Interactions of arterial cells. Studies on the mechanisms of endothelial cell modulation of cholesterol metabolism in co-cultured smooth muscle cells

Fluid phase interactions between arterial endothelial cells (EC) and smooth muscle cells (SMC) have been studied in vitro to assess the regulation of lipid metabolism in SMC (Hajjar, D. P., Falcone, D. J., Amberson, J. B., and Hefton, J. M. (1985) J. Lipid Res. 26, 1212-1223; Davies, P. F., Truskey, G. A., Warren, H. B., O'Connor, S. E., and Eisenhaure, B. H. (1985) J. Cell Biol. 101, 871-879). To identify EC-derived agonists which may modulate cholesterol metabolism in co-cultured SMC, we assessed the role of EC-derived eicosanoids and platelet-derived growth factor (PDGF) in the regulation of cholesteryl ester (CE) hydrolysis in SMC. The major eicosanoids synthesized by EC include PGI2 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and, to a lesser extent, prostaglandin E2. Exogenously added PGI2 and 12-HETE stimulated CE hydrolytic activity in SMC by 49 and 35%, respectively, when co-cultured with aspirin-treated EC. Aspirin-treated EC when co-cultured with SMC did not stimulate CE hydrolytic activity in SMC, as was the case with non-aspirin-treated EC, suggesting a role of eicosanoids in the regulation of cholesterol metabolism. Other humoral agents derived from EC such as PDGFc stimulated CE hydrolytic activity almost 2-fold in SMC cultured alone or co-cultured with EC. Aspirin-treated EC, incubated with 10 ng/ml PDGF, did not stimulate CE hydrolytic activity in co-cultured SMC. These results suggest that growth factor-promoting activity may enhance CE hydrolysis via the PGI2-cyclic AMP-CE hydrolysis cascade. This hypothesis supports our observations that PDGF stimulates PGI2 production in SMC. Elevated PGI2, in turn, can stimulate CE hydrolysis in these cells. Our findings suggest that the regulation of cholesterol metabolism in SMC can involve, at least in part, growth factors and EC-derived eicosanoids. These may play a central role in the regulation of hemostasis and the inflammatory response.     

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)     

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.     

Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

Effects of ammonium ion derived from bovine endothelial cells upon low density lipoprotein degradation in cultured vascular smooth muscle cells

Low density lipoprotein (LDL) metabolism in bovine arterial smooth muscle cells (SMC) was increased upon exposure to endothelial cell conditioned medium. The mass of LDL degraded in the SMC lysosomal system was increased, and kinetic analysis demonstrated that the rate constant for LDL degradation arising from receptor-mediated endocytosis was unchanged. The effects on LDL metabolism were accompanied by stimulation of DNA synthesis in the SMC. These results are in contrast to reports concerning a porcine endothelial cell system where LDL degradation was inhibited by endothelial-derived NH4+. We show that bovine endothelial cells produce insufficient NH4+ to inhibit LDL degradation and conclude that endothelial cell-derived NH4+ is unlikely to be a factor affecting LDL metabolism in the bovine vascular cell culture system.     

Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of TNF to the culture medium for 24-48 h resulted in an increase of [3H]oleic acid uptake and esterification into lipids. The effect could be seen already with 0.3 ng/ml and was maximal with 30 ng/ml. The effect of TNF was mainly on the incorporation of [3H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3H]cholesteryl ester. In the human SMC there was a 40% increase in [3H]oleic acid into total lipids, while the rise in [3H]triacylglycerol ranged between 60-90%. TNF did not modulate cellular triacyglycerol synthesis in cultured mouse peritoneal macrophages. Since TNF was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triacylglycerol accumulation.     

Evidence for cytokine regulation of cholesterol metabolism in herpesvirus-infected arterial cells by the lipoxygenase pathway

Cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and gamma-interferon (IF) are produced by activated hematopoietic cells. They possess antiviral activity and have other biological activities such as induction of cell proliferation and hemorrhagic necrosis of tumors. Since herpes simplex virus (HSV) infection of human vascular cells is known to produce a biochemical and cytopathological effect virtually indistinguishable from atherosclerosis, we hypothesized that these cytokines many prevent cholesteryl ester (CE) accumulation in arterial smooth muscle cells (SMC) that is seen with herpesvirus infection. We now report that TNF and IL-1 but not gamma-IF prevent CE accumulation in HSV-infected arterial SMC by induction of cyclic AMP-dependent CE hydrolysis. This effect is mediated through the arachidonate 12-lipoxygenase pathway via 12-HETE since pretreatment of cells with several lipoxygenase inhibitors abolishes the antiviral effect and 12-HETE is the major (greater than 99%) lipoxygenase metabolite produced by these cells. This conclusion is further based on our observations that TNF and IL-1 enhance 12-HETE production in SMC and that 12-HETE significantly increases both intracellular cyclic AMP and lysosomal CE hydrolysis. Moreover, dibutyryl cyclic AMP restored a normal phenotype in these virally infected cells. Collectively, these findings identify for the first time a biochemical mechanism involved in the reduction of lipid accumulation in virally infected arterial SMC by these potent cytokines.     

