Cigarette smoking and ambulatory blood pressure: a case-control study in normotensives.

The aim of our study was the evaluation of the effects of cigarette smoking on ambulatory blood pressure (ABP) in normotensive subjects participating to a cardiovascular prevention program. All subjects were followed up for an average time of 97+/-42 months to assess the event of hypertension development. Prevalence of hypertension development was higher in smokers even if regression logistic analysis was not able to predict hypertension development.

Imaging of myocardial infarction using a 64-slice MDCT scanner: correlation between infarcted region and status of territory-dependent coronary artery.

PURPOSE: This study was performed to evaluate the ability of 64-slice multidetector computed tomography (MDCT) to detect previous myocardial infarctions (MIs) in patients referred for the assessment of the coronary arteries. In patients with regional changes of left ventricular wall myocardial density, the territory-dependent coronary vessel status was examined. MATERIALS AND METHODS: We retrospectively assessed 202 consecutive patients referred for 64-slice MDCT of the coronary arteries. In all cases, detailed, clinical, serological and electrocardiograph (ECG) data were collected to identify patients with a previous diagnosis of MI. An initial qualitative evaluation of MDCT images was performed in all patients to identify areas of suspected myocardial necrosis, which were defined as regions of lower density within normally enhanced left ventricular myocardium. Thereafter, in all patients with suspected MIs, attenuation values and left ventricular wall thickness were also measured at the level of the normal myocardium and within the hypodense regions. Each MI was also assigned to the distribution territory of a coronary vessel, and morphological data were combined with MDCT angiographic findings. RESULTS: After clinical assessment, MI was found in 27 patients (six acute).; 64-slice MDCT was able to detect the presence of MI in 24/27 cases, showing sensitivity and specificity of 89% and 95%, respectively, and an overall diagnostic accuracy of 95%. Quantitative analysis showed a significant difference (p<0.01) between attenuation values of normal vs. infarcted myocardium (124.5+/-19 HU vs. 56.1+/-23 HU, respectively); wall thinning was exclusively observed in chronic MIs (p<0.01). In 23/24 detected cases, analysis of territory-dependent arteries showed findings compatible with presence of MI. CONCLUSIONS: The presence of MI is well depicted with retrospective 64-slice MDCT. The main advantage of 64-slice MDCT is that it allows to evaluate and relate the status of a vessel and its dependent myocardial region in a single exam.

Evaluation of systolic blood pressure control in elderly patients with isolated systolic hypertension.

The aim of this retrospective study was the evaluation of systolic blood pressure (SBP) control in elderly patients (pts) with isolated systolic hypertension (ISH). We assessed SBP control (i.e. average of 2 clinic BP measurements < 140 mmHg) in 152 pts (44 M, 108 F, 75 +/- 6 years) with ISH (149/84 +/- 17/6 mmHg), treated for at least 3 months by general practitioners or specialists with treatments of their choice. Most antihypertensive drugs were used at starting doses in monotherapy or combination treatment, as usual in clinical practice. ECG was abnormal in 82/152 pts (54.0%). All pts were divided in 2 groups according to SBP control. The 2 groups were compared by chi-square test for categorical variables and by Mann-Whitney test for quantitative variables. A p value < 0.05 was considered statistically significant. The global SBP control rate was 41.4% (63/152 pts). BP was higher in pts with poor SBP control, as expected, but the 2 groups were similar for sex distribution, age, prevalence of other cardiovascular risk factors and type of care (general practitioner or specialist). Pts with poor SBP control had a higher prevalence of abnormal ECG tracings (p = 0.003), a lower prevalence of combination regimes (p = 0.007) and prescriptions of dihydropyridine calcium antagonists or thiazide diuretics (p = 0.006). Global SBP control rate in our retrospective study in pts with ISH was unsatisfactory. Use of dihydropyridines or thiazides, drugs of choice in the management of ISH according to ESH/ESC and JNCVII guidelines, as single drugs or in combination regimes, can improve BP control and prevent cardiac damage.

Noninvasive assessment of coronary artery stenoses by multidetector-row spiral computed tomography: comparison with conventional angiography.

