Regulation of NADPH oxidase subunit p22(phox) by NF-kB in human aortic smooth muscle cells.

Accumulating evidence demonstrates the involvement of oxidative stress in the pathophysiology of cardiovascular diseases. The molecular mechanisms accountable for the increased production of reactive oxygen species remain uncertain. Among others, NADPH oxidase is one of the most important sources of superoxide in vascular cells. Here we investigate the role of NF-kB in the regulation of p22(phox) subunit and NADPH oxidase activity, in human aortic smooth muscle cells. Overexpression of p65/RelA or IKKbeta up-regulated p22(phox) gene promoter activity. Transcription factor pull-down assays demonstrated the physical interaction of p65/RelA protein with predicted NF-kB binding sites. Real time PCR and Western blotting analysis showed that p22(phox) mRNA and protein expression are significantly down-regulated by NF-kB decoy oligodeoxynucleotides and N-alpha-tosyl-l-phenylalanine chloromethyl ketone (TPCK). Lucigenin-enhanced chemiluminescence assay revealed that NF-kB inhibitors reduce the NADPH-dependent superoxide production. Regulation of NADPH oxidase by NF-kB may represent a possible mechanism whereby pro-inflammatory factors induce oxidative stress in atherosclerosis, hypertension, diabetes, stroke or heart failure.

Protamine inhibits tissue factor-initiated extrinsic coagulation.

The enhanced extrinsic coagulation in response to inflammation could contribute to disseminated intravascular coagulation, often manifesting cardiovascular complications. The complex mechanism remains unclear and effective management is not well established. The ability of protamine to offset bacterial endotoxin (LPS)-induced tissue factor (TF)-initiated extrinsic coagulation was demonstrated in human peripheral blood monocytes and cultured human leukaemia THP-1 monocytes, which was consistent with the inhibition of rabbit brain thromboplastin (rbTF) procoagulant activity in a cell-free in vitro model. Protamine significantly prolonged prothrombin time, further confirming the downregulation of the extrinsic pathway. However, thrombin time remained unaltered. Chromogenic assays were performed to dissect the extrinsic pathway, identifying inhibitory site(s). Protamine significantly inhibited factor VII (FVII) activation but not the dissected FX activation. The amidolytic activities of FVIIa and FXa were unaffected. The inhibited FVII activation in the presence of protamine was confirmed by the diminished FVIIa formation on Western blot analyses. Protamine preferentially inhibited TF-catalysed FVII activation, downregulating the extrinsic cascade. Protamine could be of anticoagulant significance in the management of the extrinsic hypercoagulation.

Deleterious effects of nifedipine on smooth muscle cells implies alterations of intracellular calcium signaling.

Nifedipine (NIF), a calcium channel blocker (CCB) from the first generation of dihydropyridines, induces detrimental effects on patients with cardiovascular diseases. We designed experiments to study, at cellular and molecular level, the mechanisms involved in the induction of deleterious effects by this drug. To this purpose, cultured human smooth muscle cells (HSMC) were used. The effect of NIF and two other CCB (FEL, AML) and inhibitors of intracellular signaling pathways (RR, TG, CAF and GEN) on intracellular calcium [Ca(2+)]I was determined by spectrofluorimetry using Fura 2 AM assay. The results showed that: (i) 10 microM NIF induced the increase of [Ca(2+)]I above the basal values (202.77 +/- 23.98 nM vs. 48.68 +/- 6.45 nM), an effect that was prevented by RR (50.45 +/- 13.9 nM) and was not induced by the two other CCB; (ii) NIF had a thapsigargin-like effect, because it induced the same release of intracellular calcium as TG (212.1 +/- 25.62 nM); (iii) The response to NIF was reduced by 40% after the inhibition of IP3 receptor (121.21 +/- 26.01 nM) and by 50% after the inhibition of tyrosine kinase (101.91 +/- 7.76 nM). Together, these data demonstrate that NIF produces a deregulation of intracellular calcium homeostasis. The abnormal increase of [Ca(2+)]I is due to the activation of store operated channels from the plasma membrane responsible for capacitative calcium entry, a process modulated by the activity of tyrosine kinase and the Ca(2+)-ATPase pump from the sarcoplasmic reticulum.

