Herpes simplex virus infection in human arterial cells. Implications in arteriosclerosis.

Herpesviruses have been implicated as etiologic factors in the pathogenesis of human arteriosclerosis. We have examined the pathobiological effects of human herpes simplex virus (HSV-1) infection in influencing lipid accumulation and metabolism in human and bovine arterial smooth muscle cells (SMC). Significantly greater amounts of saturated cholesteryl esters (CE) and triacylglycerols (TG) accumulate in HSV-1-infected human and bovine arterial SMC than uninfected cells. This CE accumulation results, in part, from decreased CE hydrolysis. Furthermore, arachidonate-stimulated, HSV-1-infected arterial SMC have a reduced capacity to produce prostacyclin (an agonist of intracellular CE hydrolytic activity) than uninfected, stimulated SMC. It appears that HSV-1 may induce lipid accumulation in arterial SMC similar, in part, to the lipid accumulation observed in vivo during human atherogenesis. Thus, herpesviruses may contribute to lipid accumulation, which is a characteristic feature of atherosclerosis.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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 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.

Enrichment of endothelial cell arachidonate by lipid transfer from high density lipoproteins: relationship to prostaglandin I2 synthesis.

We have previously shown that plasma high density lipoproteins (HDL) stimulate release of prostacyclin, measured as its stable metabolite, 6-keto-PGF1 alpha, by cultured porcine aortic endothelial cells. The present experiments were designed to elucidate the contribution of HDL lipids to endothelial cellular phospholipid pools and to prostacyclin synthesis. In experiments with reconstituted HDL, both the lipid and protein moieties were required to stimulate prostacyclin release in amounts equivalent to the native HDL particle. Endothelial cells incorporated label from reconstituted HDL containing cholesteryl [1-14C]arachidonate into the cellular neutral and phospholipid pools as well as into 6-keto-PGF1 alpha and PGE2. Labeled arachidonate incorporated into endothelial cell lipids from reconstituted HDL containing cholesteryl [1-14C]arachidonate was also metabolized to prostaglandins after the cells were exposed to the calcium ionophore, A-23187. Both rat and human HDL which stimulated 6-keto-PGF1 alpha release (rat greater than human) increased the weight percentage of arachidonate in endothelial cell phospholipids; phospholipid arachidonate in the enriched cells fell after exposure to the phospholipase activator, A-23187, with release of 6-keto-PGF1 alpha which was greater than in control cells. Rat HDL that was depleted of cholesteryl arachidonate (achieved by incubation with human low density lipoproteins (LDL) in the presence of cholesteryl ester transfer protein) stimulated 6-keto-PGF1 alpha release less than native rat HDL. LDL enriched in cholesteryl arachidonate stimulated 6-keto-PGF1 alpha release more than native LDL. ApoE-depleted HDL also stimulated 6-keto-PGF1 alpha release more than apoE-rich HDL suggesting the apoE receptor was not involved in the response.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Stimulation of vascular smooth muscle cell prostacyclin and prostaglandin E2 synthesis by plasma high and low density lipoproteins.

We studied the effects of plasma high density and low density lipoproteins upon the synthesis of prostacyclin and prostaglandin E2 by vascular smooth muscle cells. Prostaglandin synthesis was measured in 24-hour cultures by radioimmunoassay of the stable metabolites of prostacyclin, 6-keto-prostaglandin F1 alpha and of prostaglandin E2. High density lipoproteins induced dose-dependent increases in the release of 6-keto-prostaglandin F1 alpha and of prostaglandin E2 from smooth muscle cells to values 14- and 50-fold above control. Incubations with low density lipoproteins at comparable cholesterol concentrations also induced dose-dependent release of 6-keto-prostaglandin F1 alpha and prostaglandin E2, but to a lesser extent. Rat high density lipoprotein, which contained 2.5 times more cholesteryl arachidonate than human high density lipoproteins, stimulated 6-keto-prostaglandin F1 alpha and prostaglandin E2 release 2- to 3-fold more than human high density lipoproteins, whereas the delipidated apoproteins of high density lipoproteins had no significant effect on prostaglandin synthesis. Recombinant high density lipoproteins containing cholesteryl-[1- 14C]arachidonate stimulated release of [14C]-6-keto-prostaglandin F1 alpha and [14C]prostaglandin E2 by smooth muscle cells. The ionophore, A 23187, released labeled 6-keto-prostaglandin F1 alpha and prostaglandin E2 from cells preincubated with recombinant high density lipoprotein containing cholesteryl-[1- 14C]arachidonate. Unlabeled high density lipoproteins, in contrast, did not cause release of radioactive prostaglandins from cells preincubated with [1- 14C]arachidonate. Phospholipase activators were synergistic (bradykinin) or additive (angiotensin II) with high density lipoprotein in stimulation of prostaglandin synthesis. The data indicate that both high and low density lipoproteins stimulate the synthesis of prostacyclin and prostaglandin E2 by vascular smooth muscle cells. The results suggest that the lipoproteins provide arachidonate to a phospholipase-sensitive pool accessible to cyclooxygenase.