Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin.

Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

Combined hyperlipidemia/hyperalphalipoproteinemia associated with premature spontaneous atherosclerosis in mice lacking hepatic lipase and low density lipoprotein receptor.

BACKGROUND AND METHODS: Hepatic lipase (HL) is an enzyme which hydrolyzes triglycerides from plasma lipoproteins and thus takes part in the metabolism of triglyceride-rich lipoprotein remnants and high density lipoproteins. The search described here concentrated on the description of the double invalidation of the HL and LDL receptor genes in mice in order to better understand the possible role of HL in combined hyperlipidemia/hyperalphalipoproteinemia and development of atherosclerosis. RESULTS: We show here that mice lacking both endogenous HL and LDL receptor (HL-/-:LDLR-/-) dramatically increased their plasma triglyceride-rich lipoproteins and their remnants as a consequence of reduced liver uptake. This result is strenghthened by the fact that HL-/-:LDLR-/- were found to overexpress LRP, LSR, and apoE genes. Interestingly, HL-/-:LDLR-/- mice showed premature spontaneous atherosclerosis and aortic lesions from 1-year-old animals were two-fold larger than those of LDLR-/- single mutants. We confirmed that HL-/- and wild-type mice did not develop atherosclerosis lesion even 1 year after birth. CONCLUSIONS: Analysis of this double HL-LDLR knockout mouse model provides in vivo evidence that HL has a major role in the clearance of TRL remnants when LDLR is deficient and in the reduction of the development of atherosclerosis.

Time course of osteopontin, osteocalcin, and osteonectin accumulation and calcification after acute vessel wall injury.

Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.

Kinetics of adventitial repair in the rat carotid model.

BACKGROUND: Discrepancies between success in experimental animals with a variety of pharmacologic strategies and failure with such agents in clinical trials have raised questions concerning the mechanism of restenosis. Recent observations suggest a potential implication for the adventitial (Adv) layer in neointimal formation. METHODS: The purpose of this study was to examine the Adv changes in the rat carotid artery subjected to balloon injury. These changes were characterized by morphometric, immunohistochemical, and electron microscopy analyses, with special attention devoted to early time-points post-injury. RESULTS: We report that the most important adventitial changes occurred in the first 48 h post-injury. Within 2 h there was extensive cell-loss by apoptosis and oncosis in the Adv and in the media; this was followed by the rapid onset of proliferation and a parallel slow increase in Adv thickening, reaching a maximum at 7 days. We further demonstrate an early migration of these Adv cells to the media and neointima. Moreover, we characterize the Adv cell phenotype with a panel of antibodies. Within 48 h after injury, a population of Adv cells expressed alpha-actin and vinculin with a maximum expression 7 days post-injury. At that time, these Adv cells started to express smooth muscle myosin heavy chain, a specific marker of smooth muscle cells. In parallel, we report an impaired production of elastic fibres in the Adv and medial layer. CONCLUSIONS: We reported a detailed time-course of adventitial changes after rat carotid injury (cell death, proliferation, migration and differentiation) that supports an important role of adventitia in neointima formation.

[The mechanisms of angiogenesis. Medical and therapeutic applications]. / Les mécanismes de l'angiogenèse. Applications médicales et thérapeutiques.

PURPOSE: Endothelial and smooth muscle cells interact with each other to form new blood vessels. In this review, the cellular and molecular mechanism underlying the formation of the primary vascular plexus (vasculogenesis), the sprouting of further blood vessels (angiogenesis) and their maturation via recruitment of smooth muscle cells (arteriogenesis) during physiological and pathological conditions are summarized. CURRENT KNOWLEDGE AND KEY POINT: The concept of angiogenesis is studied in tumoral and cardiovascular pathology. Promoting the formation of new collateral vessels in ischemic tissues using angiogenic growth factors (therapeutic angiogenesis) is a promising approach in cardiovascular diseases. Conversely, inhibition of the action of key regulators of angiogenesis is a new pathway for the treatment of solid tumors and metastasis. FUTURE PROSPECTS AND PROJECTS: These concepts are being tested now in clinical trials in the oncology or cardiovascular fields. Some trials are reported in this review with their potential adverse effects, limits and developments in the future.

Freeze-drying allows double nonradioactive ISH and antigenic labeling.

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.

Elastin variations implicating in vascular smooth muscle cells phenotype in human tortuous arteries.

The aim of the present work was to study the morphological implications between the elastin and the phenotypic expression of the vascular smooth muscle cells. For this purpose, sixty human tortuous arteries from different territories have been studied. We have measured the morphometric indexes Intimal Thickening Index and Elastolyse Index and they have been quantified with computer system analysis, image-colour corresponding to the orcein and Verhoëff reactions for detecting elastin and the alpha-actin in the smooth muscle cells. We compared both territorial arteries from the cranial and from abdominal origin. The elastin concentration was similar in both territories, but not its morphology according to its spatial distribution. We have observed a relationship between the elastin structural organisation from the media of arteries and of the internal elastic lamina in these territories and the variation of reactivity to the smooth muscle alpha-actin as a marker of the phenotypic state. Our results confirm the hypothesis that elastin, besides intervening in the architecture of the arterial wall, is a factor implicated in the phenotypic variability of the smooth muscle cells and in the development and evolution of the intimal thickenings in human atherosclerosis.

