Interaction endothelial LYNN

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Departments of Anesthesia, Internal Medicine, University of Iowa, Iowa City, Iowa 52242 Stoll, Lynn L., Helgi J. Oskarsson, and Arthur A. Spector. Interaction of lysophosphatidylcholine with aortic endothelial cells. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H1853-H1860, 1992.-To better understand the vascular actions of lysophosphatidylcholine (lysoPC), we studied the interaction of [ l-14C]palmitate-labeled 1ysoPC with bovine aortic endothelial cells. These cells took up 1ysoPC from media containing albumin, low-density lipoproteins (LDL), or acetylLDL. Uptake occurred faster than conversion to phosphatidylcholine (PC), leading to some 1ysoPC accumulation in endothelial lipids. Endothelial cell monolayers grown on micropore filters took up 1ysoPC from both apical and basolateral surfaces, preventing substantial amounts from passage across the endothelial monolayer. However, 1ysoPC present in the interstitial medium of an endothelial-smooth muscle coculture was incorporated primarily by the smooth muscle cells. Endothelial cells grown on filters released 1ysoPC into both the apical and basolateral medium in the presence of albumin or lipoproteins. Exposure to 50 PM 1ysoPC produced no evidence of endothelial cytotoxicity, but prostaglandin (PG)12 production was reduced. These studies suggest that the endothelium can participate in the processing of circulating 1ysoPC and, through basolateral uptake, can facilitate the removal of 1ysoPC formed within the arterial wall. By decreasing PG12 output, however, exposure to high concentrations of 1ysoPC may reduce the antithrombotic and vasodilator capacity of the endothelium. phosphatidylcholine; fatty acid; low-density lipoproteins; albumin; cell polarity; micropore filters; permeability; cytotoxicity LYSOPHOSPHATIDYLCHOLINE (1ysoPC)hasanumberof important actions that may affect the properties and function of the arterial wall. It impairs endotheliumderived relaxing factor (EDRF) -mediated arterial vasodilation (8)) modulates smooth muscle contractility (l3), induces a conformational change in calmodulin that affects its interaction with the calmodulin binding protein (5), and influences the activity of protein kinase C (10). LysoPC also is a chemotactic factor for human monocytes and probably mediates their entry into the developing atherosclerotic lesion (12). Furthermore, it stimulates the guanosinetriphosphatase (GTPase) activity of the ras protooncogene product p21 (20). Because of these effects, there is increasing interest in the disposition and metabolism of 1ysoPC in vascular cells and tissues. There are several ways in which 1ysoPC can accumulate in the arterial wall. LysoPC is formed when cholesteryl esters are synthesized in the plasma in the lecithincholesterol acyltransferase reaction (1). Some of the 1ysoPC generated in this way may be available to the endothelium at its apical surface. Furthermore, when low-density lipoproteins (LDL) are oxidized, as much as 60% of the phosphatidylcholine (PC) content is converted to 1ysoPC (8). Much of this 1ysoPC probably is taken up by the macrophages that incorporate the oxidized LDL. However, it is possible that the other cells 0363-6135/92