Cholesterol metabolism is altered by hydrolytic metabolites of prostacyclin in arterial smooth muscle cells

Cholesteryl esters are the major lipids that accumulate in arteries during atherogenesis. The mechanisms responsible for this lipid accretion have not been completely defined. Our previous experiments have shown that prostacyclin (PGI2) enhances cholesteryl ester catabolism by increasing cyclic AMP in cultured arterial smooth muscle cells. However, PGI2 is rapidly degraded under physiologic conditions and endogenous levels of PGI2 in the human circulation are extremely low. These findings suggest that it is not a circulating hormone. We tested the hypothesis that stable PGI2 metabolites alter cholesteryl ester metabolism and cellular lipid accumulation. Ten to 100 nM dinor-6-keto PGF1 alpha, 13,14-dihydro-6,15-diketo PGF1 alpha, and 6,15-diketo PGF1 alpha increased cyclic AMP levels significantly two- to threefold with a concomitant enhancement of both lysosomal and cytoplasmic cholesteryl ester hydrolytic activities. Cholesteryl ester synthesis was unchanged by the PGI2 metabolites. When cyclic AMP concentrations were maintained at basal levels by an adenylate cyclase inhibitor, no effect on cholesteryl ester hydrolysis was observed following addition of PGI2 metabolites to the cells. Furthermore, addition of PGI2 metabolites during a 1-week culture period reduced free and esterified cholesterol by 50%. These data suggest that PGI2 metabolites: 1) decrease intracellular cholesterol accumulation by increasing cholesteryl ester catabolism; 2) act via enhancement of cyclic AMP; and, 3) may represent circulating regulators of arterial cholesteryl ester metabolism.     

Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.     

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.     

Metabolic activity of cholesteryl esters in aortic smooth muscle cells is altered by prostaglandins I2 and E2

Among the biochemical processes associated with the atherogenic process are increased aortic cholesteryl ester (CE) accumulation and altered prostaglandin (PG) production. The precise physiological role of PG, particularly prostacyclin (PGI2), in the control of CE metabolism in intact aortic smooth muscle cells remains to be fully elucidated. We report here that cytosolic neutral cholesteryl ester hydrolytic activity (NCEH) in intact cultured aortic smooth muscle cells is significantly increased by 75-250 nM PGI2 at the end of a 2-hr incubation period. The effect was mediated by increased intracellular cAMP levels since the effect of PGI2 on NCEH activity was abolished in the presence of an inhibitor of adenylate cyclase activity, viz., dideoxyadenosine (DDA0. Although the addition of 20-100 microM dibutyryl cAMP (Bt2cAMP) and 50-100 microM sodium arachidonate also increased NCEH activity twofold, 6-keto PGF1 alpha, PGE1, and PGE2 did not increase the activity of this enzyme. In contrast to these findings, 75-250 nM PGE2 significantly inhibited CE synthetic activity (ACAT) approximately 60%. Arachidonate or Bt2cAMP did not affect ACAT activity. This decrease in ACAT activity induced by PGE2 does not appear to be mediated by cAMP. Taken together, these findings suggest that PGI2, a well known potent vasodilator and inhibitor of platelet aggregation, and PGE2 may have an important regulatory role in aortic CE metabolism.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress

Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm² for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p ≤ 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC–SMC interactions affect EC responses to shear stress.     

Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell-cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum-free ECCM with antiplatelet-derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF-like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.     

Water filtration rate and infiltration/accumulation of low density lipoproteins in 3 different modes of endothelial/smooth muscle cell co-cultures

Using different endothelial/smooth muscle cell co-culture modes to simulate the intimal structure of blood vessels, the water filtration rate and the infiltration/accumulation of LDL of the cultured cell layers were studied. The three cell culture modes of the study were: (i) The endothelial cell monolayer (EC/Φ); (ii) endothelial cells directly co-cultured on the smooth muscle cell monolayer (EC-SMC); (iii) endothelial cells and smooth muscle cells cultured on different sides of a Millicell-CM membrane (EC/SMC). It was found that under the same condition, the water filtration rate was the lowest for the EC/SMC mode and the highest for the EC/Φ mode, while the infiltration/accumulation of DiI-LDLs was the lowest in the EC/Φ mode and the highest in the EC-SMC mode. It was also found that DiI-LDL infiltration/accumulation in the cultured cell layers increased with the increasing water filtration rate. The results from the in vitro model study therefore suggest that the infiltration/accumulation of the lipids within the arterial wall is positively correlated with concentration polarization of atherogenic lipids, and the integrity of the endothelium plays an important role in the penetration and accumulation of atherogenic lipids in blood vessel walls.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.     

Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine,MCP-1 and activation of TLR4 and Gi/o proteins signaling

The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.