Clotrimazole inhibits smooth muscle cell proliferation and has a vasodilator effect on resistance arteries.

Clotrimazole (CLT) is a drug known to interfere with cellular calcium homeostasis, which in turn is reported to intervene in cell proliferation and in the reactivity of small blood vessels. Experiments were designed to test the influence of CLT on the proliferative and vasorelaxant effect of bradykinin (BK) and on calcium homeostasis in smooth muscle cells (SMC). To this purpose two model systems were employed: (i) cultured human smooth muscle cells (HSMC), and (ii) isolated resistance arteries maintained in an organ bath. The effect of various concentrations of CLT (2-15 microM) on BK-induced proliferation of HSMC was quantitated by spectrometry following [3H]-thymidine incorporation, and intracellular calcium [Ca+]i was determined by spectrofluorimetry using Fura 2-AM assay. In other experiments the roles of BK receptor (AB2) and of thapsigargin were assessed. The reactivity of the resistance arteries was measured by the myograph technique, and the effects of BK, CLT, and NO synthase blocker, L-NAME were evaluated. The results showed that 10 microM CLT: (i) inhibits the BK-induced proliferation of HSMC by 45-50%: (ii) prevents the rise of [Ca2+]i induced by BK (120.8 +/- 12.4 nM vs. 235.8 +/- 34.1 nM), an cffect similar to that of "classic" L-type calcium channels blockers: (iii) reduces the release of Ca2+ entry induced by thapsigargin suggesting a possible inhibition of the capacitative Ca2+ entry. Organ bath assays showed that CLT enhanced the BK-induced relaxation of the resistance arteries by an endothelium NO-independent pathway. Together, these data suggest that the mechanism of action of CLT on SMC implies mainly a modification of intracellular calcium homeostasis, with a minor contribution of BK B2 receptors. These new distinctive features of CLT effects suggest the potential use of this drug in the therapy of cardiovascular diseases associated with SMC increased proliferation and impeded relaxation in small arteries, such as atherosclerosis and restenosis.

Endothelial cell-derived foam cells fail to express adhesion molecules (ICAM-1 and VCAM-1) for monocytes.

The purpose of this study was to assess the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cell-derived foam cells. Hamster aortic endothelial cells (HAEC) in culture were exposed to hypercholesterolemic or normal homologous serum for 24 h. At the end of the incubation period, HAEC exposed to hypercholesterolemic serum exhibited numerous lipid droplets and had a general aspect of foam cells. When examined for the expression of ICAM-1 and VCAM-1 (by indirect immunofluorescence) normal HAEC expressed constitutively (to low level) on their surface these adhesion molecules; however HAEC-derived foam cells failed to display any labeling. To further assess these results, HAEC were first incubated with normal or hypercholesterolemic sera (as above) and then exposed to freshly isolated normal hamster blood monocytes. These experiments showed that monocytes adhered in small number to normal cells and failed to adhere to the surface of HAEC-derived foam cells. Together these data indicate that endothelial cell-derived foam cells: a) do not express ICAM-1 and VCAM-1 on their surface; b) have low or no adhesion properties for monocytes and c) may represent an appropriate experimental model to study the cellular alterations that take place in the advanced stages of atherosclerosis.

Establishment of a pure vascular endothelial cell line from human placenta.

