G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras.

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 2. Role of protein kinase C-delta in the regulation of eicosanoid production.

PGI2 generation by the vessel wall is an agonist for cyclic-AMP-dependent cholesteryl ester hydrolysis. The process of enhanced PGI2 synthesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-protein-mediated PGI2 synthesis. In the studies reported herein, cells generated PGI2 in response to AlF4-, GTPgammaS, and ATP in a dose-dependent manner. G-protein agonists stimulated eicosanoid production principally by activating phospholipase A2, but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A2 and PLCgamma activities. Galphai subunits mediate G-protein agonist-induced PGI2 synthesis, since ATP- and PDGF-induced PGI2 synthesis was inhibited by pertussis toxin. Although cholesterol enrichment reduced arachidonic acid- and PDGF-induced PGI2 synthesis, cholesterol enrichment enhanced PGI2 release in response to AlF4-, GTPgammaS, and ATP. The enhancement of PGI2 release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits. Since cholesterol enrichment inhibited PDGF and AlF4--induced MAP kinase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J. D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36, 9523-9531] (the major mechanism by which phospholipase A2 is activated), these results suggest that cholesterol enrichment induces other alternative signaling pathways leading to phospholipase A2 activation. A PKC-dependent pathway is described herein that is involved in enhanced eicosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI2 production is inhibited by calphostin, and (2) cholesterol enrichment augments the specific translocation of the delta-isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol ester. These data support the concept that, in cells possessing normal levels of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid synthesis in response to G-protein activation; however, under conditions of high cellular cholesterol levels, augmented G-protein-linked eicosanoid production results from enhanced PKCdelta activity.

Cholesterol enrichment enhances expression of sterol-carrier protein-2: implications for its function in intracellular cholesterol trafficking.

Cholesterol enrichment of vascular smooth muscle cells, as occurs under conditions of hypercholesterolemia and atherosclerosis, is accompanied by specific changes in cholesterol metabolism and in intracellular cholesterol trafficking. Sterol-carrier protein-2 (SCP2), an intracellular lipid binding protein, enhances the activation of enzymes involved in cholesterol metabolism. It may also enhance cholesterol efflux by regulating the size of the "fast" cholesterol pool available for efflux to high density lipoproteins. However, a definitive role for SCP2 in arterial cholesterol metabolism is unclear. Therefore, we examined the expression of SCP2 (13.1 kD), SCPx (58 kD), and p30 (30.8 kD) in cultured arterial smooth muscle cells under conditions of cholesterol enrichment. We found that SCP2, SCPx, and p30 are localized principally in the cytosolic fraction, with lesser amounts associated with the nuclear/peroxisomal fraction; the expression of SCP2 protein and mRNA, but not SCPx, is increased after exposure of smooth muscle cells to cationized LDL. In contrast to the increased expression of SCP2, the expression of p30 decreases after cholesterol enrichment of smooth muscle cells. Coupled with previous studies demonstrating enhanced cholesterol efflux from cholesterol-enriched smooth muscle cells in response to high density lipoproteins, our results suggest that increased expression of SCP2 may partly mediate the cholesterol trafficking process.

Altered cholesterol trafficking in herpesvirus-infected arterial cells. Evidence for viral protein kinase-mediated cholesterol accumulation.

Herpesvirus infection of arterial smooth muscle cells has been shown to cause cholesteryl ester (CE) accumulation. However, the effects of human herpes simplex virus type 1 (HSV-1) infection on cholesterol binding and internalization, intracellular metabolism, and efflux have not been evaluated. In addition, the effects of viral infection on signal transduction pathways that impact upon cholesterol metabolism have not been studied. We show in studies reported herein that HSV-1 infection of arterial smooth muscle cells enhances low density lipoprotein (LDL) binding and uptake which parallels an increase in LDL receptor steady state mRNA levels and transcription of the LDL receptor gene. HSV-2 also increases CE synthesis and 3-hydroxy- 3-methylglutaryl-CoA reductase activity but concomitantly reduces CE hydrolysis and cholesterol efflux. Interestingly, this viral infection was associated with a time-dependent decrease in protein kinase A activity and an increase in viral-induced protein kinase (VPK) activity commensurate with the accumulation of esterified cholesterol. The relationship between increased VPK activity and alterations in CE accumulation in virally infected cells was explored using an HSV-1 VPK- mutant in which the portion of the HSV-1 genome encoding VPK had been deleted. Cholesteryl ester accumulation was significantly increased (> 50-fold) in HSV-1-infected cells compared to uninfected cells. However, the HSV-1 VPK- mutant had no significant effect on CE accumulation. The relationship between VPK activity and these alterations in cholesterol metabolism was further supported by the observation that staurosporine and calphostin C (protein kinase inhibitors) reduced protein kinase activity in HSV-1-infected cells. These results suggest several potential mechanisms by which alterations in kinase activities in response to HSV-1 infection of vascular cells may alter cholesterol trafficking processes that eventually lead to CE accumulation.

