Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta.

Prostaglandin synthesis in aortic smooth muscle cells originating from healthy an atherosclerotic rabbits was studied by incubating [14C]arachidonic acid with intact confluent cells and cell homogenates. In spite of a reduced 6-keto prostaglandin F1 alpha formation, no potentiating effect on the prostaglandin E2 generation occurred. Indeed, both cyclooxygenase and prostaglandin I2 synthetase activities appear to be reduced. These results suggest that an impaired arachidonic acid utilisation in aortic smooth muscle cells may be involved in the course of the atherosclerotic process.

Effect of VLDL on the inhibition of arachidonic acid transformation by dexamethasone in cultured smooth muscle cells.

Very-low-density lipoproteins (VLDL) induce a dose-dependent reduction (up to 55%) in the number of specific binding sites and about a 2-fold increase in binding affinity for [3H]dexamethasone in human and rat smooth muscle cells (SMC). Maximal effect of VLDL was achieved within 3-5 h at a lipoprotein concentration 60 micrograms protein/ml. Lipoprotein-mediated reduction in the number of [3H]dexamethasone binding sites resulted in partial loss of cellular sensitivity to hormone action: dexamethasone (1 x 10(-6) M) inhibited the transformation of [14C]arachidonic acid (AA) into metabolites to a lesser extent in SMC preincubated with VLDL (11.5%) than in untreated cells (29.0%). In particular, under these conditions the inhibitory effect of dexamethasone on prostaglandin I2 (PGI2) formation in VLDL-treated SMC was lower than in untreated cells (42.1% vs. 60%). We propose that VLDL is able to counteract the inhibitory effect of glucocorticoids on AA release and PGI2 formation in vascular SMC by reduction of cellular specific glucocorticoid binding sites.

Effects of hypercholesterolaemic serum on aortic explants from normal rats.

Aortic explants obtained from normal adult rats and composed of all 3 tunics were cultured in a semi-synthetic gelosed medium supplemented by 10% serum. Explants cultured with normocholesterolaemic serum kept their in vivo characteristics for more than 12 days at electron microscope and histometabolic levels. However, explants showed considerable differences when cultured in a medium with hypercholesterolaemic serum. Cell proliferation in the tunica media and enhanced synthesis of macromolecular components of the extracellular matrix were observed. These laterations occurring inside the explants, which had kept all their tissular relationships, suggest that this culture system may be valuable for further studies on atherosclerotic processes.

Phenotypic expression of surface antigens of rabbit aortic smooth muscle cells in culture. Monoclonal antibody, 2P1A2, characteristic of smooth muscle cells present in atherosclerotic plaque, is not correlated with cell proliferation.

The expression of smooth muscle cell (SMC) antigens was studied in culture by immunofluorescence and immunoelectron microscopy. As specific SMC markers, we used 2 monoclonal antibodies (MAb), 1PC1 and 2P1A2 which are able to detect atherosclerotic plaques in the rabbit. MAb 1PC1 recognizes an antigen expressed on the cell surface, starting on the 7th day in primary culture after serum activation, and then secreted. On a confluent SMC monolayers this antigen appears outside the cell as an important filamentous network. The kinetics of secretion of this external protein recognized by 1PC1 corresponds to the kinetics of the secretory phenotype described by Chamley-Campbell and Campbell (Atherosclerosis, 40 (1981) 347). 2P1A2 MAb is specific for SMCs exclusively present in the rabbit atherosclerotic plaque. We studied the degree of reactivity of 2P1A2 with SMCs during primary cell culture. This "atherosclerotic" antigen of SMCs recognized by 2P1A2 is expressed in culture conditions by SMCs from rabbit normal media. This antigen appears after 3 days of serum activation, and heparin growth inhibition does not interfere with its expression. 2P1A2 recognized antigen is expressed during all cell cycle phases without amplification. 3 days after fetal calf serum (FCS) stimulation of cells which are in G0/G1, 89% are labelled by 2P1A2, 4 days later G0/G1 positive cells constitute 49%. We conclude that 2P1A2 immunolabelling on the SMC surface reflects an activated state which is not correlated with SMC proliferation.

