better at using their contralesional, stroke-affected arm than controls at both 3 and 10 weeks. Saline-treated animals had a debilitating spatial neglect at 3 weeks with residual signs evident at 10 weeks. NXY-059 treatment substantially attenuated neglect at 3 weeks, with no deficit being seen at 10 weeks. NXY-059 reduced the overall infarct size by 28% (saline, 324±46 mm
3
; NXY-059, 234±30 mm
3
) with protection to the cortex, white matter, and subcortical structures.

Conclusions—

NXY-059 is an effective neuroprotective agent when administered 4 hours after pMCAO in a primate species, attenuating both motor and spatial neglect. The compound also substantially lessened the volume of cerebral damage.
Experimental Intracerebral Hemorrhage in the Mouse
Authors: Belayev Ludmila , Saul Isabel , Curbelo Karell , Busto Raul , Belayev Andrey , Zhang Yongbo , Riyamongkol Panomkhawn , Zhao Weizhao , Ginsberg Myron D.
Published: August 2003

Background and Purpose—

A major limitation of intracerebral hemorrhage (ICH) research is the lack of reproducible animal models. The present study was conducted to validate in the mouse the double-injection method of ICH initially developed in the rat. We investigated the effect of intrastriatal injection of blood or cerebrospinal fluid (CSF) on cerebral blood flow (CBF), neurological score, hematoma volume, and brain swelling.

Methods—

Male C57BL/6 mice were anesthetized with halothane/nitrous oxide delivered by face mask. Rectal and cranial temperatures were regulated at 37°C to 37.5°C. Mice were placed in a stereotactic frame, and a 30-gauge stainless steel cannula was introduced through a burr hole into the left striatum. Each mouse received a 5-μL injection of either whole blood or CSF (over 3 minutes), followed 7 minutes later by 10 μL injected over 5 minutes. The injection cannula was slowly withdrawn 10 minutes after the second injection. Control mice had only cannula insertion. CBF was studied by laser Doppler perfusion imaging. Neurological status was evaluated on days 1 and 2. After 2 days, hematoma volume and brain swelling were calculated.

Results—

Physiological values were stable. Mice with ICH but not those with CSF or cannula alone had a marked, persistent neurological deficit and a highly reproducible hematoma, whose mean±SEM volume was 2.0±0.2 mm
3
compared with a lesion size of 0.2±0.1 mm
3
in mice with CSF. Residual swelling of the ipsilateral hemisphere at 48 hours was 5.7% in the hematoma and 1.5% in the CSF groups. Relative CBF in the neocortex ipsilateral to the injection site declined by ∼45% to 60% during the first 20 minutes after cannula insertion/injection in all groups but began to renormalize at ∼25 to 30 minutes in the CSF and cannula-only groups; in the hematoma group, cortical hypoperfusion of ∼35% to 50% persisted during the 90-minute measurement period.

Conclusions—

The present ICH model in mice produces a consistent neurological deficit, hypoperfusion, hematoma volume, and brain swelling. This model closely mimics human hypertensive basal ganglionic ICH and should be useful for the evaluation of pharmaceutical therapies. Laser Doppler perfusion imaging is a useful new technique to quantify relative CBF changes and can be used for studies of dynamic changes of CBF in this in vivo model of ICH in mice.
Predication and Equation
Authors: Adger David , Ramchand Gillian
Published: August 2003

In this article, we argue that a structural distinction between predicational and equative copular clauses is illusory. All semantic predicational relationships are constructed asymmetrically via a syntactic predicational head; differences reduce to whether this head bears an event variable or not. This allows us to maintain a restrictive view of the syntax-semantics interface in the face of apparently recalcitrant data from Scottish Gaelic.