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with aortic

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within the arterial wall, including the smooth muscle and the basolateral surface of the endothelium, are exposed to 1ysoPC under these conditions. The purpose of these studies is to determine how endothelial cells interact with 1ysoPC. For these studies the 1ysoPC is presented at the apical or basolateral surfaces of an endothelial monolayer, using a recently developed system in which bovine aortic endothelial cells are cultured on micropore filters (17). In addition, 1ysoPC effects on endothelial permeability and function were examined. METHODS Cell culture. Bovine aortic endothelial cells and porcine pulmonary artery smooth muscle cells were grown as described previously (18). Briefly, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), Eagle’s minimum essential medium (MEM) nonessential amino acids (GIBCO), MEM vitamin solution (GIBCO), 15 mM N2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma, St. Louis, MO), 2 mM L-glutamine (Sigma), and 50 PM gentamicin (Schering, Kenilworth, NJ). Cultures were grown at 37°C in a humidified atmosphere containing 5% COe. Stocks were subcultured weekly by trypsinization. For experiments, endothelial cells were used between passages 7 and 12; smooth muscle cells were used between passages 5 and 12. For some experiments, endothelial cells were grown on Nuclepore polycarbonate filters (0.8 pm pore size, 9 pm thick) that had previously been impregnated with gelatin and glued onto 8-mm-diameter polystyrene chambers (ADAPS, Dedham, MA) and then coated with laminin (17). Cocultured smooth muscle cells were incorporated into this system (17) to examine the distribution and metabolism of 1ysoPCwhen both cell types are present. For these experiments, the smooth muscle cells were grown separately in 24-well plates, whereas endothelial cells were grown on filters as previously described. The filters with confluent endothelial monolayers were then suspended over the smooth muscle cells; the clearance between the smooth muscle cells and the bottom surface of the filter was 0.5 mm. Incubations and Zipid analysis. 1-Palmitoyl-L-a-lysophosphatidylcholine was obtained from Sigma. 1- [ 1-14C]palmitoylL-lysophosphatidylcholine ([ 14C]1ysoPC; 56 mCi/mmol) was obtained from Amersham. Unless otherwise noted, cultures were incubated with 1ysoPCcomplexed to fatty acid-free bovine serum albumin (BSA; Pentex/Miles, Kankakee, IL) at a lysoPC-to-BSA molar ratio of 2 in Dulbecco’s phosphate-buffered saline (DPBS) containing (in mM) 137 NaCl, 2.7 KCl, 1 CaCl,, 0.5 MgCl,, 1.5 KH,PO,, and 8.0 Na2HP04, pH 7.4. After the incubation, cells were washed thoroughly with an albuminbuffer solution, followed by two washes with buffer solution to remove any adherent 1ysoPC before measurement of cell-associated radioactivity by liquid scintillation spectrometry. Quenching was monitored with the external standard. For separation and analysis of 1ysoPCand its metabolites by thin-layer chromatography (TLC), cells and media were extracted with chloroform:methanol, 2:l (vol/vol). After this

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extraction procedure, 299% of the radiolabeled 1ysoPC is found in the chloroform phase, 5 1% in the aqueous phase. Aliquots were spotted on LK 60 Linear-K Silica Gel Plates (Whatman, Clifton, NJ) impregnated with 1.2% boric acid in 50% ethanol (6), and separation was obtained with a solvent system of CHC1,:CH30H:NH,0H:H,0, 100:90:3:6 (vol/vol/vol/vol). After development, the TLC plates were analyzed with a radio TLC model RS plate scanner (Radiomatic Instruments, Tampa, FL), which provides automatic peak search and integration. Metabolites were identified by comparison with radiolabeled standards. Neutral lipids were analyzed by TLC using LK 60 Linear-K Silica Gel Plates and a solvent system of mixed hexanes:diethyl ether:methanol:acetic acid, l70:40:4:4 (vol/vol/vol/vol). The free fatty acid was identified by comigration with a radiolabeled standard and by a shift in TLC migration from a retardation factor (&) of 0.38 to Rf equal to 0.65 after a 15-min incubation with diazomethane in methanol (16). Lipoprotein preparations. LDL and high-density lipoproteins (HDL) were isolated from the serum of a normal volunteer by ultracentrifugation (7). Acetylation of the LDL was performed as described by Basu et al. (2). Integrity of the lipoproteins was confirmed with agarose gel electrophoresis. For the experiment measuring uptake of 1ysoPC from LDL and acetyl-LDL, the lipoproteins were incubated with 2 PM [14C]lysoPC in DPBS for 16 h. After this incubation, the LDL and acetyl-LDL were reisolated from the buffer solution by ultracentrifugation and then dialyzed. Endothelial monolayers were then incubated with 1 ml of DPBS containing 75 rug of protein of one of the [ 14C]lysoPC-labeled lipoproteins. Prostuglandin formation. Prostaglandin I2 (PG12) production was determined by radioimmunoassay of the stable end product, 6-keto-PGF,,. Anti-6-keto-PGF,, was purchased from Advanced Magnetics (Cambridge, MA). This antibody has a 7.8% cross-reactivity with PGF1,, 6.8% with 6-keto-PGE,, 2.2% with PGF2,, and ~1.0% with all other prostaglandins. 6-Keto-[3H]PGFlcv was purchased from Amersham (Arlington Heights, IL); the 6-keto-PGF,, standard was obtained from Cayman Chemicals (Ann Arbor, MI). All solutions were made up in DPBS containing 0.2% bovine gamma globulin. Confluent cultures of endothelial cells were washed twice with warm DPBS and then incubated for 2 h in serum-free DMEM containing increasing concentrations of 1ysoPC; controls were incubated with serum-free DMEM. After this preincubation, the medium was removed, and the cells were washed once with buffer and then incubated for 20 min with 2 PM ionophore A23187 or 7.5 PM arachidonic acid. The medium was collected and centrifuged. Aliquots (100 ~1) of samples or standards were incubated for 30 min with 50 ~1 of the antibody; 50 ~1 of radiolabeled 6-keto-PGF,, was then added to all tubes. After the samples were incubated overnight at 4°C 400 ~1of 1% dextrancoated charcoal was added to each tube, and the contents were centrifuged for 10 min at 4°C. A 500-~1 aliquot of the supernatant solution was added to 5 ml Budget Solve scintillation cocktail, and the radioactivity was measured in a Beckman LS 2800 scintillation spectrometer. A complete standard curve was run with each assay; the detection limits were 0.25-50 pmol/ml. RESULTS

