bluuebunny/crossref_metadata_2025 huggingface dataset:
ACE and α-Adducin Polymorphism as Markers of Individual Response to Diuretic Therapy
Authors: Sciarrone Maria Teresa , Stella Paola , Barlassina Cristina , Manunta Paolo , Lanzani Chiara , Bianchi Giuseppe , Cusi Daniele
Published: March 2003

Renin-angiotensin system reactivity and the constitutive capacity of the renal tubule to reabsorb sodium play a role in the individual response to diuretic therapy; therefore we evaluated the blood pressure (BP) response to hydrochlorothiazide in 87 never-treated individuals with mild essential hypertension, according to ACE gene I/D and α-adducin Gly460Trp polymorphism. These genotypes where chosen because previous data showed their interaction in determining the BP response to salt probably was the result of their involvement in the activation of the renin-angiotensin system (ACE) and in the constitutive capacity of the kidney to reabsorb sodium (α-adducin) (treatment for 2 months). BP was measured after 3 run-in visits and after the first and second months of treatment by means of a standardized procedure. Data were analyzed by ANOVA,
t
test, and multivariate ANOVA for repeated measures (covarying for gender, age, and body mass index). Although basal mean BP (MBP) was similar in the different ACE and α-adducin genotypes, patients carrying at least one I allele of ACE and one 460Trp allele of α-adducin had the largest MBP decrease with treatment (12.7±1.9 mm Hg), the effect of the combination of genotypes being additive but not epistatic. These patients had an odds ratio of 15.75 of being a responder to hydrochlorothiazide compared with patients with Gly460Gly+DD, with the least MBP decrease (3.4±1.7 mm Hg). α-Adducin and ACE I/D polymorphism may be useful to predict the interindividual degree of response to hydrochlorothiazide; the analysis of the combination of the 2 genotypes increases the accuracy of the prediction of response to the drug.
Outcomes With Nifedipine GITS or Co-Amilozide in Hypertensive Diabetics and Nondiabetics in Intervention as a Goal in Hypertension (INSIGHT)
Authors: Mancia Giuseppe , Brown Morris , Castaigne Alain , de Leeuw Peter , Palmer Christopher R. , Rosenthal Talma , Wagener Gilbert , Ruilope Luis M.
Published: March 2003

To investigate the impact of treatment on cardiovascular mortality and morbidity, we assessed outcomes in patients with hypertension and diabetes who received co-amilozide or nifedipine in the International Nifedipine GITS Study: Intervention as a Goal in Hypertension. Participants had to be 55 to 80 years of age, with hypertension (≥150/95 or ≥160 mm Hg) and at least one additional cardiovascular risk factor. Patients received 30 mg nifedipine once daily or co-amilozide (25 mg hydrochlorothiazide and 2.5 mg amiloride) daily. Doses were doubled if target blood pressures (<140/90 mm Hg) were not achieved. Primary (composite of cardiovascular death, myocardial infarction, heart failure, and stroke) and secondary outcomes (composite of primary outcomes, including all-cause mortality and death from vascular and nonvascular causes) were assessed by means of intent-to-treat analyses. There was no significant difference in the incidence of primary outcomes between nifedipine-treated and co-amilozide–treated patients with diabetes at baseline (n=1302) (8.3% versus 8.4%; relative risk, 0.99, 95% CI, 0.69 to 1.42;
P
=1.00). A significant benefit for nifedipine-treated patients was seen for the composite secondary outcome (14.2% versus 18.7%; relative risk, 0.76, 95% CI, 0.59 to 0.97;
P
=0.03). Among patients without diabetes at baseline (n=5019), there was a significant difference in the incidence of new diabetes (nifedipine 4.3% versus co-amilozide 5.6%,
P
=0.023). Nifedipine GITS once daily is as effective as diuretic therapy in reducing cardiovascular complications in hypertensive diabetics. Nifedipine-treated patients were also less likely to have diabetes or have secondary events (a composite of all-cause mortality, death from a vascular cause, and death from a nonvascular cause) than co-amilozide recipients. Our results suggest that nifedipine could be considered as first-line therapy for hypertensive diabetics.
Vitamins Reverse Endothelial Dysfunction Through Regulation of eNOS and NAD(P)H Oxidase Activities
Authors: Ülker Sibel , McKeown Pascal P. , Bayraktutan Ulvi
Published: March 2003