Fibrous long spacing collagen in aortic explants of normal rabbit cultured in hypercholesterolemic serum.

Aortic tissues consisting of all three tunics were removed from normal adult rabbits and cultured in a semisynthetic gelosed medium supplemented by 10% serum obtained either from normal or hypercholesterolemic rabbits. Fibrillar cross-striated aggregates appeared with a high frequency (50%) in the extracellular space of explants cultured from four to eight days in medium supplemented by serum from hypercholesterolemic rabbits, but did not appear in explants cultured in serum from control animals (3%). The electron-dense segment was ruthenium red positive and digested by testicular hyaluronidase. The electron-lucent segment, composed of ruthenium red negative thin filaments, was not modified after hyaluronidase treatment but was strongly digested after collagenase treatment. It is believed that this material was fibrous long spacing collagen synthetized under culture conditions, as shown after tritiated proline incorporation.

Detection of atherosclerotic plaque with two monoclonal antibodies. 2P1A2 monoclonal antibody is specific for smooth muscle cells in atherosclerotic plaque.

We investigated two monoclonal antibodies (MAbs) which recognize rabbit atherosclerotic tissues. In particular, one antibody, 2P1A2, is specific for rabbit aortic smooth muscle cells (RASMCs). We used RASMCs in primary culture to produce and screen MAbs directed towards the cell surface. The specificity of the described antibodies was tested on a battery of tissue cryosections of different origin (rabbit, rat and human) by immunological staining. 2P1A2 shows an exclusive immunolabeling for SMCs present inside rabbit atherosclerotic plaque. This MAb shows inside the fibrous plaque a staining similar to two other SMC-specific antibodies (anti-desmin and anti-alpha-actin). In an early stage of atherosclerosis, close to the internal elastic lamina, underlying a fibrous plaque, 2P1A2 detects some SMCs; in contrast, anti-desmin and anti-alpha-actin fail to stain such SMCs. This antibody may be therefore considered as directed specifically against SMCs in an activated state. The other antibody which we describe, 1PC1, stains a pericellular antigen expressed by cultured SMCs and shows a specificity for smooth muscle tissues. 1PC1 MAb strongly stains the fibrous plaque of atherosclerotic rabbit aorta and the recognized epitope is present inside the aortic media. These two antibodies may be useful in the recognition of vascular SMCs during the atherosclerotic process.

Prostacyclin synthesis by proliferative aortic smooth muscle cells. A kinetic in vivo and in vitro study.

The capacity of arterial SMCs to produce PGI2 when stimulated by exogenous AA was studied in proliferative and confluent cultured cells and at different periods following endothelial denudation in vivo. PGI2 production per cell was doubled during the exponential growth-phase in culture. By contrast, increased PGI2 formation did not correlate with mitotic activity in intimal regeneration tissue but with the presence of SMCs in a synthetic phenotype. The present results suggest a potential role for PGI2 in SMC differentiation and proliferation.

15-Lipoxygenase expression in smooth muscle cells from atherosclerotic rabbit aortas.

