bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo
klotho
Gene Transfer Ameliorates Angiotensin II-Induced Renal Damage
Authors: Mitani Haruo , Ishizaka Nobukazu , Aizawa Toru , Ohno Minoru , Usui Shin-ichi , Suzuki Toru , Amaki Toshihiro , Mori Ichiro , Nakamura Yasushi , Sato Misako , Nangaku Masaomi , Hirata Yasunobu , Nagai Ryozo
Published: July 2002

The
klotho
gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal
klotho
gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the
klotho
gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal
klotho
gene expression. Long-term infusion of angiotensin II downregulated renal
klotho
gene expression at both the mRNA and protein levels. This angiotensin II-induced renal
klotho
downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse
klotho
gene (ad-klotho, 3.3×10
10
plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal
klotho
gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the
klotho
gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.
Ambulatory Blood Pressure in 12-Year-Old Children Born Small for Gestational Age
Authors: Rahiala Eero , Tenhola Sirpa , Vanninen Esko , Herrgård Eila , Tikanoja Tero , Martikainen Anneli
Published: July 2002

An association between low birth weight and subsequent elevated blood pressure has been demonstrated in a large number of studies, but the number of subjects born small for gestational age in these studies has been negligible. The inverse relationship between birth weight and blood pressure in children has been evaluated previously with an ambulatory blood pressure device, but only in children with normal birth weights. In this prospective case-control study from birth to the age of 12, we evaluated the ambulatory blood pressures in 50 children born at term but small for gestational age and in 50 full-term children born appropriate for gestational age. Children born small for gestational age had similar mean±SD systolic (117.5±8.5 mm Hg versus 115.3±7.4 mm Hg,
P
=0.221), and diastolic (69.2±5.3 mm Hg versus 67.3±4.4 mm Hg,
P
=0.075) 24-hour ambulatory blood pressure compared with the values of the children born appropriate for gestational age. However, 24-hour systolic blood pressure in the small-for-gestational-age children was higher (3.90 mm Hg; 95% confidence interval, 0.65 to 7.15) after adjusting for current body mass index. The difference in current body mass index was the only determinant for the difference in systolic blood pressure between the groups. Birth weight had no direct association with the blood pressure values. Impaired fetal growth may have a relationship with higher later blood pressure, but in 12-year-old children, blood pressure differences between small for gestational age and appropriate for gestational age children are much more dependent on current body size.
Ultrasonic Backscatter and Serum Marker of Cardiac Fibrosis in Hypertensives
Authors: Maceira Alicia M. , Barba Joaquín , Varo Nerea , Beloqui Oscar , Díez Javier
Published: July 2002

Elevations in the serum concentration of the carboxy-terminal propeptide of procollagen type I (PIP) >127 μg/L have been found to predict severe myocardial fibrosis in hypertensive patients. This study was designed to assess whether ultrasonic reflectivity, evaluated by a real-time integrated backscatter analysis, was related to the severity of myocardial fibrosis as estimated by serum PIP. Thirty-four subjects were included in the study. Serum PIP was measured by specific radio immunoanalysis. Backscatter cyclic variation and maximal intensity were measured in 6 regions throughout the left ventricle. The subjects were divided into 3 groups: 14 normotensives with PIP <127 μg/L (group 1), 12 hypertensives with PIP <127 μg/L (group 2), and 8 hypertensives with PIP >127 μg/L (group 3). The highest cyclic variation was found in group 1 and the lowest in group 3 (5.78±0.25 versus 4.70±0.33 dB,
P
<0.05), with intermediate values in group 2 (5.10±0.27 dB). No differences in maximal intensity were found among the 3 groups of subjects. Using receiver operating characteristics curves, we observed that a cutoff of 2.90 dB for cyclic variation measured in the apex provided 75% sensitivity and 63% specificity for predicting PIP >127 μg/L in hypertensives, with a relative risk of 2.50 (95% CI, 0.72 to 34.70). These results show an association between diminished cyclic variation of backscatter and increased serum concentration of PIP in hypertension. Thus, the combination of these 2 parameters may be useful for the diagnosis of severe myocardial fibrosis associated with hypertension.
Eight-Year Blood Pressure Change in Middle-Aged Men
Authors: Stamler Jeremiah , Liu Kiang , Ruth Karen J. , Pryer Jane , Greenland Philip
Published: July 2002