Antioxidant vitamins C and E have protective properties in genetic hypertension associated with enhanced oxidative stress. This study investigated whether vitamins C and/or E modulate vascular function by regulating enzymatic activities of endothelial nitric oxide synthase (eNOS) and NAD(P)H oxidase using thoracic aortas of 20- to 22-week-old male spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar-Kyoto rats (WKY). SHR aortas had impaired relaxant responses to acetylcholine but not to sodium nitroprusside, despite an ≈2-fold increase in eNOS activity and NO release. The levels of superoxide anion (O
2
−
), a potent NO scavenger, and NAD(P)H oxidase activity were also 2-fold higher in SHR aortas. Mechanical but not pharmacological inactivation of endothelium (by rubbing and 100 μmol/L L-NAME, respectively) significantly abrogated O
2
−
in both strains. Treatments of SHR aortas with NAD(P)H oxidase inhibitors, namely diphenyleneiodinium and apocynin, significantly diminished O
2
−
production. The incubation of SHR aortas with different concentrations of vitamin C (10 to 100 μmol/L) and specifically with high concentrations of vitamin E (100 μmol/L) improved endothelial function, reduced superoxide production as well as NAD(P)H oxidase activity, and increased eNOS activity and NO generation in SHR aortas to the levels observed in vitamin C- and E-treated WKY aortas. Our results reveal endothelial NAD(P)H oxidase as the major source of vascular O
2
−
in SHR and also show that vitamins C and E are critical in normalizing genetic endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities.
Gα
12
- and Gα
13
-Protein Subunit Linkage of D
5
Dopamine Receptors in the Nephron
Authors: Zheng Shaopeng , Yu Peiying , Zeng Chunyu , Wang Zheng , Yang Zhiwei , Andrews Peter M. , Felder Robin A. , Jose Pedro A.
Published: March 2003

The roles of the G-protein α-subunits, G
s
, G
i
, and G
q/11
, in the signal transduction of the D
1
-like dopamine receptors, D
1
and D
5
, have been deciphered. Gα
12
and Gα
13
, members of the 4th family of G protein subunits, are not linked with D
1
receptors, and their linkage to D
5
receptors is not known. Therefore, we studied the expression of Gα
12
and Gα
13
and interaction with D
5
dopamine receptors in the kidney from normotensive Wistar-Kyoto (WKY) rats and D
5
receptor-transfected HEK293 cells. Gα
12
and Gα
13
were found in the proximal tubule, distal convoluted tubule, and artery and vein in the WKY rat kidney. Whereas Gα
12
was expressed in the ascending limb of Henle, Gα
13
was expressed in the collecting duct and juxtaglomerular cells. In renal proximal tubules, Gα
12
and Gα
13
, as with D
5
receptors, were expressed in brush border membranes. Laser confocal microscopy revealed the colocalization of D
5
receptors with Gα
12
and Gα
13
in rat renal brush border membranes, immortalized rat renal proximal tubule cells, and D
5
receptor-transfected HEK293 cells. In these cells, a D
1
-like agonist, fenoldopam, increased the association of Gα
12
and Gα
13
with D
5
receptors, results that were corroborated by immunoprecipitation experiments. We conclude that although both D
1
and D
5
receptors are linked to Gαs, they are differentially linked to Gα
12
and Gα
13
. The consequences of the differential G-protein subunit linkage on D
1
- and D
5
-mediated sodium transport remains to be determined.
Cardiac Transcriptome Analysis in Obesity-Related Hypertension
Authors: Philip-Couderc Pierre , Smih Fatima , Pelat Michel , Vidal Cyril , Verwaerde Patrick , Pathak Atul , Buys Sophie , Galinier Michel , Senard Jean-Michel , Rouet Philippe
Published: March 2003

Obesity is associated with volumetric arterial hypertension and with early increase in heart rate and decreased heart rate variability. The consequences of obesity-related hypertension on heart gene regulation are poorly known and were investigated in a model of obesity-related hypertension induced by high fat diet in dogs. When compared with control animals (n=6), a 9-week high fat diet (n=6) provoked significant weight gain and increased blood pressure load and heart rate but failed to significantly change left ventricular mass assessed by echocardiography. Subtractive hybridization of dog heart cDNA libraries were used to generate sublibraries containing differentially expressed cDNAs that were in turn spotted onto membranes to create custom microarrays. Hybridizations of these microarrays with complex probes representing mRNAs expressed in right atria and left ventricles from obese hypertensive and control dogs were performed. Thirty-eight differentially expressed genes were identified; altered expression was confirmed by Northern blot analysis in 15. In addition, real-time quantitative polymerase chain reaction confirmed differential expression for 80% of the randomly chosen tested genes. Once identified, transcripts were categorized into groups involved in metabolism, cell signaling, ionic regulation, cell proliferation, protein synthesis, and tissue remodeling. In addition, we found a set of 11 cDNAs encoding proteins with unknown functions. This study clearly shows that obesity-related hypertension, lasting for only 9 weeks, causes marked changes in gene expression in right atrium as well as the left ventricle that may contribute to early functional changes in heart function and to long-term structural changes such as left ventricular hypertrophy and remodeling.
Left Ventricular Concentric Geometry as a Risk Factor in Gestational Hypertension
Authors: Novelli Gian Paolo , Valensise Herbert , Vasapollo Barbara , Larciprete Giovanni , Altomare Francesco , Di Pierro Giuseppe , Casalino Beniamino , Galante Alberto , Arduini Domenico
Published: March 2003