To determine the extent and origin of the stimulation of 15-lipoxygenase activity in atherosclerotic aortas, formation of hydroxy-derivatives from arachidonic acid was measured by HPLC-analysis and 15-lipoxygenase mRNA expression was investigated by RNA blot and in situ hybridization in atherosclerotic and normal rabbit aortic tissues. The synthesis of hydroxy-eicosatetraenoic acids (HETE) from exogenously added [14C]arachidonic acid was unchanged in atherosclerotic aortas in comparison with healthy aortas, but pretreatment with indomethacin demonstrated that 15-HETE production resulted essentially (75%) from cyclooxygenase activity in healthy aorta and from lipoxygenase activity in atherosclerotic aorta. The RNA blot and in situ hybridization with radiolabelled oligonucleotide probe demonstrated that 15-lipoxygenase mRNA was strictly localized in intimal thickening of atherosclerotic aortas. The immunostaining using anti-alpha smooth muscle actin, revealed that smooth muscle cell rich areas of the intimal thickening expressed 15-lipoxygenase mRNA. In addition, RNA blot hybridization indicated that cultured smooth muscle cells from atherosclerotic aortas expressed strongly 15-lipoxygenase mRNA. These results demonstrate that augmentation of 15-lipoxygenase activity in atherosclerotic aortas is correlated with 15-lipoxygenase mRNA expression in atherosclerotic plaque, and that intimal smooth muscle cells were involved, in addition to macrophages, in the expression of 15-lipoxygenase.

Megakaryopoiesis disturbances in atherosclerotic rabbits.

Rabbits given a hypercholesterolemic diet (500 mg/day) for 6 months and then maintained for another 6 months on a normal diet were found to have developed fibrous lipidic lesions in the aorta. Although circulating platelet levels in these animals were normal there was a reduction in mean megakaryocyte ploidy. The high concentrations of megakaryoblasts in all the sedimentation fractions collected by the 'STAPUT' system suggested an increase in megakaryocyte turnover with activation of committed stem cells. In addition, other defects in maturation of megakaryocytes were observed, such as abnormalities in the demarcation membrane system and granule number. These data reveal that defects in megakaryocyte maturation and turnover may occur during the process of reparative fibrosis of the arterial tree following a period of moderate hypercholesterolemic diet in the rabbit.

Hemorheological changes in elderly subjects--effect of pentosan polysulfate and possible role of leucocyte arachidonic acid metabolism.

The effects of pentosan polysulfate (PPS) on various hemorheological parameters were studied in a group of very elderly subjects in good general health. Alterations in blood viscosity and filterability were detected in these patients, without any concomitant changes in factors which are known to affect these parameters: notably hematocrit, fibrinogen and plasma lipid levels. The hemorheological abnormalities were considerably improved by twice daily treatment with 50 mg of PPS (i.m.). Apart from its anticoagulant activity, PPS has been shown to have an anti-inflammatory action. We were interested to investigate its effects on metabolism of exogenous arachidonic acid (AA) by both platelets and leucocytes. It is becoming increasingly recognized that metabolites of AA via the 5 LO pathway appear to play a role in inflammatory processes. In this study, PPS was found to inhibit leucocyte 5 LO activity. Reduction in the levels of these metabolites may therefore have an effect on whole blood rheology.

Activation of prostacyclin synthesis in cultured aortic smooth muscle cells by 'diuretic-antihypertensive' drugs.

In cultured smooth muscle cells of rat aorta, four diuretic agents, furosemide, bumetanide, cicletanide and piretanide (all at 10(-6)-10(-5) M), significantly enhanced the transformation of exogenously added arachidonic acid (AA) to prostacyclin. Studies with cultured smooth muscle cells and human leukocytes revealed that these same agents failed to inhibit lipoxygenase pathways. Taken together, these results indicate that the diuretic properties of these agents might be associated with a general activation of the AA cascade.

Acute erythroblastic leukemia presenting as acute undifferentiated leukemia: a report of two cases with ultrastructural features.

This report describes two elderly patients with acute leukemia in which blast cells were undifferentiated with conventional light microscopy (L.M.) and cytochemistry. Blast cells were identified as belonging to the erythroblastic line by their ultrastructural features: glycogen deposits, lipidic vacuoles, cytoplasmic ferritin molecules and rhopheocytotic invagination. Moreover, blast cells were surrounding a central macrophage. Thus, these two patients had acute erythroblastic leukemia which differs from erythroleukemia (M6 of FAB classification) in which blast cells present myeloblastic characteristics.

Effects of monohydroxylated fatty acids on arterial smooth muscle cell properties.