Relationships of nutrients, alcohol intake, and change in weight to change in blood pressure over 8 years in 1714 employed middle-aged men from the Chicago Western Electric Study were explored. At first and second annual examinations, 2 in-depth interviews were performed to assess usual intake of foods and beverages during the preceding 28 days. Annual follow-up data through examination year 9 were used to determine change in weight and blood pressure. Averages of nutrients from 2 interviews were related to annual blood pressure change from baseline by use of the Generalized Estimating Equation, with control for confounders. In analyses of dietary variables considered individually, total and animal protein; total, saturated, monounsaturated, and polyunsaturated fatty acids; cholesterol; Keys dietary lipid score; calcium; alcohol; and average annual change in weight were positively and significantly related to average annual change in systolic pressure; vegetable protein, total carbohydrate, beta-carotene, and an antioxidant vitamin score based on vitamin C and beta-carotene were inversely and significantly related to average annual change in systolic pressure. In analyses of combinations of dietary factors, cholesterol, Keys score, and alcohol were positively related to change in systolic pressure (eg, Z-scores 2.21, 2.05, and 2.50); vegetable protein and antioxidant index were inversely related to change in systolic and diastolic pressure. Change in weight was directly related to change in systolic and diastolic pressure. These findings support the concept that multiple macro- and micronutrients, alcohol intake, and calorie imbalance relate prospectively to blood pressure change.
Angiotensin 1-9 and 1-7 Release in Human Heart
Authors: Jackman Herbert L. , Massad Malek G. , Sekosan Marin , Tan Fulong , Brovkovych Viktor , Marcic Branislav M. , Erdös Ervin G.
Published: July 2002

Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A’s presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B
2
receptor in Chinese hamster ovary cells transfected to express human ACE and B
2
(CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B
2
receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC
50
s) with ACE. They probably induce conformational changes in the ACE/B
2
receptor complex via interaction with ACE.
Aldosterone Breakthrough During Angiotensin II Receptor Antagonist Therapy in Stroke-Prone Spontaneously Hypertensive Rats
Authors: Naruse Mitsuhide , Tanabe Akiyo , Sato Atsuhisa , Takagi Sachiko , Tsuchiya Ken , Imaki Toshihiro , Takano Kazue
Published: July 2002

Aldosterone breakthrough during ACE inhibitor therapy has been reported. This study investigates changes in plasma aldosterone concentration (PAC) and its mechanism and effects on target organ damage during long-term angiotensin II type 1 (AT1) receptor antagonist (AT1A) therapy in hypertensive rats. An AT1A (candesartan, 1 mg/kg per day PO) was administered in stroke-prone spontaneously hypertensive rats from 4 weeks of age for 34 weeks. PAC was significantly decreased during the first 4 weeks but showed aldosterone breakthrough after 8 weeks of AT1A administration. Plasma angiotensin II concentration was significantly elevated, whereas no change was seen in plasma ACTH or serum potassium. The mechanism(s) of aldosterone breakthrough were investigated by giving high doses of candesartan (3 mg/kg per day PO), dexamethasone (200 μg/kg per day IP), or the AT2 antagonist (PD123319, 10 mg/kg per day SC) during the last week of the 24-week AT1A treatment period. Dexamethasone and AT2 antagonist but not high-dose AT1A produced a significant decrease in PAC, with a larger decrease produced by the AT2 antagonist. To clarify the effects of the residual aldosterone, effects of coadministration of low-dose spironolactone (10 mg/kg per day SC), an aldosterone antagonist, on left ventricular hypertrophy and expression of brain natriuretic peptide mRNA were determined. Low-dose spironolactone further improved left ventricular hypertrophy and brain natriuretic peptide mRNA expression despite no additional depressor effect. These results suggest that aldosterone breakthrough occurs during long-term AT1A therapy, mainly by an AT2-dependent mechanism. Residual aldosterone may attenuate the cardioprotective effects of AT1A.
Effect of Pravastatin on Proteinuria in Patients With Well-Controlled Hypertension
Authors: Lee Tsung-Ming , Su Sheng-Fang , Tsai Chang-Her
Published: July 2002