In the past, an adverse prognostic significance of an altered left ventricular geometry in essential hypertension has been demonstrated. There are no data on the prognostic significance of an altered cardiac structure during pregnancy. The present study was designed to evaluate the prognostic impact on the outcome of pregnancy of an altered geometry of the left ventricle in mild gestational hypertension. One hundred forty-eight consecutive, pregnant, mild gestational hypertensive women (systolic and diastolic blood pressure, 140 to 150 mm Hg and 90 to 99 mm Hg, respectively) were included in the study. Patients were monitored until term to detect subsequent fetal and/or maternal adverse outcomes (preeclampsia, preterm delivery, abruptio placentae, other maternal medical problems, fetal distress, neonatal low birth weight, admittance to neonatal intensive care unit). One hundred one gestational hypertensive patients (68.2%) had an uneventful pregnancy; 47 patients (31.8%) showed a subsequent development of maternal and/or fetal complications. Concentric geometry was prevalent among patients with the subsequent development of complicated gestational hypertension (37 out of 47 patients) compared with the uneventful gestational hypertensive patients (31 out of 101 patients; 78.7% versus 30.1%;
P
=0.0001). The multivariate analysis showed concentric geometry as an independent predictor of adverse outcomes (odds ratio, 3.65; 95% confidence interval, 1.30 to 10.27;
P
=0.014). In patients with gestational hypertension, blood pressure values alone appear to be insufficient to identify the effective risk of adverse events. Ventricular geometry gives additional prognostic information, possibly improving our clinical ability to follow and eventually treat these patients.
Role of the Subfornical Organ in the Chronic Hypotensive Response to Losartan in Normal Rats
Authors: Collister John P. , Hendel Michael D.
Published: March 2003

Angiotensin II is known to act at a unique set of brain regions known as the circumventricular organs. These structures lack the normal blood–brain barrier and are therefore thought to participate in the central nervous system processing of neuroendocrine signals. We have reported that chronic treatment with the angiotensin type 1 (AT
1
) receptor antagonist, losartan, decreases arterial pressure in normotensive rats. Furthermore, this hypotension is attenuated in area postrema–lesioned rats, suggesting a role of endogenous angiotensin II at this circumventricular organ. Another circumventricular organ, the subfornical organ (SFO), has also been shown to mediate actions of angiotensin II. The present study tested the hypothesis that the SFO is a central site of action of endogenous angiotensin II at AT
1
receptors. Adult male Sprague-Dawley rats were anesthetized and placed in a stereotaxic apparatus, and the SFO was sham or electrolytically lesioned. One week later, rats were instrumented with venous catheters and radiotelemetry pressure transducers for continuous infusion and monitoring of mean arterial pressure, respectively. After 3 days of control, losartan was administered intravenously (10 mg · kg
−1
· d
−1
) for 10 days in both SFO-lesioned and sham rats. By day 4 of losartan administration, mean arterial pressure had decreased to 75±2 mm Hg in sham rats (n=9) but had only fallen to 83±2 mm Hg in lesioned rats (n=10). This attenuated hypotensive response in SFO-lesioned rats continued through day 10 of losartan treatment. These results support the hypothesis that the SFO mediates part of the hypotensive effects of chronic AT
1
receptor blockade in the normotensive rat.
Short-Versus Long-Term Effects of Different Dihydropyridines on Sympathetic and Baroreflex Function in Hypertension
Authors: Grassi Guido , Seravalle Gino , Turri Carlo , Bolla GianBattista , Mancia Giuseppe
Published: March 2003

Antihypertensive treatment with dihydropyridines may be accompanied by sympathetic activation. Data on whether this is common to all compounds and similar in the various phases of treatment are not univocal, however. In 28 untreated essential hypertensives (age, 56.4±1.8 years; mean±SEM) finger blood pressure (BP, Finapres), heart rate (HR, ECG), plasma norepinephrine (NE, high-performance liquid chromatography), and muscle sympathetic nerve traffic (MSNA, microneurography) were measured at rest and during baroreceptor manipulation (vasoactive drugs) in the placebo run-in period and after randomization to double-blind acute and chronic (8 weeks) felodipine (10 mg/d, n=14) or lercanidipine (10 mg/d, n=14). Acute administration of both drugs induced pronounced BP reductions and marked increases in HR, NE, and MSNA. After 8 weeks of treatment, BP reductions were similar to those observed after acute administration, whereas HR, NE, and MSNA responses were markedly attenuated (−7%, −32%, and −14%, respectively;
P
<0.05). There was a small residual increase in sympathetic activity in the felodipine group, whereas in the lercanidipine group, all adrenergic markers returned to baseline values. Baroreflex control of HR and MSNA was markedly impaired (−42% and −48%, respectively) after acute drug administration, with a recovery and complete resetting during chronic treatment. Thus, the sympathoexcitation induced by 2 different dihydropyridines is largely limited to the acute administration. The 2 drugs have, nevertheless, a different chronic sympathetic effect, indicating that dihydropyridines do not homogeneously affect this function. The acute sympathoexcitation, but not the small between-drugs differential chronic adrenergic effect, is accounted for by baroreflex impairment.
Characterization of Renal Angiotensin-Converting Enzyme 2 in Diabetic Nephropathy
Authors: Tikellis Christos , Johnston Colin I. , Forbes Josephine M. , Burns Wendy C. , Burrell Louise M. , Risvanis John , Cooper Mark E.
Published: March 2003