Inhibition of cholesterol esterification in macrophages and vascular smooth muscle foam cells: evaluation of E5324, an acyl-CoA cholesterol acyltransferase inhibitor.

Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N'-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methyl-phenyl]urea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for efflux in vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.

Specific inhibition of eIF-5A and collagen hydroxylation by a single agent. Antiproliferative and fibrosuppressive effects on smooth muscle cells from human coronary arteries.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.

Signal transduction in atherosclerosis: second messengers and regulation of cellular cholesterol trafficking.

The data summarized in this review demonstrate that the regulation of intracellular cholesterol trafficking is mediated not only by extracellular lipoprotein concentrations and transcriptional responses to alterations in intracellular free cholesterol content. Rather, the modulation of cholesterol trafficking is also regulated by the products synthesized following activation of signal transduction pathways originating at the cell surface. Furthermore, we have identified those cell-derived factors which utilize these signal transduction pathways to elicit alterations in cholesterol trafficking, and demonstrated the importance of the generation of second messengers, most notably eicosanoids, and cyclic AMP in promoting a modulatory influence on specific pro-atherogenic effects of mitogens.

Eicosanoid metabolism in cholesterol-enriched arterial smooth muscle cells. Evidence for reduced posttranscriptional processing of cyclooxygenase I and reduced cyclooxygenase II gene expression.

Eicosanoid biosynthetic activity by the cyclooxygenase pathway is reduced in smooth muscle cell-derived foam cells [Pomerantz, K.B., & Hajjar, D.P. (1989) J. Lipid Res. 30, 1219-1231; Pomerantz, K.B., & Hajjar, D.P. (1990) Biochemistry 29, 1892-1899]. The present study identifies those mechanisms which contribute to reduced production of cyclooxygenase products following cholesterol enrichment of arterial smooth muscle cells. Cyclooxygenase activity, as assessed by the conversion of exogenous arachidonate to 6-keto-PGF1 alpha, was reduced approximately 8-fold in intact lipid-laden cells relative to untreated cells. Microsomes from cholesterol-enriched cells also converted less [3H]arachidonic acid to 6-keto-PGF1 alpha and PGE2 relative to microsomes from untreated cells. The reduction in cyclooxygenase activity paralleled the reduced mass of the constitutive form of cyclooxygenase (COX-1) and PGI2 synthase by approximately 80% and 33%, respectively. Northern blot hybridization analyses of COX-1 mRNA steady-state levels revealed no differences between normal and cholesterol-enriched cells under basal conditions, indicating that cholesterol enrichment did not alter COX-1 gene expression. Furthermore, cholesterol enrichment did not alter the relative levels of COX-1 mRNA expression over time following exposure of the cells to actinomycin D, indicating that cholesterol enrichment did not significantly alter the rate of COX-1 mRNA degradation. Recovery of PGI2 biosynthesis in untreated cells exposed to serum following the inactivation of COX occurred within 12 h, while the recovery of COX activity in lipid-enriched cells did not return to levels observed in untreated cells even after up to 48 h, suggesting that the induction of COX-2 (inducible form of cyclooxygenase) synthesis by growth factors or cytokines is impaired. Indeed, cholesterol enrichment attenuated IL-1 beta-, PDGF-, and TNF alpha-induced PGI2 synthesis relative to controls and was consistent with the results of in vitro labeling experiments demonstrating that cholesterol enrichment reduced the incorporation of [35S]methionine into immunoprecipitable COX-1 and COX-2 following induction by PDGF. Cholesterol enrichment also reduced the induction of COX-2 mRNA steady-state levels following exposure to PDGF. Taken together, these data demonstrate that reduced eicosanoid synthesis in smooth muscle-derived foam cells is due, in part, to impaired transcription of mRNA for COX-1 and COX-2 as well as fatty acid remodeling in membrane phospholipids. These findings support the hypothesis that cholesterol enrichment alters posttranscriptional processing of COX-1 expression, as well as altering COX-2 gene expression.