Immunohistological and ultrastructural analysis of the intimal thickening in coarctation of human aorta.

OBJECTIVES: The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. METHODS: In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. RESULTS: Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson's trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: alpha-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only alpha-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. CONCLUSIONS: These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.

Vitronectin expression and interaction with receptors in smooth muscle cells from human atheromatous plaque.

Vitronectin (VN) is a plasma glycoprotein that promotes cell attachment and induces migration of human smooth muscle cells (SMCs) in culture. VN has been observed to accumulate in human atherosclerotic plaques, although its origin and role in atherosclerosis are not yet established. In the present experiments, synthesis of VN by intimal cells and its colocalization with receptors, alphavbeta3 and alphavbeta5, were studied by in situ hybridization and immunohistochemistry on 15 human atherosclerotic plaques from carotid arteries obtained after surgery. Strong VN protein and mRNA expression was observed in the intima and in the media. In the intima, VN mRNA expression was colocalized with SMCs, indicating that these cells produce VN, which may account for its accumulation in atherosclerotic plaques. In SMCs in culture, immunoprecipitation after metabolic labeling demonstrated that human SMCs do synthesize vitronectin. Confocal microscopic examination showed that VN colocalized with its receptors, alphavbeta3 and alphavbeta5, in the atherosclerotic intima. However, the distribution of the VN receptors on SMCs in culture in contact with VN was different. These observations suggest that VN plays various parts in atherogenesis via different SMC membrane receptors.

Glucocorticoids stimulate cholesteryl ester formation in human smooth muscle cells.

The aim of the present study was to investigate the effect of synthetic glucocorticoid dexamethasone (Dex) on cholesterol esterification in cultured human smooth muscle cells (SMC). In labeled SMC, Dex stimulated the esterification of [3H]cholesterol in a dose-dependent manner. This effect was specific for glucocorticoid hormones and could be inhibited by cycloheximide (3 ng/mL), actinomycin D (10(-5) mol/L), and the specific glucocorticoid antagonist RU 486 (10(-8) mol/L). When plasma membrane was selectively labeled with trace quantities of [3H]cholesterol (0.25 microCi/mL, 1 hour, 10 degrees C), Dex (10(-8) mol/L) caused a net flux of free [3H]cholesterol into the cells. Moreover, Dex (10(-8) mol/L, 24 hours) stimulated the esterification of sterols, newly synthesized from [14C]mevalonate (10 microCi/mL, 4 hours) and lowered the amount of [14C]sterols susceptible for cholesterol oxidase. The incorporation of [14C]oleic acid into cholesteryl esters was markedly higher in Dex-pretreated SMC than in the control cells (2.1 +/- 0.07 and 1.4 +/- 0.1 pmol/h/microgram protein, respectively, P < .01). At the time, cholesteryl ester hydrolysis in Dex-treated cells was reduced (72 +/- 8 pmol cholesteryl esters/h per milligram versus 130 +/- 10 in the control cells). HDL3-mediated [3H]cholesterol efflux was also inhibited in Dex-treated cells; moreover, HDL3 (40 micrograms/mL, 24 hours) had practically no effect on [3H]cholesteryl ester content in Dex-treated SMC but caused a 50% reduction of [3H]cholesteryl esters in the control cells. Thus, in human SMC glucocorticoids alter the redistribution of cholesterol between the pools of free and esterified cholesterol, paralleled by the change in acyl coenzyme A: cholesteryl acyltransferase and neutral cholesteryl ester hydrolase activities, leading to the impaired HDL3-mediated cholesterol efflux.

Reduced basal NO-mediated dilation and decreased endothelial NO-synthase expression in coronary vessels of spontaneously hypertensive rats.

Basal vasomotor tone in coronary vessels is, in part, maintained by nitric oxide (NO) production by endothelial constitutive NO synthase (ecNOS). Alteration of coronary circulation observed in left ventricular hypertrophy secondary to hypertension could be associated with a decrease in NO production. The aim of this study was to measure: (1) coronary flow in the Langendorff-perfused heart model at baseline, after maximum vasodilation in response to adenosine (10(-5) M), after endothelium-dependent vasodilation in response to bradykinin (10(-8) M) and after ecNOS inhibition by nitro-L-arginine methyl ester (L-NAME) (10(-4) M); (2) medial thickening of coronary microvessels and perivascular collagen on histological heart sections; and (3) ecNOS expression by immunohistochemical staining in these vessels using 20-week-old spontaneously hypertensive (SHR) and Wistar-Kyoto control rats (WKY). These measurements were determined by computer-directed color analysis. When SHR were compared with WKY rats, we found: (1) a decrease in basal flow (10.1+/-0.6 v 15.3+/-1.2 ml/min/g, n=10, P<0.0001), in maximum flow (15.4+/-0.7 v 24.3+/-1.3 ml/min/g, n=10, P<0.001), in bradykinin-induced flow increment (1.5+/-0.3 v 2.6+/-0.3 ml/min/g, n=5, P<0.05) and in L-NAME-sensitive flow (3.3+/-0.6 v 6.3+/-0.9 ml/min/g, n=7, P<0.05); (2) an increase in medial thickness (9.4+/-0.6 v 5.4+/-0.3 microm, n=8, P<0.001) and in perivascular collagen area (1509+/-311 v 462+/-120 microm2, n=8, P<0.01) of coronary arterioles; and (3) a decrease in ecNOS expression in the endothelium (ecNOS-stained cross-sectional area in arterioles: 40.0+/-9.1 v 84.6+/-9.0 microm2, n=7, P