ACE2, initially cloned from a human heart, is a recently described homologue of angiotensin-converting enzyme (ACE) but contains only a single enzymatic site that catalyzes the cleavage of angiotensin I to angiotensin 1–9 [Ang(1–9)] and is not inhibited by classic ACE inhibitors. It also converts angiotensin II to Ang(1–7). Although the role of ACE2 in the regulation of the renin-angiotensin system is not known, the renin-angiotensin system has been implicated in the pathogenesis of diabetic complications and in particular in diabetic nephropathy. Therefore, the aim of this study was to assess the possible involvement of this new enzyme in the kidney from diabetic Sprague-Dawley rats to compare and contrast it to ACE. ACE2 and ACE gene and protein expression were measured in the kidney after 24 weeks of streptozocin diabetes. ACE2 and ACE mRNA levels were decreased in diabetic renal tubules by ≈50% and were not influenced by ACE inhibitor treatment with ramipril. By immunostaining, both ACE2 and ACE protein were localized predominantly to renal tubules. In the diabetic kidney, there was reduced ACE2 protein expression that was prevented by ACE inhibitor therapy. The identification of ACE2 in the kidney, its modulation in diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of various angiotensin peptides provides a rationale to further explore the role of this enzyme in various pathophysiological states including diabetic complications.
Gene Transfer of Extracellular Superoxide Dismutase Reduces Arterial Pressure in Spontaneously Hypertensive Rats
Authors: Chu Yi , Iida Shinichiro , Lund Donald D. , Weiss Robert M. , DiBona Gerald F. , Watanabe Yoshimasa , Faraci Frank M. , Heistad Donald D.
Published: March 2003

Oxidative stress may contribute to hypertension. The goals of this study were to determine whether extracellular superoxide dismutase (ECSOD) reduces arterial pressure in spontaneously hypertensive rats (SHR) and whether its heparin-binding domain (HBD), which is responsible for cellular binding, is necessary for the function of ECSOD. Three days after intravenous injection of an adenoviral vector expressing human ECSOD (AdECSOD), mean arterial pressure (MAP) decreased from 165±4 mm Hg (mean±SE, n=7) to 124±3 mm Hg (n=7) in adult anesthetized SHR (
P
<0.01) but was not altered in normotensive Wistar-Kyoto rats. Cardiac output was not changed in SHR 3 days after AdECSOD. Gene transfer of ECSOD with deletion of the HBD (AdECSODΔHBD) had no effect on SHR MAP, even though plasma SOD activity was greater after AdECSODΔHBD than after AdECSOD. Immunohistochemistry revealed intense staining for ECSOD in blood vessels and kidneys after AdECSOD but not after AdECSODΔHBD. Impaired relaxation of the carotid artery to acetylcholine in SHR was significantly improved after AdECSOD. Cumulative sodium balance in SHR was reduced by AdECSOD compared with AdECSODΔHBD. Gene transfer of ECSOD also reduced MAP in conscious SHR, although the effect was not as profound as in anesthetized SHR. In summary, gene transfer of ECSOD, with a strict requirement for its HBD, reduces systemic vascular resistance and arterial pressure in a genetic model of hypertension. This reduction in arterial pressure may be mediated by vasomotor and/or renal mechanisms.
VEGFR-1–Selective VEGF Homologue PlGF Is Arteriogenic
Authors: Pipp Frederic , Heil Matthias , Issbrücker Katja , Ziegelhoeffer Tibor , Martin Sandra , van den Heuvel Joop , Weich Herbert , Fernandez Borja , Golomb Gershon , Carmeliet Peter , Schaper Wolfgang , Clauss Matthias
Published: March 2003