Induction of basic fibroblast growth factor mRNA and protein synthesis in smooth muscle cells by cholesteryl ester enrichment and 25-hydroxycholesterol.

Basic fibroblast growth factor (bFGF) is a potent smooth muscle cell mitogen. Smooth muscle cell and macrophage-derived foam cells, resulting from cholesteryl ester accretion, are hallmark characteristics of atherosclerosis. We wanted to determine if bFGF synthesis is altered during cholesteryl ester accumulation in smooth muscle cells. Cholesteryl ester enrichment causes a 3-fold increase in bFGF in cellular lysates and a 3-fold increase in steady state mRNA levels for bFGF, as compared with control cells. Conditioned media from cholesteryl ester-enriched smooth muscle cells contains 6 times more mitogenic activity than conditioned media from control cells; this activity is neutralized by an antibody directed against bFGF but not by an antibody directed against platelet-derived growth factor. These results suggest that cholesteryl ester enrichment also enhances bFGF release. Since oxysterols have been implicated in the pathogenesis of atherosclerosis, we determined if oxysterols could affect bFGF production and release. 25-Hydroxycholesterol also increases the release of bFGF-like mitogens from smooth muscle cells, as well as increasing mRNA transcript levels for bFGF. Cholesteryl ester enrichment and 25-hydroxycholesterol did not promote bFGF release secondary to cell injury. In conclusion, these data define a basic mechanism for smooth muscle cell hyperplasia during atherogenesis involving the generation of bFGF by smooth muscle cell-derived foam cells.

Cholesterol enrichment of arterial smooth muscle cells upregulates cytokine-induced nitric oxide synthesis.

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. NO is also produced by vascular smooth muscle (VSMC) where it can inhibit proliferation. Since cytokine-activated VSMC proliferation is a major event in the development of atherosclerosis, we investigated the influence of cholesterol (CE)-enrichment of VSMC on cytokine-induced NO synthesis. Treatment of VSMC with native LDL for one week did not promote CE-accretion or alter NO production following exposure to endotoxin (LPS). In contrast, CE-enrichment by cationized LDL augmented LPS-induction of NO synthesis 2-5-fold. While TNF-alpha promoted little NO synthesis in control VSMC, it was very potent after CE-enrichment. Similarly, CE-enrichment augmented IL-1 alpha-induced NO synthesis. However, CE-enrichment did not affect the synergistic induction of NO synthesis by cytokines in combination with IFN-gamma. Our findings suggest that CE-enrichment of VSMC upregulates signal transduction pathways which mediate cytokine and LPS induction of NO synthase activity.

Signal transduction in atherosclerosis: integration of cytokines and the eicosanoid network.

Atherosclerosis can be defined in broad terms as a vascular disease accompanied by dysregulation of cholesterol metabolism and the accumulation of smooth muscle cells and macrophages within the vessel wall. At the interface of the blood and the vessel wall is the endothelium, which actively participates in a plethora of critical homeostatic functions in addition to affecting cholesterol trafficking in the underlying smooth muscle cell and macrophage. These events include: 1) inflammation resulting in release of cytokines, 2) changes in vascular reactivity causing release of endothelial cell derived relaxing factor (EDRF) and PGI2, and 3) control of vascular smooth cell proliferation via release of growth factors and growth suppressor molecules. Each process has been linked to the regulation of cholesterol accretion in the arterial cell. Furthermore, each homeostatic process is regulated by transmembrane signaling mechanisms at the lipid-protein interface of the membrane. Data have emerged recently indicating that biological response modifiers that trigger transmembrane signaling work in a sequential manner to control cell function. We review studies of the regulatory mechanisms of transmembrane signal transduction that advance the concept that phosphorylation of the specific protein components of the receptor machinery may result in a cooperative cellular response to ligands that will ultimately affect cholesterol delivery and trafficking within cells. We review recent data demonstrating that eicosanoids and cytokines released from one cell activate their receptors on neighboring cells, and interact with each other during this "cross-talk phenomenon." Cross-talking among phosphorylation reactions involving, for example, protein kinases A and C and tyrosine protein kinase, coupled with the highly regulated eicosanoid pathways and the diacylglycerol-phosphatidyl inositol (DG-PI) system, are discussed in terms of their metabolic impact on cholesterol delivery, intracellular processing, and efflux.