Proteinuria is an important risk factor for cardiovascular and renal morbidity and mortality. The effects of 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitor (statin) therapy on proteinuria in normolipidemic patients with well-controlled hypertension have not been studied. A total of 63 normolipidemic (total cholesterol <240 mg/dL) and proteinuric (300 to 3000 mg/d) patients with well-controlled blood pressure (<140/90 mm Hg) were randomized to receive either placebo (n=32) or pravastatin (10 mg/d; n=31) after a 3-month placebo period. Pravastatin lowered proteinuria after 6 months by 54% (
P
<0.0001). Creatinine clearance was stable throughout the study in the 2 groups. Despite unchanged plasma endothelin-1 levels throughout the study, urinary excretion of the peptide was decreased and significantly correlated with improvement in urinary protein excretion in pravastatin-treated patients (
r
=0.64,
P
=0.001). The urinary excretion of retinol-binding protein decreased after pravastatin administration, probably reflecting an improvement in tubular function. In contrast, the urinary excretion of IgG did not change significantly throughout the study in either group. Multivariate analysis revealed that proteinuria was only significantly correlated with statin use (
P
<0.0001,
R
2
= 0.66). Linear regression analysis in the statin-treated group did not show any correlation between changes in lipid profiles and proteinuria regression. Thus, in addition to their primary function of antilipidemia, the addition of pravastatin to treatment for well-controlled hypertension may have an additive effect on reducing proteinuria independent of hemodynamics and lipid-lowering effects, possibly through inhibiting renal endothelin-1 synthesis and improving tubular function.
Role of Heteromultimers in the Generation of Myocardial Transient Outward K
+
Currents
Authors: Guo Weinong , Li Huilin , Aimond Franck , Johns David C. , Rhodes Kenneth J. , Trimmer James S. , Nerbonne Jeanne M.
Published: July 2002

Previous studies have demonstrated a role for Kv4 α subunits in the generation of the fast transient outward K
+
current,
I
to,f
, in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular
I
to,f
. Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in
I
to,f
density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3 selectively attenuate
I
to,f
in mouse ventricular cells. Adenoviral-mediated coexpression of Kv4.2 and Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces transient outward K
+
currents with properties distinct from those produced on expression of Kv4.2 or Kv4.3 alone, and the gating properties of the heteromeric Kv4.2/Kv4.3 channels in ventricular cells are more similar to native
I
to,f
than are the homomeric Kv4.2 or Kv4.3 channels. Biochemical studies reveal that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from adult mouse ventricles. In addition, most of the Kv4.2 and KChIP2 are associated with Kv4.3 in situ. Taken together, these results demonstrate that functional mouse ventricular
I
to,f
channels are heteromeric, comprising Kv4.2/Kv4.3 α subunits and KChIP2. The results here also suggest that Kv4.2 is the primary determinant of the regional heterogeneity in
I
to,f
expression in adult mouse ventricle.
Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of K
ATP
Channels
Authors: Matsushita Kenji , Kinoshita Kengo , Matsuoka Tetsuro , Fujita Akikazu , Fujikado Takashi , Tano Yasuo , Nakamura Haruki , Kurachi Yoshihisa
Published: July 2002

ATP-sensitive K
+
(K
ATP
) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K
ATP
channels in SUR-subtypes. By examining ADP-effect on K
ATP
channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K
ATP
channels.
Mitochondrial PKCε and MAPK Form Signaling Modules in the Murine Heart
Authors: Baines Christopher P. , Zhang Jun , Wang Guang-Wu , Zheng Yu-Ting , Xiu Joanne X. , Cardwell Ernest M. , Bolli Roberto , Ping Peipei
Published: July 2002

Although activation of protein kinase C (PKC) ε and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCε signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCε and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCε, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCε greatly increased mitochondrial PKCε expression and activity, which was concomitant with increased mitochondrial interaction of PKCε with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCε activity, as the interactions were also observed in mice expressing inactive PKCε. However, although both active and inactive PKCε bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCε but not in mice expressing inactive PKCε. Examination of potential downstream targets of mitochondrial PKCε-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCε forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCε-ERK modules appears to play a role in PKCε-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.
Matrix Metalloproteinases
Authors: Spinale Francis G.
Published: July 2002