UNLABELLED: Endothelial cells (EC) from various sectors of the circulatory system have distinct characteristics, some of which have only been identified in cultures upon their isolation from specific organs or tissues. Cultured vascular EC, derived from the human placenta (HPEC), may be helpful for studying their specific function in the fetoplacental unit, such as in the control of maternofetal traffic. In this paper we report an improved method for isolation, purification and culture of HPEC, that implies an enzymatic perfusion of the term placenta, followed by separation of resulting cells on a Percoll density gradient. The inoculated starting suspension was purified by a two-step selection procedure, based on differential trypsinization, leading to a pure population of about 8x10(7)cells/placenta, with 2.7-3.4 population doublings. The average population doubling time during eight passages was 60-65 h and the life span of HPEC was approximately 45-50 population doublings. The cell morphology at optical and electron microscopical level revealed a good differentiation of HPEC, which were endowed with numerous plasmalemmal vesicles (caveolae) and Weibel-Palade bodies. The transendothelial electrical resistance of the HPEC monolayer varied between 22 and 52 Ohm/cm(2). The cultures were mycoplasma free, as revealed by fluorescence microscopy using DNA dyes and the polymerase chain reaction (PCR). The negative immunofluorescent reaction for keratin confirmed that the HPEC were not contaminated with either type of placenta cells, as syncytiotrophoblast. Cultured HPEC demonstrated a strong reaction for von Willebrand factor antigen (by fluorescence microscopy), took up AcLDL-DiI and expressed active angiotensin converting enzyme. These characteristics substantiate the endothelial nature of cultured cells. The interactions with different lectins (BS-I, SBA, RCA, UEA and WGA) assessed by fluorescence microscopy and blotting reveal a strong reaction of HPEC with UEA and a negligible reaction with BS-I lectin. WGA lectin displayed a marked fluorescence staining in subconfluent HPEC, and at the level of intracellular clefts in post-confluent cultures. IN CONCLUSION: (i) we have obtained a pure line of cultured EC originating from the human placental venous side of the circulatory tree; (ii) the cells have the general characteristics and markers ascribed to EC; (iii) as opposed to large human placental vessels, HPEC do not react to BS-I lectin and, unlike human umbilical vein EC, have a much higher proliferation rate and a long lifespan; (iv) HPEC expressed a characteristic glycosylated coat particularly rich in alpha- L -fucose and beta-GlcNAc containing glycocompounds.

Exposure to hypercholesterolemic serum modifies the expression of cytoskeletal proteins in cultured endothelia.

Arterial endothelial layer dysfunction is considered to be one of the most important events which initiate the development of the atherosclerotic plaque and the cell cytoskeleton plays an essential role in maintaining the integrity of the endothelium exposed continuously to haemodynamic forces. The aim of this work was to study the modifications of the cytoskeletal proteins in the vascular endothelium exposed to atherogenic conditions. A hamster aortic endothelial cell line (HAEC) grown on glass coverslips was exposed for 24 h to hypercholesterolemic or normal homologous serum. Upon staining with Oil Red O and examination by phase contrast and fluorescence microscopy, HAEC incubated with hypercholesterolemic serum appeared heavily loaded with lipid droplets that showed a yellow autofluorescence in UV light and the general aspect of a foam cell. HAEC were incubated with: a) anti-actin serum; b) anti-vinculin monoclonal antibody (MoAb); c) anti-alpha actinin MoAb, and d) anti-talin MoAb, followed by appropriate secondary antibodies coupled with FITC or rhodamine. As compared to normal HAEC, the cells exposed to hypercholesterolemic serum showed a modified pattern for actin and vinculin localization. Actin appeared as a weakly stained network around the nuclear zone whereas vinculin was distributed as small granules throughout the cell cytoplasm. These experimental data suggest that in advanced atherosclerosis, some of the endothelial cytoskeletal proteins undergo modifications which could represent one of the important factors involved in further development of the atheromatous plaque. In addition they indicate that HAEC exposed to hypercholesterolemic serum could represent an in vitro working model for studying the events occurring in the endothelium at advanced stages of atherosclerosis.

The effect of ACE inhibitors on atheroma formation is potentiated by association with a calcium channel blocker. A biochemical and ultrastructural study.

The effect of two angiotensin converting enzyme (ACE) inhibitors, enalapril maleate and captopril, on the progression of atherosclerosis was investigated. Golden Syrian hamsters were divided into five groups: controls (C), fed a standard chow diet; hypercholesterolemic animals (HH) induced by supplementing the diet with 3% cholesterol and 15% butter; HH treated with enalapril (20 mg/kg/day); HH treated with captopril (60 mg/kg/day) and HH treated simultaneously with enalapril and a calcium channel blocker, diltiazem (45 mg/kg/day). The drugs were administered for one month, concomitantly with the atherogenic diet. As compared to controls, in HH group a significant increase in serum cholesterol (approximately 5 fold) and ACE activity (approximately 3 fold) was found. In HH-treated animals, both drugs maintained the serum ACE activity within the normal values. However, the effect upon serum cholesterol was different: enalapril and its combination with diltiazem had a significant hypocholesterolemic effect (128.8 +/- 25 mg/dl), whereas captopril had no effect on the cholesterol values (326.6 +/- 41.48 mg/dl). Electron microscopical examination of the coronary arteries and aortic valve in all experimental groups indicated a good correlation between the high levels of cholesterol, ACE activity and the development of the atherosclerotic lesions. Captopril treatment inhibits the early phases of atherosclerosis at level of the coronary artery but has no influence upon the lesion development in the aortic valve. By comparison, enalapril and enalapril-diltiazem co-administration impede the development of fatty streaks by decreasing the accumulation of lipids and calcium deposits in the lesion-prone areas examined. These data indicate that: 1) captopril does not have a hypocholesterolemic potential and cannot prevent atheroma formation in heart valves; 2) enalapril, especially combined with diltiazem, has a hypocholesterolemic effect and impedes the development of atheromatous plaque; 3) the anti-atherosclerosis therapy may benefit from the co-administration of an ACE-inhibitor with a calcium antagonist.