Two signaling receptors for vascular endothelial growth factor (VEGF) in the vasculature are known with not yet well-understood roles in collateral vessel growth (arteriogenesis). In this study, we examined the involvement of the two VEGF receptors in arteriogenesis. Therefore, we used the VEGF homologue placenta growth factor (PlGF), which only binds to VEGFR-1 and VEGF-E, which only recognizes VEGFR-2. These peptides were locally infused over 7 days after ligation of the femoral artery in the rabbit. Evaluation of collateral growth by determining collateral conductance and angiographic scores demonstrated that the VEGFR-1–specific PlGF contributed significantly more to arteriogenesis than the VEGFR-2 specific VEGF-E. The combination of VEGF-E and PlGF did not exceed the effect of PlGF alone, indicating that cooperation of the two VEGF receptors in endothelial cell signaling is not required for arteriogenesis. In an in vitro model of angiogenesis, VEGF and VEGF-E were comparably active, whereas PlGF displayed no activity when given alone and did not further increase the effects of VEGF or VEGF-E. However, PlGF was as potent as VEGF when monocyte activation was assessed by monitoring integrin surface expression. In addition, accumulation of activated monocytes/macrophages in the periphery of collateral vessels in PlGF-treated animals was observed. Furthermore, in monocyte-depleted animals, the ability of PlGF to enhance collateral growth in the rabbit model and to rescue impaired arteriogenesis in PlGF gene–deficient mice was abrogated. Together, these data indicate that the arteriogenic activity observed with the VEGFR-1–specific PlGF is caused by its monocyte-activating properties.
Posttranscriptional Control of Renin Synthesis
Authors: Skalweit Angela , Doller Anke , Huth Antje , Kähne Thilo , Persson Pontus B. , Thiele Bernd-Joachim
Published: March 2003

Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3′-untranslated region (3′-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed. The following six proteins were unambiguously identified as hREN mRNA 3′-UTR binding proteins: hnRNP E1 (synonyms α-CP or PCBP), hnRNP K, dynamin, nucleolin, YB-1, and MINT-homologous protein. All proteins contain various RNA binding motifs, and most have been described in the context of mRNA binding and mRNA stabilization. Four proteins for which antibodies were available were verified by immunological techniques (dynamin, nucleolin, hnRNP E1, and YB-1). Forskolin, an activator of cAMP synthesis, considerably stimulates renin synthesis via inhibition of REN mRNA decay. Functionally, this cAMP-based mRNA stabilization is accompanied by a 3- to 6-fold upregulation of REN mRNA binding proteins. RNase degradation assays confirm that 3′-UTR binding proteins are able to protect and stabilize REN mRNA in vitro.
Remodeling of Gap Junctional Channel Function in Epicardial Border Zone of Healing Canine Infarcts
Authors: Yao Jian-An , Hussain Wajid , Patel Pravina , Peters Nicholas S. , Boyden Penelope A. , Wit Andrew L.
Published: March 2003

The epicardial border zone (EBZ) of canine infarcts has increased anisotropy because of transverse conduction slowing. It remains unknown whether changes in gap junctional conductance (G
j
) accompany the increased anisotropy. Ventricular cell pairs were isolated from EBZ and normal hearts (NZ). Dual patch clamp was used to quantify G
j
. At a transjunctional voltage (V
j
) of +10 mV, side-to-side G
j
of EBZ pairs (9.2±3.4 nS, n=16) was reduced compared with NZ side-to-side G
j
(109.4±23.6 nS, n=14,
P
<0.001). G
j
of end-to-end coupled cells was not reduced in EBZ. Steady-state G
j
of both NZ and EBZ showed voltage dependence, described by a two-way Boltzmann function. Half-maximal activation voltage in EBZ was shifted to higher V
j
in positive and negative directions. Immunoconfocal planimetry and quantification showed no change in connexin43 per unit cell volume or surface area in EBZ. Decreased side-to-side coupling occurs in EBZ myocytes, independent of reduced connexin43 expression, and is hypothesized to contribute to increased anisotropy and reentrant arrhythmias.
Cx43 and Dual-Pathway Electrophysiology of the Atrioventricular Node and Atrioventricular Nodal Reentry
Authors: Nikolski Vladimir P. , Jones Sandra A. , Lancaster Matthew K. , Boyett Mark R. , Efimov Igor R.
Published: March 2003

Fluorescent imaging has revealed that posterior nodal extensions provide the anatomical substrate for the dual-pathway electrophysiology of the atrioventricular (AV) node during normal conduction and reentry. The reentry can be intranodal, or as well as the posterior nodal extensions, it can involve an endocardial layer of atrial/atrial-nodal (A/AN) cells as part of the AV nodal reentry (AVNR) circuit. Using fluorescent imaging with a voltage-sensitive dye and immunolabeling of Cx43, we mapped the electrical activity and structural substrate in 3 types of AVNR induced by premature atrial stimulation in 8 rabbit hearts. In 6 cases, the AVNR pathway involved (1) a fast pathway (FP), (2) the A/AN layer, and (3) a slow pathway (SP). In 4 cases, reentry took the path (1) SP, (2) A/AN layer, and (3) FP. In 2 cases, reentry was intranodal, propagating between the 2 posterior nodal extensions. Immunolabeling revealed that the FP and SP are formed by Cx43-expressing bundles surrounded by tissue without Cx43. Cx43-expressing posterior nodal extensions are the substrate of AVNR during both intranodal and extranodal reentry.
Coordinate Control of Proliferation and Migration by the p27
Kip1
/Cyclin-Dependent Kinase/Retinoblastoma Pathway in Vascular Smooth Muscle Cells and Fibroblasts
Authors: Díez-Juan Antonio , Andrés Vicente
Published: March 2003

Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27
Kip1
against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27
Kip1
-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27
Kip1
inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27
Kip1
mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27
Kip1
/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders.
Alveolar Type 1 Cells Express the α2 Na,K-ATPase, Which Contributes to Lung Liquid Clearance
Authors: Ridge K.M. , Olivera W.G. , Saldias F. , Azzam Z. , Horowitz S. , Rutschman D.H. , Dumasius V. , Factor P. , Sznajder J.I.
Published: March 2003

The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent ≈95% and ≈5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the α1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na
+
transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the α1 and α2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the α2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The β-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the α2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase α2 gene (Adα2), which resulted in the overexpression of the α2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Adα2, which increased α2 protein abundance and resulted in a ≈250% increase in lung liquid clearance. These studies provide the first evidence that α2 Na,K-ATPase in AT1 cells contributes to most of the active Na
+
transport and lung liquid clearance, which can be further increased by stimulation of the β-adrenergic receptor or by adenovirus-mediated overexpression of the α2 Na,K-ATPase.
Structural Adaptation of the Nuclear Pore Complex in Stem Cell–Derived Cardiomyocytes
Authors: Perez-Terzic Carmen , Behfar Atta , Méry Annabelle , van Deursen Jan M.A. , Terzic Andre , Pucéat Michel
Published: March 2003

Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell–derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle–isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell–derived cardiomyocytes. Stem cell–derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell–derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell–derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.
Induction of Glutathione Synthesis in Macrophages by Oxidized Low-Density Lipoproteins Is Mediated by Consensus Antioxidant Response Elements
Authors: Bea Florian , Hudson Francesca N. , Chait Alan , Kavanagh Terrance J. , Rosenfeld Michael E.
Published: March 2003

The uptake of oxidized low-density lipoproteins (oxLDL) by macrophages leading to conversion into foam cells is a seminal event in atherogenesis. Excessive accumulation of oxLDL can cause oxidative stress in foam cells leading to cell death and the progression and destabilization of atherosclerotic lesions. Oxidative stress induces a protective compensatory increase in the synthesis of the endogenous antioxidant glutathione (GSH). Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis and is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM), which are products of separate genes. Treatment of RAW 264.7 mouse macrophages and mouse peritoneal macrophages with oxLDL (30 μg/mL) induces increased expression of both
Gclc
and
Gclm
in vitro. The increase in mRNA occurs in part via increased transcription as demonstrated with luciferase reporter constructs. The promoters for both
GCLC
and
GCLM
contain consensus antioxidant response elements (AREs). Electrophoretic mobility shift assays revealed induction of nuclear factor binding to these AREs after treatment of RAW 264.7 cells and mouse peritoneal macrophages with oxLDL. Nuclear factor binding to the AREs is diminished by a single base pair substitution in the core sequence. Site-directed mutagenesis of the AREs within the
Gclc
and
Gclm
promoters resulted in a decrease of oxLDL-induced luciferase activity. Supershift analyses revealed that oxLDL stimulates binding of the transcription factors Nrf1, Nrf2, and c-jun to the AREs. These data suggest that AREs play a direct role in mediating the induction of GSH synthesis by oxLDL and in protecting macrophages against oxidized lipid-induced oxidative stress.
Familial Hypertrophic Cardiomyopathy-Linked Mutant Troponin T Causes Stress-Induced Ventricular Tachycardia and Ca
2+
-Dependent Action Potential Remodeling
Authors: Knollmann Björn C. , Kirchhof Paulus , Sirenko Syevda G. , Degen Hubertus , Greene Anne E. , Schober Tilmann , Mackow Jessica C. , Fabritz Larissa , Potter James D. , Morad Martin
Published: March 2003