Molecular motions and thermotropic phase behavior of triacylglycerols and cholesteryl esters in herpesvirus-infected arterial smooth muscle cells: a deuterium nuclear magnetic resonance study.

The physical state of lipids in arterial smooth muscle cells (SMC) may contribute to lipid accumulation following injury. We have previously demonstrated that herpes simplex virus (HSV) infection alters the physical state of the neutral lipid accumulating in arterial SMC, as determined by differential scanning calorimetry (Biochem. J. 268 (1990) 693-697). To more precisely determine the molecular packing of neutral lipids in HSV-infected cells, the influence of HSV-infection on the thermotropic and phase-behavior of the lipids in intact arterial smooth muscle cells and in cell-free lipid extracts was evaluated using [2H]-NMR, employing U-[2H]-oleic acid incorporated into cells. Inspection of the [2H]-line-widths indicate that the lipid of HSV-infected cells exhibited more restricted motion or a greater chemical shift dispersity than lipids from uninfected cells, as evidenced by significant broadening of the -CD = CD- signals at 25 degrees C and 45 degrees C. Fatty acid compositional analysis of the neutral lipids of control and HSV-infected cells following C18:1 supplementation (an amount added similar to the NMR experiments) reveals that: (1) there is approximately 55-fold more triacylglycerols (TG) than cholesteryl esters (CE) in control cells and 40-fold more TG than CE in the HSV-infected cells; (2) HSV infection significantly increases the C18:1 content of CE, and C18:3 and C20:4 in TG; and (3) HSV-infection does not alter the ratio of TG to CE. These data support the hypothesis that the greater restriction of the neutral lipids in HSV-infected cells may be due to the rigidifying effects of C18:1 on lipid mobility. Thus, alterations in the physical state of neutral lipids in HSV-infected cells may lead to reduced CE hydrolysis which, in turn, may contribute to or exacerbate lipid accumulation.

Dihydropyridine calcium antagonist modulates cholesterol metabolism and eicosanoid biosynthesis in vascular cells.

Recent clinical studies have shown that calcium channel blockers can retard and possibly reduce the angiographic progression of coronary artery disease. Calcium channel blockers also inhibit dietary-induced atherosclerosis in animal models of this disease. In this study, we delineate potential cellular and molecular mechanisms by which nicardipine, a dihydropyridine calcium antagonist, may alter lipoprotein and cholesterol trafficking, affect the regulatory signal transduction pathways involved in accelerating cholesteryl ester (CE) catabolism in vascular smooth muscle cells, and modulate cell-cell interactions of vascular and inflammatory cells. We demonstrate in arterial smooth muscle cells that nicardipine increases 1) LDL binding, uptake, and degradation, 2) RNA transcript levels for the LDL receptor, 3) CE catabolic activity, 4) PGI2 release, and 5) RNA transcript levels for cyclooxygenase. Furthermore, nicardipine blocked cytokine-induced monocyte adhesion to endothelial cells and smooth muscle cells. Taken together, these findings support the hypothesis that nicardipine may function as an anti-atherosclerotic agent by promoting CE catabolism and cholesterol clearance and by reducing monocyte adhesion to the activated endothelium.

Analysis of the physical state of cholesteryl esters in arterial-smooth-muscle-derived foam cells by differential scanning calorimetry.

The physical state of cholesteryl esters (CE) in the arterial-smooth-muscle-derived foam cells may contribute to the documented reduction in CE hydrolysis. The physical state of CE may also provide a potential enhancing mechanism for increased CE accumulation. To explore these concepts, we therefore examined the influence of alterations in CE and triacylglycerol (TG) content and their fatty acid composition on the thermotropic behaviour of these lipids by differential scanning calorimetry (d.s.c.). After exposure to cationized LDL (cLDL) or after infection with herpes simplex virus type I (HSV), smooth-muscle cells accumulated significant amounts of CE. The CE/TG ratio was significantly higher in cells treated with cLDL compared with HSV infection. TG content was unaffected by either treatment. However, the fatty acid profile of both CE and TG was significantly different between treatment groups, with the polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio being significantly higher in cLDL-treated cells than in HSV-infected cells. The d.s.c.-generated thermograms of intact cells revealed that neutral lipids of both treatment groups were in the isotropic-liquid state, similar to the state of lipids derived from 'fatty streak' types of atherosclerotic lesions. Differences in the thermograms between HSV-infected and cLDL-treated cells can be ascribed to differences in the CE content and the fatty acid composition of CE and TG (PUFA/SFA ratio). Polarizing optical microscopy revealed the presence of isotropic lipids in both groups. Biochemical and physicochemical data confirm the lysosomal localization of engorged CE, and indicate that the cellular isotropic CE in these foam cells are in a physical state which favours enzymic hydrolysis.