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible for myocardial extracellular protein degradation. Several MMP species identified within the human myocardium may be dysregulated in congestive heart failure (CHF). For example, MMPs that are expressed at very low levels in normal myocardium, such as collagenase-3 (MMP-13) and the membrane-type-1 MMPs, are substantially upregulated in CHF. However, MMP species are not uniformly increased in patients with end-stage CHF, suggesting that a specific portfolio of MMPs are expressed in the failing myocardium. With the use of animal models of CHF, a mechanistic relationship has been demonstrated with respect to myocardial MMP expression and the left ventricular (LV) remodeling process. The tissue inhibitors of the MMPs (TIMPs) are locally synthesized proteins that bind to active MMPs and thereby regulate net proteolytic activity. However, there does not appear to be a concomitant increase in myocardial TIMPs during the LV remodeling process and progression to CHF. This disparity between MMP and TIMP levels favors a persistent MMP activation state within the myocardium and likely contributes to the LV remodeling process in the setting of developing CHF. The elucidation of upstream signaling mechanisms that contribute to the selective induction of MMP species within the myocardium as well as strategies to normalize the balance between MMPs and TIMPs may yield some therapeutic strategies by which to control myocardial extracellular remodeling and thereby slow the progression of the CHF process.
Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP
3
Receptors
Authors: Gobeil Fernand , Dumont Isabelle , Marrache Anne Marilise , Vazquez-Tello Alejandro , Bernier Sylvie G. , Abran Daniel , Hou Xin , Beauchamp Martin H. , Quiniou Christiane , Bouayad Asmaa , Choufani Sanaa , Bhattacharya Mousumi , Molotchnikoff Stephane , Ribeiro-da-Silva Alfredo , Varma Daya R. , Bkaily Ghassan , Chemtob Sylvain
Published: July 2002

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE
2
in tissues and presence of perinuclear PGE
2
receptors (EP). We presently studied mechanisms by which PGE
2
induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE
2
and selective EP
3
receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP
3
immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP
3
receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear K
Ca
channels were detected, and their functionality corroborated by NS1619-induced Ca
2+
signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca
2+
chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as K
Ca
channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-κB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca
2+
transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE
2
through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP
3
receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K
Ca
channels, protein kinases, and NF-κB; the roles for nuclear EP
3
receptors seem different from those on plasma membrane.
Optical Mapping of Arrhythmias Induced by Strong Electrical Shocks in Myocyte Cultures
Authors: Fast Vladimir G. , Cheek Eric R.
Published: July 2002

Strong electrical shocks can induce arrhythmias, which might explain why shocks fail to defibrillate. In this work, the localization of arrhythmia source and the relationship with local changes of transmembrane potential (V
m
) were determined in geometrically defined cell cultures using optical mapping technique. Uniform-field shocks with strength (E) of 10 to 50 V/cm were applied across cell strands with width of 0.2 and 0.8 mm. The threshold for arrhythmia induction was dependent on the strand width: in the 0.8- and 0.2-mm strands, arrhythmias were induced at E≥20.6±1.8 V/cm (n=8) and E≥30.3±1.8 V/cm (n=8), respectively. At the same shock strength, the arrhythmia rate and duration were larger in the wider strands. During shocks that induced arrhythmias, the V
m
waveforms on the anodal side revealed a positive V
m
shift that followed the initial large hyperpolarization and postshock elevation of the diastolic V
m
. These V
m
changes were absent during failed shocks. To determine the localization of the arrhythmia source, arrhythmias were induced in narrow cell strands containing regions of local expansion. Optical mapping of the first extrabeat with a coupling interval of 315±60 ms revealed that in the majority of cases (9 out of 13) the source of arrhythmias was localized in the areas of shock-induced hyperpolarization. Thus, (1) induction of postshock arrhythmias, their rate, and their duration strongly depend on the tissue structure; (2) arrhythmia induction coincides with the appearance of a positive V
m
shift in the areas of hyperpolarization; and (3) the source of postshock arrhythmias is located in the areas of shock-induced hyperpolarization.
Nucleotide Receptors Involved in UTP-Induced Rat Arterial Smooth Muscle Cell Migration
Authors: Pillois Xavier , Chaulet Hervé , Belloc Isabelle , Dupuch Françoise , Desgranges Claude , Gadeau Alain-Pierre
Published: July 2002