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and high incidence of sudden death, despite causing little or no cardiac hypertrophy in patients. Transgenic mice expressing mutant human TnT (I79N-Tg) have increased cardiac contractility, but no ventricular hypertrophy or fibrosis. Enhanced cardiac function has been associated with myofilament Ca
2+
sensitization, suggesting altered cellular Ca
2+
handling. In the present study, we compare cellular Ca
2+
transients and electrophysiological parameters of 64 I79N-Tg and 106 control mice in isolated myocytes, isolated perfused hearts, and whole animals. Ventricular action potentials (APs) measured in isolated I79N-Tg hearts and myocytes were significantly shortened only at 70% repolarization. No significant differences were found either in L-type Ca
2+
or transient outward K
+
currents, but inward rectifier K
+
current (IK1) was significantly decreased. More critically, Ca
2+
transients of field-stimulated ventricular I79N-Tg myocytes were reduced and had slow decay kinetics, consistent with increased Ca
2+
sensitivity of I79N mutant fibers. AP differences were abolished when myocytes were dialyzed with Ca
2+
buffers or after the Na
+
-Ca
2+
exchanger was blocked by Li
+
. At higher pacing rates or in presence of isoproterenol, diastolic Ca
2+
became significantly elevated in I79N-Tg compared with control myocytes. Ventricular ectopy could be induced by isoproterenol-challenge in isolated I79N-Tg hearts and anesthetized I79N-Tg mice. Freely moving I79N-Tg mice had a higher incidence of nonsustained ventricular tachycardia (VT) during mental stress (warm air jets). We conclude that the TnT-I79N mutation causes stress-induced VT even in absence of hypertrophy and/or fibrosis, arising possibly from the combination of AP remodeling related to altered Ca
2+
transients and suppression of IK1.
Chlamydia pneumoniae
Induces Tissue Factor Expression in Mouse Macrophages via Activation of Egr-1 and the MEK-ERK1/2 Pathway
Authors: Bea Florian , Puolakkainen Mirja H. , McMillen Timothy , Hudson Francesca N. , Mackman Nigel , Kuo Cho Chou , Campbell Lee Ann , Rosenfeld Michael E.
Published: March 2003

Recent studies have suggested that infection with
Chlamydia pneumoniae (C pneumoniae)
may contribute to the instability of atherosclerotic plaques and thrombosis and is associated with acute coronary events. Tissue factor (TF), a potent prothrombotic molecule, is expressed by macrophages and other cell types within atherosclerotic lesions and plays an essential role in thrombus formation after plaque rupture. Therefore the effects of
C pneumoniae
on induction of TF expression in macrophages were investigated. Infection of RAW mouse macrophages with
C pneumoniae
induced a time-dependent increase in procoagulant activity, expression of TF protein, and TF mRNA.
C pneumoniae
infection stimulated increased binding of nuclear proteins to the consensus DNA sequence for Egr-1, a key response element within the TF promoter, and increased the expression of Egr-1 protein. Transient transfections of RAW cells with mutated TF promoter constructs showed that the Egr-1 binding region is an important transcriptional regulator of
C pneumoniae
–induced TF expression. Furthermore,
C pneumoniae
–stimulated phosphorylation of ERK1/2 and Elk-1 and pharmacological inhibition of mitogen-activated protein kinase activity reduced the expression of TF and Egr-1. Antibody and polymyxin B blocking of the Toll-like receptor 4 (TLR4) partially reduced the
C pneumoniae
–induced expression of TF and Egr-1. In conclusion, the
C pneumoniae
–induced increase in TF expression in macrophages is mediated in part by Egr-1, signaling through TLR4, and activation of the MEK-ERK1/2 pathway.
Activation of RhoA and Inhibition of Myosin Phosphatase as Important Components in Hypertension in Vascular Smooth Muscle
Authors: Seko Tetsuya , Ito Masaaki , Kureishi Yasuko , Okamoto Ryuji , Moriki Nobuyuki , Onishi Katsuya , Isaka Naoki , Hartshorne David J. , Nakano Takeshi
Published: March 2003

Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca
2+
sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in
N
ω
-nitro-
l
-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.
Is Depressed Myocyte Contractility Centrally Involved in Heart Failure?
Authors: Houser Steven R. , Margulies Kenneth B.
Published: March 2003

This review examines the evidence for and against the hypothesis that abnormalities in cardiac contractility initiate the heart failure syndrome and drive its progression. There is substantial evidence that the contractility of failing human hearts is depressed and that abnormalities of basal Ca
2+
regulation and adrenergic regulation of Ca
2+
signaling are responsible. The cellular and molecular defects that cause depressed myocyte contractility are not well established but seem to culminate in abnormal sarcoplasmic reticulum uptake, storage, and release. There are also strong links between Ca
2+
regulation, Ca
2+
signaling pathways, hypertrophy, and heart failure that need to be more clearly delineated. There is not substantial direct evidence for a causative role for depressed contractility in the initiation and progression of human heart failure, and some studies show that heart failure can occur without depressed myocyte contractility. Stronger support for a causal role for depressed contractility in the initiation of heart failure comes from animal studies where maintaining or improving contractility can prevent heart failure. Recent clinical studies in humans also support the idea that beneficial heart failure treatments, such as β-adrenergic antagonists, involve improved contractility. Current or previously used heart failure treatments that increase contractility, primarily by increasing cAMP, have generally increased mortality. Novel heart failure therapies that increase or maintain contractility or adrenergic signaling by selectively modulating specific molecules have produced promising results in animal experiments. How to reliably implement these potentially beneficial inotropic therapies in humans without introducing negative side effects is the major unanswered question in this field.
Spatial Microstimuli in Endothelial Mechanosignaling
Authors: Davies Peter F. , Zilberberg Jenny , Helmke Brian P.
Published: March 2003