The hydroxylated derivatives of polyunsaturated fatty acids may be potent modulators of basic biological responses involved in pathological processes, including atherosclerosis. The object of the present investigation was to study the effects of monohydroxylated fatty acids (namely 12-HETE) on the properties of aortic smooth muscle cells (SMC) in culture. The changes in cell expression of differentiation antigen alpha-SM actin and 2P1A2 was followed by computerized morphometry, using specific monoclonal antibodies and the activation of cells by measuring cell motility. In addition, intracellular [Ca2+]i mobilization and IP3 formation were studied. Finally, the metabolic routes of monohydroxylated compounds and their effects on PGI2 secretion were reported. The results demonstrate that 12-HETE is able to stimulate the phenotypic modulation. PGI2 production and motility of arterial SMCs, despite any detectable activity in increasing [Ca2+]i or IP3 formation. By contrast with parent compounds 15-HETE and 13-HODE, which appear as potent prodifferentiating molecules, 12-HETE is specifically metabolized via a 10-11 reductase pathway in addition to the classical beta-oxidation pathway. Taken together, our results suggest that cellular metabolism of 12-HETE, produced by platelets in the vicinity of the arterial intima, and also by cells present inside the atherosclerotic intima, or associated with modified LDL may play a key role in the atherosclerotic process.

Modulation of the transcellular metabolism of 12(S)HETE by 10-11 reductase activity in cultured rat aortic smooth muscle cells.

Cultured rat aortic smooth muscle cells (SMC) metabolize 12(S)hydroxyeicosatetraenoic acid (12(S)HETE) by two different pathways; beta-oxidation leading to 16:3(8-OH), and 10-11 reductase activity producing 20:3(12-OH) which is beta-oxidized to 16:2(8-OH). In this work, we demonstrate that 10-11 reductase activity is modulated in cultured rat aortic SMC as a function of cell state (proliferating vs quiescent) and stimulated by serum. Most of the 20:3(12-OH) is recovered in the incubation medium but a significant part is esterified into phospholipids. By comparison with its parent compound, 12(S)HETE, 20:3 (12-OH) is mainly incorporated into phosphatidyl-choline and phosphatidyl-ethanolamine, suggesting that it may affect cellular functions. Taken together, these findings may be relevant to the effects of 12(S)HETE on vascular SMC functions related to atherosclerotic development.

Action of some salicylate derivatives on in vitro platelet aggregation. Inhibitory and inhibition antagonistic effects.

Twenty salicylate derivatives were tested for their antagonistic activity on the inhibitory effect of aspirin on platelet aggregation. The blocking effect was not limited to the salicylate but also characterised some of its substituted compounds. The substituant influence did not seem to be related to electronic or size parameters. This antagonistic activity of these derivatives decreased as concentrations increased, owing to the emergence of their own inhibitory activity: several salicylate derivatives showed dual inhibitory and inhibition antagonistic activity, with both properties present at the same concentration. A mechanism involving dissociated activities on the two enzymatic sites of cyclooxygenase is proposed.

Estimation of seric and parietal inhibiting power of elastase; its changes with experimental conditions.

The existence of a parietal elastase and its physiological role in the pathology of the arterial wall remain unknown: it is probably difficult to demonstrate its specific activity because of existing tissular inhibitors, homologus to seric inhibitors. The change of seric inhibitor power when elastase is injected to animals with or without a cholesterol diet is discussed. Moreover, an aortic inhibiting power against elastase is shown. Our results emphasize some new aspects of elastase in its role in arterial wall pathology.

Involvement of endothelium-derived NO in the basal tone and in the vasodilator responses to muscarinic agonists in the rat isolated mesenteric arterial bed.