Many factors have been shown to be involved in the development of hyperplasic lesions of vessels, but the role of extracellular nucleotides remains largely unknown. The presence of P2Y and P2X nucleotide receptors on arterial endothelial and smooth muscle cells suggests a potential role for nucleotides in the vessel pathophysiology. Although the role of P2X in physiology of vessels is well documented, that of P2Y is not completely understood. We recently demonstrated that extracellular nucleotides, and particularly UTP, induced migration of cultured arterial smooth muscle cells (ASMCs). This migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. An important question is to determine the specific role of the different P2Y receptors of rat ASMCs in the UTP-induced migration process. Therefore, we first quantified mRNA levels of P2Y
2
, P2Y
4
, and P2Y
6
nucleotide receptors in cultured rat ASMCs by a competitive RT-PCR approach and demonstrated that P2Y
2
is the most highly expressed among these receptors potentially involved in the UTP-mediated response. In addition to UTP, UDP also induced ASMC migration even when UTP regeneration was inhibited, suggesting the involvement of UDP receptor P2Y
6
. Moreover, suramin, a specific antagonist of rat P2Y
2
receptor, acted as an inhibitor of UTP-induced migration. Taken together, these results suggest a prominent role for the UTP receptor, P2Y
2
, and for the UDP receptor, P2Y
6
, in UTP-induced rat ASMC migration.
CADASIL Notch3 Mutant Proteins Localize to the Cell Surface and Bind Ligand
Authors: Haritunians Talin , Boulter Jim , Hicks Carol , Buhrman Jonathon , DiSibio Guy , Shawber Carrie , Weinmaster Gerry , Nofziger Donna , Schanen Carolyn
Published: July 2002

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor–like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.
Phosphorylation of Troponin I Controls Cardiac Twitch Dynamics
Authors: Pi YeQing , Kemnitz Kara R. , Zhang Dahua , Kranias Evangelia G. , Walker Jeffery W.
Published: July 2002

The cardiac myofilament protein troponin I (cTnI) is phosphorylated by protein kinase C (PKC), a family of serine/threonine kinases activated within heart muscle by a variety of agonists. cTnI is also a substrate for cAMP-dependent protein kinase (PKA) activated during β-adrenergic signaling. To investigate the role of cTnI phosphorylation in contractile regulation by these pathways, we generated transgenic mice harboring a mutated cTnI protein lacking phosphorylation sites for PKC (serine
43/45
and threonine
144
mutated to alanine) and for PKA (serine
23/24
mutated to alanine). Transgenic mice were interbred with cTnI-knockout mice to ensure the absence of endogenous phosphorylatable cTnI. Here, we report that regulation of myocyte twitch kinetics by β-stimulation and by endothelin-1 was altered in myocytes containing mutant cTnI. In wild-type myocytes, the β-agonist isoproterenol decreased twitch duration and relaxation time constant (τ) by 37% to 44%. These lusitropic effects of isoproterenol were reduced by about half in nonphosphorylatable cTnI mutant myocytes and were absent in cTnI mutants also lacking phospholamban (generated by crossing cTnI mutants with phospholamban-knockout mice). These observations are consistent with important roles for both cTnI and phospholamban phosphorylation in accelerating relaxation after β-adrenergic stimulation. In contrast, endothelin-1 increased twitch duration by 32% and increased τ by 58%. These endothelin-1 effects were substantially blunted in nonphosphorylatable cTnI myocytes, indicating that PKC phosphorylation of cTnI slows cardiac relaxation and increases twitch duration. We propose that β-agonists and endothelin-1 regulate cardiac twitch dynamics in opposite directions in part through phosphorylation of the myofilament protein cTnI on distinct sites.
Double-Edged Role of Statins in Angiogenesis Signaling
Authors: Urbich Carmen , Dernbach Elisabeth , Zeiher Andreas M. , Dimmeler Stefanie
Published: July 2002