Differential effect of two calcium channel blockers--nifedipine and diltiazem--on atherogenesis in hypercholesterolemic hamster.

We compared the effect of two different calcium channel blockers (CCB), Nifedipine (1,4-dihydropyridine calcium antagonist) and Diltiazem (a benzothiazepine agent) on plasma components and the development of atherosclerotic plaque in experimental hypercholesterolemia. Golden male Syrian hamsters were divided into four groups: atherogenic animals (AT) induced by standard diet supplemented with 3% cholesterol and 15% butter; AT animals treated with Nifedipine (20 or 60 mg/kg/day); AT hamsters treated with Diltiazem (45 mg/kg/day) and controls (C), fed a standard chow diet. For one month, the drugs were administered concomitantly with the atherogenic diet. During the experiment, serum cholesterol, free calcium and angiotensin-converting enzyme (ACE) activity values were determined. Specimens from the lesion-prone areas: aortic valves, coronary arteries, and aortic arch, were collected and processed for light and electron microscopy. The results show that the atherogenic diet induces a significant increase of serum cholesterol (389 +/- 67.47 mg/dl), free calcium (13.44 +/- 0.84 mg/dl) and ACE activity (78.46 +/- 9.25 mU/ml) as compared to controls (cholesterol 73.76 +/- 3.31 mg/dl; calcium 8.9 +/- 0.5 mg/dl; ACE 33.68 +/- 2.6 mU/ml). Administration of Diltiazem reduced significantly these parameters (cholesterol, 196.25 +/- 22 mg/dl; calcium, 8.41 +/- 0.6 mg/dl) while Nifedipine had no effect (cholesterol, 283.03 +/- 44.7 mg/dl; calcium, 11.13 +/- 1.25 mg/dl) and increased the ACE activity (100.28 +/- 36.9 mU/ml). At the structural level, a significant correlation between the apparition and progression of the atherosclerotic lesions and the biochemical parameters detected, was observed. Diltiazem treated animals showed a reduction in the lesion severity, at the level of aortic valves, coronary arteries and aortic arch; we assume that Diltiazem acts on the early phases of atherosclerosis by blocking the lipid transport and accumulation into the subendothelial space. In contradistinction, Nifedipine treatment failed to suppress the atherogenic effect of fat-rich diet, and as in AT hamsters, the plaques developed in all lesion-prone areas. The latter were characterised by numerous lipid-laden cells (in aorta and aortic valves) and calcification and necrotic centres, in all locations, including coronary arteries. The results suggest a different mechanism of action and the ensuing effects of various CCB on atherogenesis.

Cellular structures involved in albumin interaction with preadipocytes in various stages of adipogenesis, in vitro.