Overexpression of P2Y2 purinoceptor in intimal lesions of the rat aorta.

Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y2 (P2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.

A human monoclonal antibody obtained from EBV-transformed B cells with specificity for myosin.

We describe the preparation of a stable human lymphoblastoid cell line obtained during ex vivo studies in which peripheral blood lymphocytes of a Glanzmann's thrombasthenia patient were transformed with Epstein-Barr virus. Somatic hybrids secreted an IgM monoclonal antibody (B7) that reacted with the myosin heavy chain of human platelets by immunoblotting. Flow cytometry showed that B7 barely recognized unstimulated intact platelets, but bound abundantly after permeabilization of fixed cells with Triton X-100. The reactivity of the antibody on thin sections of human myocardium and aorta was studied by immunohistochemistry. B7 specifically stained myosin of myocytes, but there was no labelling of aortic smooth muscle cells. The epitope was conserved in cardiac or skeletal myosin prepared from pig or rabbit. Measurement of the dissociation constant in a competitive ELISA showed that B7 bound with high affinity (10(-8) M). Purified Fab fragments retained their ability to bind to myosin, suggesting that B7 may be useful in the imaging of myocardial necrosis after myocardial infarction, myocarditis, cardiac drug toxicosis or graft rejection. This work also shows that EBV transformation of B cells may uncover naturally occurring autoantibodies which under normal circumstances are inhibited by the immune surveillance system.

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.

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.

Synthesis of monohydroxylated fatty acids from linoleic acid by rat aortic smooth muscle cells and tissues: influence on prostacyclin production.

We have investigated whether cellular metabolism of linoleic acid (18:2) can influence prostacyclin (PGI2) production by cultured rat aortic smooth muscle cells (SMC) and tissues. Incubation of rat SMC homogenates with [1-14C]18:2 results in the enzymatic synthesis of [14C]13-HODE (hydroxyoctadecadienoic acid) and to a lesser extent [14C]9-HODE as defined by gas-liquid chromatography-mass spectrometry (GLC-MS). The observed changes, in percent enzymatically synthesized 13-HODE in the presence of indomethacin, aspirin, metyrapone, 15-HPETE (hydroperoxyeicosatetraenoic acid), and NDGA, suggest that it is formed from the PGH (prostaglandin endoperoxide) synthase pathway. Incubation of intact adherent SMC with [14C]linoleic acid demonstrates that the monohydroxylated compounds are predominantly esterified within the membrane phospholipids and not released into the incubation medium. The simultaneous incubation or a short-term preincubation of 18:2 and arachidonic acid (20:4) do not modify the enzymatic profile of 20:4 transformation. By contrast, long-term preincubation of cells with 18:2 or 13-HODE stimulates the transformation of exogenously added [14C]20:4 to [14C]6-keto PGF1 alpha. However, exogenous 13-HODE does not enhance [14C]6-keto PGF1 alpha recovery from [14C]20:4 prelabeled SMCs. Our results demonstrate that 18:2 is a substrate for PGH-synthase in rat aortic SMC and tissues. The 13-HODE formed is essentially esterified in cell phospholipids and remains without any significant effects on the release of [14C]6-keto PGF1 alpha from [14C]20:4 prelabeled SMC.

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.

Dual metabolic pathways of 12-HETE in rat aortic smooth muscle cells.

12(S)-HETE, a major lipoxygenase-derived compound from arachidonic acid is incorporated and metabolized by vascular smooth muscle cells via beta-oxidation. We have now identified for the first time in this cell type 12(S)-HETE metabolites formed by a combination of reductase and oxidation pathways. HPLC and GC-MS analysis of time-course experiments allow us to characterize two different metabolic pathways: a direct peroxisomal beta-oxidation of 12(S)-HETE leading to the formation of 16:3 (8-OH) which accumulates first and a reduction of one of the conjugated double bonds of 12(S)-HETE giving the dihydro-intermediate 20:3(12-OH) that transiently accumulates before being converted itself by peroxisomal beta-oxidation to 16:2(8-OH). Taken together these results may suggest that the transient accumulation of 20:3(12-OH) through transcellular metabolism of 12(S)-HETE may represent a part of the modulatory effect of 12(S)-HETE on vascular function.

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.

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.