Uptake and metabohvn. The cultured bovine aortic endothelial cells took up a substantial amount of the [ 14C]lysoPC available in the extracellular fluid. Figure 1 (top) shows the time dependence of 1ysoPC incorporation from a medium containing albumin. The initial uptake was very rapid; 37% of the available radioactivity was incorporated in the first 2 min of incubation. TLC analysis of the cell lipids showed that most of the radioactiv-

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Fig. 1. Time dependence of 1-[1-14C]palmitoyl-~-lysophosphatidylcholine ([ 14C]1ysoPC) uptake by endothelial cells. Cells were grown to confluency in 6-well plates. Confluent monolayers were gently washed twice with warm phosphate-buffered saline (PBS). Cultures were then incubated with 1 PM [ 14C]lysoPC in serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing 0.5 PM albumin. At intervals, medium was removed from sets of 3-6 cultures, and cells were washed with albumin and buffer and harvested by scraping into ice-cold methanol. Cell lipids were extracted into chloroform:methanol, and aliquots of cell lipid extracts and incubation media were assayed for radioactivity. Distribution of radioactivity in cell lipid extracts was analyzed by thin-layer chromatography (TLC), using a solvent system of CHCl,:CHSOH:NH40H:H20, 100:90:3:6 (vol/vol/vol/vol). Radioactive peaks-were-identified by comparison with radiolabeled standards. Each point represents 3-6 individual cultures pooled for TLC analysis. PC, phosphatidylcholine; lysoPC, lysophosphatidylcholine; dpm, disintegrations per minute.

ity taken up during the first 20 min remained in the form of 1ysoPC (Fig. 1, bottom). Subsequently, the amount of labeled 1ysoPC decreased, and labeled PC gradually accumulated in the cell lipids. Although total uptake slowed considerably after 45 min under these conditions, the conversion of 1ysoPC to PC continued throughout the incubation; after 2 h, 85% of the incorporated radioactivity was converted to PC. In addition, a small amount of the radioactivity that accumulated in the cells,

Interaction of lysophosphatidylcholine with aortic endothelial cells.

To better understand the vascular actions of lysophosphatidylcholine (lysoPC), we studied the interaction of [1-14C]palmitate-labeled lysoPC with bovi...
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