Albumin is the major carrier of long chain free fatty acids, that are known to be heavily utilized by fat cells. To investigate the cellular structures involved in the interaction of albumin with preadipocytes, precursor fat cells isolated from rat epididymal fat pads were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and antibiotics. Cell differentiation was induced by 1.7 nM insulin added to the culture medium. The ensuing cells in various stages of differentiation were incubated with albumin, alone or bearing oleic acid, adsorbed to 5 nm gold particles (Alb-Au or Alb-OA-Au) for 10 min at 37 degrees C. As control, polyethyleneglycol-gold and IgG-gold complexes were used in same conditions. After extensive washing, the cell monolayer was fixed in 2.5% glutaraldehyde, and processed for electron microscopy. Examination of thin sections and morphometric analysis showed that: a) in the process of adipogenesis induced in vitro four cell types were simultaneously present: fibroblast-like cells (F), early adipocytes (EA), adipocytes (A) and aged adipocytes (AA); b) upon incubation with Alb-Au, the probe labelled preferentially coated pits and coated vesicles and only some uncoated vesicles; in time the tracer was found in endosomes, occasionally, multivesicular bodies and lysosome-like structures. The binding and uptake was dependent on the stage of cell differentiation, being less pronounced in fibroblast-like cells and increased in later stages (A and AA); c) Alb-OA-Au labelled the same structural features like the Alb-Au (i.e. coated pits and vesicles); however, the binding to the cell membrane was fourfold higher than that of Alb-Au in F and EA stages and was maintained at the same level in A and AA stages; d) control probes were taken up only by adipocytes and aged adipocytes, cells that express a strong phagocytic function for all tracers used. These results indicated that albumin bound to gold and especially albumin-bearing fatty acids-gold, bind preferentially to coated pits and vesicles of preadipocytes. Further internalization of albumin-gold particles seems to be non-specific, since both the probes and the controls adsorbed to gold were delivered to the same endosomal-lysosomal compartment.

Radiation damage to endothelial cells in vitro, as judged by the micronucleus assay.

The aim of our study was to optimize the cytokinesis-blocked (CB) micronucleus (MN) assay for the evaluation of radiation induced chromosomal damage in endothelial cells (EC) in vitro. After irradiation of confluent monolayers of rat and bovine aortic EC, with various doses of 250 kV X rays (0-3 Gy), the cells were trypsinized, resuspended in medium with cytochalasin B and then replated. After 3 days of growth they were again trypsinized and after fixing and staining of the cells, 1000 CB cells were scored for MN. The MN dose-response curves showed a rapid increase in the MN yield after low doses (< 0.25 Gy) of irradiation. This points to the high radiosensitivity of EC, with rat EC being more radiosensitive than bovine EC. A further slow increase (< 1 Gy) was observed after the initial fast increase. The occurrence of a fast and a slow component can be attributed to differences in radiosensitivity of EC in different stages of the cell cycle. For doses higher than 1 Gy, no further increase occurred due to severe damage at the spindle apparatus, as a result of which the division of many cells was inhibited.

Albumin binding sites are expressed on the abluminal plasma membrane of capillary endothelium.

The presence of albumin binding sites on the abluminal front of vascular endothelium was examined on the capillaries of the adipose tissue. The experimental procedure consisted in injecting interstitially within the rat epididymal fat and epicardial fat, albumin (alone or bearing oleic acid) either conjugated with gold particles (Alb-Au or Alb-OA-Au) or radioiodinated [( 125I]-Alb). In controls, polyethyleneglycol-gold complex (PEG-Au) and [125I]-IgG were used as tracers. The results revealed that: a) albumin binding sites are expressed on the abluminal front of endothelium especially concentrated on plasmalemmal vesicles; b) the retrotranscytosis of albumin conjugates from the interstitium to the capillary lumen is a poorly represented process; c) the binding of the tracers used appears to be time and concentration dependent; d) albumin conjugates do not bind significantly to plasmalemmal vesicles of adipocytes, pericytes and smooth muscle cells; e) PEG-Au and [125I]-IgG do not show a binding pattern similar to that of albumin conjugates.

[Studies of dysenteric infection in a closed children's community before and after anti-dysentery vaccination. II. Data on the response to antibiotics and chemotherapy]. / Cercetari privind infectia dizenterica într-o colectivitate închisa de copii, înainte si dupa vaccinarea antidizenterica. Nota II. Date privind comportamentul la antibiotice si chimioterapice.

Study of the behaviour to 7 antibiotics and chemotherapeutics of the Shigella and E. coli strains, isolated from the feces samples of children and adults in a closed community, revealed the high incidence of multiple resistance, especially to the currently used drugs tested. The existence of the phenomenon of plasmid transferable resistance in commensal or pathogenic E. coli strains is an important factor in the appearance of epidemic foci caused by Shigella and T. coli strains, multiply resistant to antibiotics.