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) exert potent vasculoprotective effects. However, the potential contribution to angiogenesis is controversial. In the present study, we demonstrate that atorvastatin dose-dependently affects endothelial cell migration and angiogenesis. In vivo relevant concentrations of 0.01 to 0.1 μmol/L atorvastatin or mevastatin promote the migration of mature endothelial cells and tube formation. Moreover, atorvastatin also increases migration and the potency to form vessel structures of circulating endothelial progenitor cells, which may contribute to vasculogenesis. In contrast, higher concentrations (>0.1 μmol/L atorvastatin) block angiogenesis and migration by inducing endothelial cell apoptosis. The dose-dependent promigratory and proangiogenic effects of atorvastatin on mature endothelial cells are correlated with the activation of the phosphatidylinositol 3-kinase–Akt pathway, as determined by the phosphorylation of Akt and endothelial NO synthase (eNOS) at Ser1177. In addition, the stimulation of migration and tube formation was blocked by phosphatidylinositol 3-kinase inhibitors. In contrast, the well-established stabilization of eNOS mRNA was achieved only at higher concentrations, suggesting that posttranscriptional activation rather than an increase in eNOS expression mediates the proangiogenic effect of atorvastatin. Taken together, these data suggest that statins exert a double-edged role in angiogenesis signaling by promoting the migration of mature endothelial cells and endothelial progenitor cells at low concentrations, whereas the antiangiogenic effects were achieved only at high concentrations.
Hypoxia Causes Downregulation of Protein and RNA Synthesis in Noncontracting Mammalian Cardiomyocytes
Authors: Casey Tammy M. , Pakay Julian L. , Guppy Michael , Arthur Peter G.
Published: July 2002

The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia (≈5 μmol/L, 0.5% O
2
) and severe hypoxia (<0.5 μmol/L, <0.05% O
2
) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by ≥40%). Our results suggest that energetic stress is the signal responsible for this downregulation.
Wall Tissue Remodeling Regulates Longitudinal Tension in Arteries
Authors: Jackson Zane S. , Gotlieb Avrum I. , Langille B. Lowell
Published: July 2002

Changes in blood pressure or flow induce arterial remodeling that normalizes mechanical loads that are imposed on arterial tissue. Arteries are also under substantial longitudinal stretch (axial strain) that may be altered by growth or atrophy of tissues to which they are attached. We therefore tested whether axial strain is also regulated in a negative feedback manner through arterial remodeling. Axial strain in rabbit carotid arteries was increased from 62±2% to 97±2% without altering other mechanical loads on wall tissues. Strain was reduced within 3 days and completely normalized by 7 days. Remodeling involved tissue elaboration, endothelial cell replication rates were increased by >50-fold and smooth muscle cell replication rates were increased by >15-fold, and substantially elevated DNA, elastin, and collagen contents were recorded. Also, increased rates of apoptosis were indicated by degradation of DNA into oligonucleosomes, and matrix remodeling was reflected in enlarged fenestrae in the internal elastic lamina and increased expression and activation of gelatinases, especially matrix metalloproteinase-2. Intriguingly, reduced axial strain was not normalized, presumably because remodeling processes, apart from cell contraction, are ineffective in decreasing strain, and arterial smooth muscle orientation precludes large effects of contraction on axial strain.
Functional Reconstitution of Endothelial Nitric Oxide Synthase Reveals the Importance of Serine 1179 in Endothelium-Dependent Vasomotion
Authors: Scotland Ramona S. , Morales-Ruiz Manuel , Chen Yan , Yu Jun , Rudic Radu Daniel , Fulton David , Gratton Jean-Philippe , Sessa William C.
Published: July 2002

Phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1179 can activate the enzyme, leading to NO release. Because eNOS is important in regulating vascular tone, we investigated whether phosphorylation of this residue is involved in vasomotion. Adenoviral transduction of endothelial cells (ECs) with the phosphomimetic S1179DeNOS markedly increased basal and vascular endothelial cell growth factor (VEGF)–stimulated NO release compared with cells transduced with wild-type virus. Conversely, adenoviral transduction of ECs with the non-phosphorylatable S1179AeNOS suppressed basal and stimulated NO release. Using a novel method for luminal delivery of adenovirus, transduction of the endothelium of carotid arteries from eNOS knockout mice with S1179DeNOS completely restored NO-mediated dilatation to acetylcholine (ACh), whereas vasomotor responses in arteries transduced with S1179AeNOS were significantly attenuated. Basal NO release was also significantly reduced in arteries transduced with S1179AeNOS, compared with S1179DeNOS. Thus, our data directly demonstrate that phosphorylation of eNOS at serine 1179 is an important regulator of basal and stimulated NO release in ECs and in intact blood vessels.
Control of Renin Secretion From Rat Juxtaglomerular Cells by cAMP-Specific Phosphodiesterases
Authors: Friis Ulla G. , Jensen Boye L. , Sethi Shala , Andreasen Ditte , Hansen Pernille B. , Skøtt Ole
Published: July 2002