To investigate the involvement of nitric oxide (NO) derived from endothelial cells in the control of vascular tone in the rat mesenteric vascular bed, the effects of different procedures known to interfere with the NO-cyclic GMP pathway were evaluated both on the basal tone and on the vasodilatory responses to four muscarinic agonists. To this aim, rat isolated mesenteric vascular beds were perfused at constant pressure. Water infusion significantly increased the resting perfusion pressure whereas L-NOARG, L-NAME and methylene blue were devoid of effect. In noradrenaline-preconstricted vascular bed, the perfusion pressure was significantly increased after water or L-NAME infusion. The vasodilator response induced by subsequent addition of acetylcholine in bolus was not significantly modified by pre-treatment with indomethacin but was significantly reduced by water infusion. Responses to acetylcholine and to three other muscarinic agonists--carbachol, oxotremorine or McNeil A 343--were assessed. Incubation with L-NAME did not modify the initial peak falls of the agonists except for McNeil A 343, whereas it significantly reduced the area under the pressure trace for all the substances. The latter effect was reversed after a subsequent incubation with L-Arginine. Finally, L-NAME strongly and significantly increased the drop in perfusion pressure and the area under the pressure trace following bolus of glyceryl trinitrate. These results suggest that in the mesenteric arterial bed of the rat, which can be considered as a resistant arteries preparation, basal tone appears to be controlled by a factor other than NO. Moreover, the vasodilator responses of muscarinic agonists are affected by L-NAME in their second late sustained phase only, which probably relies on a de novo synthesis of endothelium derived-NO. Finally, endothelium derived-NO exerts inhibitory effects both on the sensitivity of the vascular smooth muscle to glyceryl trinitrate and on the magnitude of its contraction in the presence of noradrenaline, two types of effects which are sensitive to L-NAME.

Association with the Syndrome "Basses Richesses" of Sugar Beet of a Phytoplasma and a Bacterium-Like Organism Transmitted by a Pentastiridius sp.

ABSTRACT The syndrome "basses richesses" of sugar beet (SBR) was first observed in 1991 in Burgundy, France. A cixiid planthopper, Pentastiridius beieri, has been proved to be involved in the transmission to sugar beet of a stolbur phytoplasma, which could be detected in some affected plants. In 2000, periwinkle and sugar beet exposed to field-collected cixiids developed symptoms similar to SBR on sugar beet. Use of 4'-6-diamidino-2-phenylindole (DAPI) staining and transmission electron microscopy confirmed the presence of phytoplasma in some of the plants, which were also positive for this pathogen in a polymerase chain reaction (PCR) analysis. A phloem-restricted gram-negative bacteria was seen in all other plants with symptoms but PCR-negative for phytoplasma. Three primer pairs reported as diagnostic for phloem-limited bacteria were tested but only primers specific for 'Candidatus Phlomobacter fragariae' gave a positive signal, which related to the presence of DAPI-stained bacteria-like objects in diseased plants. Although phytoplasma and bacterium-like organisms were associated with the same macroscopic symptoms on sugar beet, histochemical analysis of phloem cells showed that phytoplasma were associated with cell necrosis and cell wall lignification, while bacteria were associated with these same abnormalities as well as deposit of phenolic compounds in the lumen of phloem cells.

Cytological Characterization of Elicitin-Induced Protection in Tobacco Plants Infected by Phytophthora parasitica or Phytoplasma.

ABSTRACT Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultrastructural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloem-restricted microorganisms.

Cicletanine and eicosanoids in cultured vascular smooth muscle cells.

Cicletanine, a new antihypertensive, slightly diuretic, drug was tested for its effects on arachidonic acid (AA) metabolism in cultured smooth muscle cells from rat aorta. Cicletanine significantly enhanced the production of prostacyclin from both exogenously added arachidonic acid and endogenous sources. This effect is not mediated through inhibition of the lipoxygenase pathways and occurs despite a slight but definite inhibition of the activity of acyl hydrolase. Taken together, these results indicate that the antihypertensive properties of cicletanine might be associated, at least in part, with activation of the AA cascade through the cyclooxygenase pathway.