BACKGROUND: Coronary artery disease (CAD) is the most common cause of hospitalization and mortality in many industrialized countries. We analysed the diagnostic accuracy of multi-detector row spiral computed tomography (MDCT) in determining mid- to high-grade coronary artery stenoses (> 50%). METHODS: Sixty-nine patients with suspected CAD were referred to MDCT coronary angiography. Patients with a heart rate above 60 bpm received 20-40 mg propranol before the scan. The left main (LM), the left anterior descending artery (LAD), the first diagonal branch (D1), the right coronary artery (RCA) and the proximal tract of the circumflex artery (LCX) were independently evaluated by two blinded observers and screened for > 50% stenoses. The mean values of MDCT coronary narrowings assessed by two observers were compared to quantitative coronary angiography. RESULTS: MDCT correctly detected 95 of 123 coronary lesions (sensitivity 77.2%) and absence of stenoses was correctly identified in 388 of 426 segments (specificity 91%). The sensitivity for the LM, LAD, RCA and the proximal tract of LCX was 100%, 86.5%, 69.8% and 80% respectively. Classification of patients as having 1-vessel, 2-vessels, 3-vessels or left main disease was accurate in 75.4% (46/61) of patients. CONCLUSIONS: MDCT technology, combined with heart rate control, allows reliable noninvasive detection of hemodynamically significant CAD.

A case of non-conducted atrial bigeminy simulating a second-degree atrioventricular block. A holter ECG diagnosis.

A 82-years-old man, symptomatic for fatigue and lypothymia, was referred to our centre in order to evaluate the opportunity of a permanent pacemaker insertion. A 12-lead ECG was described as a Mobitz type II second-degree atrioventricular block with 2:1 conduction. This surface ECG revealed some sinus beats with normal AV conduction, everyone followed by a bizarre, non-conducted P' wave, inscripted in the previous T wave. The Holter ECG showed a sinus rhythm with a mean rate of 70 beats/minute: during the night and at 06:39 pm of the following day, ECG strip showed some sinus beats, everyone followed by an atrial non-conducted ectopic beat, characterized by prematurity and abnormal shape (P' wave), that appears as a small deformation on the preceding T wave. At the beginning and the end of the strip we can estimate respectively 9 and 4 sinus beats, that represent the normal rhythm of this patient. So, it is possible to compare the normal P-P interval (P-P = 0.84 sec) to the shorter P-P' interval (P-P = 0.40 sec) and make the correct diagnosis of non-conducted atrial bigeminy simulating a second-degree AV block with 2:1 conduction.

Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury.

Tumor necrosis factor alpha induces apoptosis in mammary adenocarcinoma cells by an increase in intranuclear free Ca2+ concentration and DNA fragmentation.

The incubation of human mammary adenocarcinoma cells (BT-20) with tumor necrosis factor alpha in the absence or presence of cycloheximide resulted in progressive DNA fragmentation. This was preceded by a sustained increase in intracellular free Ca2+ concentration and was not detected in cells pretreated with intracellular Ca2+ chelators, calmodulin antagonists, or activators of protein kinase C. Image analysis of fura-2-loaded BT-20 cells treated with tumor necrosis factor alpha revealed that, in many cells, the initial increase in Ca2+ level occurred in a cellular region that corresponded to the localization of the nucleus. Our findings suggest that tumor necrosis factor alpha can promote an increase in intranuclear free Ca2+ which, in turn, may stimulate Ca(2+)-dependent endonuclease activity, resulting in DNA fragmentation and apoptosis.

Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation.

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features.

Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2-methyl-1,4-naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament- and the microtubule-enriched fractions from menadione-treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione-dependent and thiol-sensitive oxidation of actin, leading to the formation of high-molecular-weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione-dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP-induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate-size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation-dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate-size filament distribution in menadione-treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate-size filaments) are actually significant targets in quinone-induced oxidative stress.

The cytoskeleton as a target in quinone toxicity.

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure known as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears to be due mainly to actin cross-linking and dissociation of alpha-actinin from the actin network. The major biochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein sulfhydryl groups and the increase in cytosolic Ca2+ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca2+ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma membrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes, and thus it does not seem to be causatively related to their appearance. Thiol reductants, such as dithiothreitol, efficiently prevent the oxidation of cytoskeletal protein thiols, the increase in cytosolic free Ca2+ concentration and cell blebbing induced by redox cycling, but not alkylating, quinones. These results demonstrate that alkylating and redox cycling quinones cause similar structural and biochemical modifications of the cytoskeleton by means of different mechanisms, namely alkylation and oxidation of critical sulfhydryl groups.

Calcium-dependent DNA fragmentation in human synovial cells exposed to cold shock.