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C
m
) with a marked increase in C
m
at 1 μmol/L, no net change at 10 μmol/L, and a decrease at 100 μmol/L cAMP. cGMP also had a dual effect on C
m
at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C
m
. Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on C
m
and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.
Differential Distribution of Cardiac Ion Channel Expression as a Basis for Regional Specialization in Electrical Function
Authors: Schram Gernot , Pourrier Marc , Melnyk Peter , Nattel Stanley
Published: July 2002

The cardiac electrical system is designed to ensure the appropriate rate and timing of contraction in all regions of the heart, which are essential for effective cardiac function. Well-controlled cardiac electrical activity depends on specialized properties of various components of the system, including the sinoatrial node, atria, atrioventricular node, His-Purkinje system, and ventricles. Cardiac electrical specialization was first recognized in the mid 1800s, but over the past 15 years, an enormous amount has been learned about how specialization is achieved by differential expression of cardiac ion channels. More recently, many aspects of the molecular basis have been revealed. Although the field is potentially vast, an appreciation of key elements is essential for any clinician or researcher wishing to understand modern cardiac electrophysiology. This article reviews the major regionally determined features of cardiac electrical function, discusses underlying ionic bases, and summarizes present knowledge of ion channel subunit distribution in relation to functional specialization.
Novel Vascular Endothelial Growth Factor Binding Domains of Fibronectin Enhance Vascular Endothelial Growth Factor Biological Activity
Authors: Wijelath Errol S. , Murray Jacqueline , Rahman Salman , Patel Yatin , Ishida Atsushi , Strand Kurt , Aziz Salim , Cardona Carlos , Hammond William P. , Savidge Geoffrey F. , Rafii Shahin , Sobel Michael
Published: July 2002

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, α
5
β
1
, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to α
5
β
1
integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.
Role of Sterol Regulatory Element Binding Proteins in the Regulation of Gα
i2
Expression in Cultured Atrial Cells
Authors: Park Ho-Jin , Begley Ulrike , Kong Dequan , Yu Haiyan , Yin Liya , Hillgartner F. Bradley , Osborne Timothy F. , Galper Jonas B.
Published: July 2002

We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M
2
muscarinic receptor; the α-subunit of the heterotrimeric G protein, Gα
i2
; and the inward rectifying K
+
channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of Gα
i2
promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the Gα
i2
promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the Gα
i2
promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative Gα
i2
SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the Gα
i2
SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of Gα
i2
promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of Gα
i2
promoter activity, suggesting that SREBP1 may play a role in the regulation of Gα
i2
expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.
Cerebral Perfusion and Cerebrovascular Reactivity Are Reduced in White Matter Hyperintensities
Authors: Marstrand J.R. , Garde E. , Rostrup E. , Ring P. , Rosenbaum S. , Mortensen E.L. , Larsson H.B.W.
Published: July 2002

Background and Purpose

—

There is growing evidence that white matter hyperintensities (WMH) should not be considered as benign age-dependent changes on MR images but indicate pathological changes with clinical consequences. Previous studies comparing subjects with WMH to normal controls have reported global reductions in cerebral blood flow (CBF) and cerebral vascular reactivity. In this study, we examined localized hemodynamic status to compare WMH to normal appearing white matter (NAWM).

Methods

—

A group of 21 normal 85-year-old subjects were studied using dynamic contrast-enhanced MRI together with administration of acetazolamide. From a combination of anatomic images with different signal weighting, regions of interest were generated corresponding to gray and white matter and WMH. Localized measurements of CBF and cerebral blood volume (CBV) and mean transit time were obtained directly within WMH and NAWM.

Results

—

When comparing WMH to NAWM, measurements showed significantly lower CBF (
P
=0.004) and longer mean transit time (
P
< 0.001) in WMH but no significant difference in CBV (
P
=0.846). The increases in CBF and CBV induced by acetazolamide were significantly smaller in WMH than in NAWM (
P
=0.026,
P
<0.001).

