J. Larrue

Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells

In addition to the well established cyclooxygenase pathway, cultured aortic smooth muscle cells convert arachidonic acid to several polar metabolites identified by high performance liquid chromatography and gaz chromatography-mass spectrometry. 15-Hydroxyeicosatetraenoic acid, 12-Hydroxyeicosatetraenoic acid and 5-Hydroxyeicosatetraenoic acid are the major products formed. These observations indicate that the rabbit aortic smooth muscle cells are a potential source of lipoxygenase products and raise the possibility that this pathway of arachidonic acid metabolism can influence the biological functions of arterial myocytes under normal and pathological conditions.

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.

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.

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.

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.

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.

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.

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.

Endogenous arachidonic acid metabolism by cultured arterial smooth muscle cells.

Abstract Cultured aortic smooth muscle cells originated from healthy and atherosclerotic rabbits produce prostaglandins (namely prostacyclin) at a basal state. Prostaglandin secretion is dramatically reduced in atherosclerotic cells. This impairment was not correlated with any alteration of acyl hydrolase activities and probably involved a decrease of cyclooxygenase activities.

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.