Descriptive and quantitative analyses of microstimuli in living endothelial cells strongly support an integrated mechanism of mechanotransduction regulated by the spatial organization of multiple structural and signaling networks. Endothelial responses to blood flow are regulated at multiple levels of organization extending over scales from vascular beds to single cells, subcellular structures, and individual molecules. Microstimuli at the cellular and subcellular levels exhibit temporal and spatial complexities that are increasingly accessible to measurement. We address the cell and subcellular physical interface between flow-related forces and biomechanical responses of the endothelial cell. Live cell imaging and computational analyses of structural dynamics, two important approaches to microstimulation at this scale, are briefly reviewed.
Glucose Regulates Monocyte Adhesion Through Endothelial Production of Interleukin-8
Authors: Srinivasan Suseela , Yeh Michael , Danziger Eric C. , Hatley Melissa E. , Riggan Anna E. , Leitinger Norbert , Berliner Judith A. , Hedrick Catherine C.
Published: March 2003

We have shown that glucose increases monocyte adhesion to human aortic endothelial cells (HAECs) in vitro.

1

In the present study, we examined mechanisms by which glucose stimulates monocyte:endothelial interactions. HAECs cultured for 7 days in 25 mmol/L glucose had a 2-fold elevation in interleukin-8 (IL-8) secretion over control cells cultured in 5.5 mmol/L glucose (
P
<0.001). Use of a neutralizing antibody to IL-8 prevented glucose-mediated monocyte adhesion. Both glucose and IL-8 activated β
1
integrin on the HAEC surface, suggesting that both activate the α
5
β
1
integrin complex on the endothelial surface. The α
5
β
1
integrin complex is important for anchoring connecting segment-1 fibronectin on the HAEC surface for monocyte adhesion. Analysis of the human IL-8 promoter revealed binding sites for NF-κB and AP-1 as well as several aligned carbohydrate response elements (also known as E-boxes). Glucose dramatically stimulated IL-8 promoter activity. Using mutated IL-8 promoter constructs and EMSA, we found that the AP-1 element and the glucose-response element were responsible for much of the glucose-mediated activation of IL-8 transcription. Interestingly, inhibition of reactive oxygen species (ROS) production through use of pharmacological uncouplers of the mitochondrial electron transport chain significantly reduced glucose-mediated induction of IL-8 expression. These data indicate that glucose regulates monocyte:endothelial interactions through stimulation of IL-8 and ROS production and activation of the α
5
β
1
integrin complex on HAECs.
Selective Neuromicrovascular Endothelial Cell Death by 8-Iso-Prostaglandin F
2α
Authors: Brault Sonia , Martinez-Bermudez Ana Katherine , Marrache Anne Marilise , Gobeil Fernand , Hou Xin , Beauchamp Martin , Quiniou Christiane , Almazan Guillermina , Lachance Christian , Roberts Jackson , Varma Daya R. , Chemtob Sylvain
Published: March 2003

Background and Purpose—

Free radical-induced peroxidation is an important factor in the genesis of hypoxic-ischemic encephalopathy, including that of the preterm infant. Isoprostanes are major peroxidation products. Since microvascular dysfunction seems to contribute to ischemic encephalopathies, we studied the cytotoxicity of 8-iso-prostaglandin F
2α
(PGF
2α
) on cerebral microvascular cells.

Methods—

Microvascular endothelial, astroglial, and smooth muscle cells from newborn brain were cultured. The cytotoxicity of 8-iso-PGF
2α
on these cells was determined by MTT assays and lactate dehydrogenase (LDH) release, propidium iodide incorporation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]). In addition, effects of intraventricular injections of 8-iso-PGF
2α
and possible involvement of thromboxane in 8-iso-PGF
2α
-induced cytotoxicity were determined.

Results—

8-Iso-PGF
2α
induced time- and concentration-dependent endothelial cell death (EC
50
=0.1 nmol/L) but exerted little effect on smooth muscle and astroglial cells; endothelial cell death seemed mostly of oncotic nature (propidium iodide incorporation and LDH release). Cell death was associated with increased endothelial thromboxane A
2
(TXA
2
) formation and was prevented by TXA
2
synthase inhibitors (CGS12970 and U63557A); TXA
2
mimetics U46619 and I-BOP also caused endothelial cell death. Intraventricular injection of 8-iso-PGF
2α
induced periventricular damage, which was attenuated by CGS12970 pretreatment.

Conclusions—

These data disclose a novel action of 8-iso-PGF
2α
involving TXA
2
in oxidant stress-induced cerebral microvascular injury and brain damage.