Exposure of confluent human synovial McCoy's cells to near-freezing temperatures followed by rewarming at 37 degrees C resulted in endonuclease activation and cell death characteristic of a suicide process known as apoptosis. Both DNA fragmentation and cell killing were dependent on a sustained increase in the cytosolic Ca2+ concentration. Sensitivity to cold shock-induced endonuclease activation was critically dependent on the cell cycle (proliferative) status and limited to confluent cells, whereas cells in the logarithmic growth phase were completely resistant. However, DNA fragmentation was promoted in the proliferating McCoy's cells pretreated with H-7 or sphingosine, inhibitors of protein kinase C. In addition, phorbol ester, known to activate PKC, inhibited DNA fragmentation in the confluent cells. Our findings indicate that cold shock-induced DNA fragmentation in McCoy's cells is dependent on a sustained Ca2+ increase, and sensitivity to the process appears to be regulated by the status of protein kinase C.

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload.

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.

Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca2+-dependent mechanisms.

The metabolism of the redox-active quinone, menadione (2-methyl-1,4-naphthoquinone), in human platelets was associated with superoxide anion production, oxidation and depletion of intracellular glutathione, and modification of protein thiols. The cytoskeletal fraction extracted from menadione-treated platelets exhibited a dose-dependent increase in the amount of cytoskeleton-associated protein and a concomitant loss of protein thiols. These alterations were associated with oxidative modifications of actin, including beta-mercaptoethanol-sensitive crosslinking of actin to form dimers, trimers, and high-molecular-weight aggregates which also contained other cytoskeletal proteins, i.e., alpha-actinin and actin-binding protein. In addition, analysis of the cytoskeletal fraction from platelets treated with high concentrations (greater than or equal to 100 microM) of menadione by polyacrylamide gel electrophoresis under reducing conditions revealed a net decrease in the relative abundance of the individual cytoskeletal polypeptides. Under the same incubation conditions the platelets exhibited a sustained increase in cytosolic Ca2+ concentration. The presence of glucose, or the omission of Ca2+ from the incubation medium, prevented both the increase in cytosolic Ca2+ and the decrease in the relative amounts of cytoskeletal proteins. The latter effect was also largely prevented in platelets loaded with Quin-2 tetraacetoxymethyl ester to buffer the menadione-induced elevation of cytosolic Ca2+. Finally, the presence of a protease inhibitor, leupeptin, in the incubation medium prevented the menadione-induced decrease in the amount of actin-binding protein but not the decrease in the other cytoskeletal proteins. Our findings demonstrate that the multiple effects of oxidative stress on the platelet cytoskeleton are mediated by oxidative as well as by Ca2+-dependent mechanisms.

Alterations in hepatocyte cytoskeleton caused by redox cycling and alkylating quinones.

Quinones may induce toxicity by a number of mechanisms, including alkylation and oxidative stress following redox cycling. The metabolism of quinones by isolated rat hepatocytes is associated with cytoskeletal alterations, plasma membrane blebbing, and subsequent cytotoxicity. The different mechanisms underlying the effects of alkylating (p-benzoquinone), redox cycling (2,3-dimethoxy-1,4-naphthoquinone), and mixed redox cycling/alkylating (2-methyl-1,4-naphthoquinone) quinones on hepatocyte cytoskeleton have been investigated in detail in this study. Analysis of the cytoskeletal fraction extracted from quinone-treated cells revealed a concentration-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols, irrespective of the quinone employed. In the case of redox cycling quinones, these alterations were associated with an oxidation-dependent actin crosslinking (sensitive to the thiol reductant dithiothreitol). In contrast, with alkylating quinones an oxidation-independent cytoskeletal protein crosslinking (insensitive to thiol reductants) was observed. In addition to these changes, a dose-dependent increase in the relative abundance of F-actin was detected as a consequence of the metabolism of oxidizing quinones in hepatocytes. Addition of dithiothreitol solubilized a considerable amount of polypeptides from the cytoskeletal fraction isolated from hepatocytes exposed to redox cycling but not alkylating quinones. Our findings indicate that the hepatocyte cytoskeleton is an important target for the toxic effects of different quinones. However, the mechanisms underlying cytoskeletal damage differ depending on whether the quinone acts primarily by oxidative stress or alkylation.

Alterations of surface morphology caused by the metabolism of menadione in mammalian cells are associated with the oxidation of critical sulfhydryl groups in cytoskeletal proteins.

Incubation of freshly-isolated (rat hepatocytes) or cultured (HeLa, GH3, and McCoy) mammalian cells with menadione (2-methyl-1,4-naphthoquinone) resulted in the appearance of numerous cell surface protrusions. The perturbation of surface structure was associated with an increase in the amount of cytoskeletal protein and the oxidation of sulfhydryl groups in actin, leading to the formation of high-molecular weight aggregates sensitive to treatment with thiol reductants. Our findings indicate that the oxidation of thiol groups in cytoskeletal proteins may be responsible for menadione-induced cell surface abnormalities in mammalian cells.