High-density-lipoprotein-induced cholesterol efflux from arterial smooth muscle cell derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis.

Eicosanoids have been implicated in the regulation of arterial smooth muscle cell (SMC) cholesteryl ester (CE) metabolism. These eicosanoids, which include prostacyclin (PGI2), stimulate CE hydrolytic activities. High-density lipoproteins (HDL), which promote cholesterol efflux, also stimulate PGI2 production, suggesting that HDL-induced cholesterol efflux is modulated by eicosanoid biosynthesis. To ascertain the role of endogenously synthesized eicosanoids produced by arterial smooth muscle cells in the regulation of CE metabolism, we examined the effects of cyclooxygenase inhibition on CE hydrolytic enzyme activities, cholesterol efflux, and cholesterol content in normal SMC and SMC-derived foam cells following exposure to HDL and another cholesterol acceptor protein, serum albumin. Alterations of these activities were correlated with cholesterol efflux in response to HDL or bovine serum albumin (BSA) in the presence or absence of aspirin. HDL stimulated PGI2 synthesis and CE hydrolases in a dose-dependent manner. Eicosanoid dependency was established by demonstrating that HDL-induced acid cholesteryl ester hydrolase (ACEH) activity was blocked by aspirin. CE enrichment essentially abrogated HDL-induced PGI2 production in cells which also exhibited decreased lysosomal and cytoplasmic CE hydrolase activities. In CE-enriched cells whose cytoplasmic CE pool was metabolically labeled with [3H]oleate or cLDL containing [3H]cholesteryl linoleate, aspirin did not alter HDL- or BSA-induced net CE hydrolysis or efflux, respectively. Finally, aspirin treatment did not alter the mass of either free or esterified cholesterol content of untreated or CE-enriched SMC following exposure to acceptor proteins. These data demonstrated that CE enrichment significantly reduced HDL-induced activation of CE hydrolytic activity via inhibition of endogenous PGI2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Eicosanoids and their role in atherosclerosis.

Our laboratory has been actively investigating the role of endogenously synthesized eicosanoids in the control of vascular cholesterol metabolism. Using an in vivo rabbit model, cholesteryl esters (CE) accumulated under the regenerating edge of the endothelium due to reductions in CE hydrolytic activity and the accumulation of LDL. In addition, the aortic neointima immediately formed after de- endothelialization synthesized little PGI2, but regained its capacity to synthesize PGI2 over time of endothelial cell regeneration. Importantly, hypercholesterolemia inhibited the recovery of PGI2-synthetic capacity by the vessel wall using this model. Using cultured arterial smooth muscle cells, PGI2 and its derivatives (but not PGE2 and PGE1) stimulated lysosomal (acid) and cytoplasmic (neutral) CE hydrolase activities and reduced cellular cholesterol content. CE-synthetic activity was unaffected by PGI2 or its stable metabolites, but was inhibited by PGE2. Eicosanoids generated from platelet-neutrophil-smooth muscle cell interactions (including platelet-generated arachidonic acid, 12-HETE, 12,20-diHETE) may be important in the role of eicosanoids in mediating vascular cholesterol metabolism. We also observed that CE-enriched arterial smooth muscle cells have reduced capacity to synthesize PGI2 and PGE2. Collectively, our data suggest that eicosanoids derived from blood-borne cells and the vascular endothelium may regulate cholesterol metabolism in smooth muscle cells, and that eicosanoid regulation of vascular CE content may be impaired during hypercholesterolemia owing to an inability of arterial tissue to generate PGI2 and related eicosanoids.

Cicletanine stimulates cholesteryl ester hydrolase activities and prostacyclin production in arterial smooth muscle cells.