Conclusion

—

These results show that a change in the hemodynamic status is present within the WMH, making these areas more likely to be exposed to transient ischemia inducing myelin rarefaction. In the future, MRI may be used to examine the effect of therapeutic strategies designed to prevent or normalize vascular changes.
Poly(ADP-Ribose) Polymerase Impairs Early and Long-Term Experimental Stroke Recovery
Authors: Goto Shozo , Xue Rong , Sugo Nobuo , Sawada Masahiko , Blizzard Kathleen K. , Poitras Marc F. , Johns David C. , Dawson Ted M. , Dawson Valina L. , Crain Barbara J. , Traystman Richard J. , Mori Susumu , Hurn Patricia D.
Published: July 2002

Background and Purpose

—

Poly(ADP-ribose) polymerase (PARP-1; Enzyme Commission 2.4.30) is a nuclear DNA repair enzyme that mediates early neuronal ischemic injury. Using novel 3-dimensional, fast spin-echo-based diffusion-weighted imaging, we compared acute (21 hours) and long-term (3 days) ischemic volume after middle cerebral artery (MCA) occlusion in PARP-1-null mutants (PARP−/−) versus genetically matched wild-type mice (WT mice). PARP−/− mice were also treated with viral transfection of wild-type PARP-1 to determine whether protection from MCA occlusion is lost with restoration of the gene product.

Methods

—

Halothane-anesthetized mice were treated with reversible MCA occlusion via intraluminal suture technique. Ischemic volumes were delineated by diffusion-weighted imaging with high spatial and temporal resolution during MCA occlusion and reperfusion. Recombinant Sindbis virus carrying β-galactosidase (lacZ) or PARP-1 was injected into ipsilateral striatum, then animals underwent MCA occlusion 3 days later. Infarction volume was measured at 22 hours of reperfusion (2,3,5-triphenyltetrazolium chloride histology).

Results

—

Reduction in regional water apparent diffusion coefficient (ADC) during occlusion or secondary ADC decline during reperfusion was not different between groups. Ischemic volume was smaller early in occlusion in PARP−/− versus WT mice and remained less at 21 hours of reperfusion. Ischemic volume then increased from 1 to 2 days in all mice, then stabilized without further change. Ischemic damage was smaller in PARP−/− than in WT mice at 3 days. Transfection of PARP-1 into PARP−/− mice increased stroke damage relative to lacZ-injected PARP−/− and increased damage to that of the WT mice. Intraischemic laser-Doppler flowmetry and physiological variables were not different among groups.

Conclusions

—

PARP-1 deficiency provides both early and prolonged protection from experimental focal stroke. The mechanism is not linked to preservation of ADC and mitigation of secondary energy depletion during early reperfusion.
Risk Factors for Progression of Aortic Atheroma in Stroke and Transient Ischemic Attack Patients
Authors: Sen Souvik , Oppenheimer Stephen M. , Lima Joao , Cohen Barry
Published: July 2002

Background and Purpose

—

Aortic atheroma is an independent risk factor for stroke and undergoes temporal progression. Clinical and risk factor associations of such progression are unknown. Hyperhomocysteinemia has been linked with atherosclerosis, including that in the cerebral vasculature. This study investigated associations between elevated homocysteine levels and other stroke vascular risk factors and the risk of aortic atheroma progression in patients with cerebrovascular disease.

Methods

—

Fifty-seven stroke and 21 transient ischemic attack patients underwent multiplanar transesophageal echocardiograms within 1 month of symptom onset and again at 9 months. Aortic atheroma was graded and stratified by use of existing criteria. Stroke risk factors; use of anticoagulant, antiplatelet, and hypolipidemic drugs; and clinical and etiological subtypes of stroke were recorded and compared in patients stratified for the presence or absence of aortic atheroma progression.

Results

—

Of the 78, 29 (37%) progressed, 32 (41%) remained unchanged, and 17 (22%) regressed. Progression was most marked at the aortic arch (
P
=0.005), followed by the ascending segment (
P
<0.04). In nearly two thirds of the patients in whom aortic atheroma remained unchanged over 9 months, no atheroma was evident on baseline transesophageal echocardiogram. Only homocysteine levels ≥14.0 μ mol/L (
P
=0.02), total anterior cerebral infarct (
P
=0.02), and large-artery atherosclerosis (
P
=0.005) significantly correlated with progression.

Conclusions

—

Among vascular risk factors, elevated homocysteine levels are associated with aortic atheroma progression. Stroke and transient ischemic attack patients with aortic atheroma should undergo assessment of homocysteine levels, which, if elevated, may be treated with vitamins in an effort to arrest aortic atheroma progression.