Cicletanine (1,3,-dihydro-6-methyl-7-hydroxy-3-(4-chlorophenyl) furo (3,4-c)pyridine) is a novel antihypertensive agent whose principal mechanisms of action may be stimulation of arterial PGI2 synthesis. Since PGI2 and related eicosanoids promote cholesteryl ester hydrolytic activities in arterial smooth muscle cells via cyclic AMP, we evaluated the effects of cicletanine on PGI2 production, the enzymes comprising the arterial cholesteryl ester cycle, and cyclic AMP generation by arterial smooth muscle cells in tissue culture. Toxicity was monitored using dye-exclusion tests. Monocyte adhesion essays were also performed to determine if cicletanine would make blood-derived monocytes less adherent to endothelium, thus less prone to initiating a pre-atherosclerotic event. Cells were exposed to a standard dose of 1.7 mM cicletanine for 2 hours. Cicletanine stimulated PGI2 production 2-3 fold over basal levels. Furthermore, cicletanine stimulated both lysosomal and cytoplasmic CE-hydrolases, with modest inhibitory effect on CE synthesis. However, cyclic AMP was not increased following cicletanine treatment. These data suggest that cicletanine may stimulate CE-hydrolase activity directly, and independently of endogenously synthesized eicosanoids. These effects were not due to toxic effects of this compound, as determined by dye exclusion assays. No effects were seen on monocyte adhesion to endothelial cells in vitro. These results suggest that in addition to being a novel, potent antihypertensive agent, cicletanine may also contribute to cholesterol mobilization in smooth muscle cells by stimulating CE hydrolysis.

Human glial cell production of lipoxygenase-generated eicosanoids: a potential role in the pathophysiology of vascular changes following traumatic brain injury.

Acute cerebrovascular changes which occur following traumatic brain injury represent a highly complex, multifactorial pathophysiologic process which is poorly understood. It is now recognized that, under normal conditions, the brain is a source of a variety of arachidonic acid metabolites which are synthesized by both cyclooxygenase and lipoxygenase. The specific cellular source of these highly vasoactive substances remains controversial. Recent work has demonstrated that lipoxygenase products were detected by immunosensitive assay in whole brain samples from a gerbil concussive injury model, yet the production of leukotrienes could not be accounted for by cerebral vessels and their contents alone. It has been theorized that the probable source for these metabolites is the cortical neuron. We sought to elucidate whether cultured human glial cells, obtained from specimens removed at the time of surgery, are a significant source of lipoxygenase products as measured by high performance liquid chromatography (HPLC). We observed that these cells consistently produced 5, 12, and 15-HETE class eicosanoids despite failure to produce significant cyclooxygenase products. These preliminary findings are of considerable interest because these lipoxygenase products are known to be highly vasoactive as well as potent mediators of increased vascular permeability. Since it is known that mechanical perturbation of cell membranes stimulates the release of arachidonic acid from membrane phospholipids, it is conceivable that the production of these eicosanoids following traumatic brain injury could account for local cerebrovascular changes including both vasospasm and interstitial edema formation.

Eicosanoid metabolism in cholesterol-enriched arterial smooth muscle cells: reduced arachidonate release with concomitant decrease in cyclooxygenase products.

A biochemical correlation between vascular cholesterol metabolism and eicosanoid biosynthesis has not been fully elucidated. To assess the effects of cholesteryl ester (CE) accretion on eicosanoid synthesis, we studied eicosanoid metabolism in cultured rabbit aortic smooth muscle cells (SMC) following lipid-enrichment by incubation with cationized LDL (cLDL). SMC exposed to cLDL synthesized 50% less immunoreactive 6-keto-PGF1 alpha than untreated cells when exposed to the calcium ionophore, A-23187. In addition, cLDL-treatment reduced arachidonate acid (AA)-induced prostacyclin (PGI2) production sevenfold. Components of cLDL decreased eicosanoid biosynthesis in the following rank-order: linoleate greater than cholesterol greater than apo-cLDL. Lipid-enriched cells incorporated amounts of [1-14C]AA into phosphatidylcholine and phosphatidylethanolamine equal to control cells, but subsequent exposure to ionophore released significantly less radioactivity as free arachidonate (AA), with proportionally less conversion to eicosanoids. Ionophore released equivalent amounts of AA from all phospholipids, suggesting specificity for uptake, but not release of AA by cellular phospholipases. Cells enriched in CE had an eightfold decrease in percentage of phospholipid-derived AA relative to linoleate as compared to controls. Taken together, our data demonstrate that SMC metabolism of cLDL leads to cholesterol and CE accretion concomitant with diminished production of eicosanoids. Potential mechanisms for this effect include competitive inhibition of eicosanoid production by linoleate derived from LDL, direct inhibition of phospholipase A2 activity by cholesterol, and decrease in cyclooxygenase activity. These findings may have pathophysiological significance in that a reduction in PGI2 synthetic capacity of arterial SMC may exacerbate CE deposition since PGI2 promotes intracellular CE hydrolysis.