American Journal ofPathology, Vol. 136, No. 6, June 1990 Copyright © American Association ofPathologists
Atherogenic Concentrations of Low-density Lipoprotein Enhance Endothelial Cell Generation of Epoxyeicosatrienoic Acid Products
Kirkwood A. Pritchard, Jr.,*t P. Y.-K. Wong,t and M. B. Stemerman* From the Departments ofMedicine* and Physiology,t New York Medical College, Valhalla, New York
To investigate the effects ofprotracted low-density lipoprotein (LDL) exposure on endothelial cell (EC) epoxyeicosatrienoic acid (EET) generation, human umbilical vein ECs were incubated in atherogenic concentrations of LDL (240 mg cholesterolper deciliter) (LDL-EC). After 4 days' incubation with LDL, EC were stimulated with human thrombin in the presence of 1-['4CJ-arachidonic acid. Substantially more EETproducts were generated by LDL-ECs than by cells not exposed to high levels of LDL (C-EC). Thrombin stimulation caused LDL-EC to producefive- to eightfold more in 14,15-EET, 11, 12-EET, 8,9-EET, and 5,6-EET, with 14,15-EET as the major product. This is the first demonstration, to date, that EETs can be induced in EC. Metapyrone (SKF-525A) markedly inhibited EC EET generation, indicating a role for the cytochrome P-450 enzyme system in human EC arachidonic acid metabolism. One EET product, 14,15EET, has been found to be chemotactic and to promote adhesion of U93 7 cells, a human monocytic lymphoma cell line, to EC. Thus, protracted exposure to atherogenic LDL concentrations increases the generation of chemotactic and adhesion factors (ie, 14,15-EET) after thrombin stimulation, possibly through the cytochrome P-450 enzyme system. (AmjPathol 1990, 136:1383-1391)
Elevated plasma low-density lipoprotein (LDL) concentrations are associated with the premature atherosclerosis1; however, the pathophysiology remains unclear. Low-density lipoprotein may affect endothelial cell (EC) function through alteration of eicosanoid production.23 Therefore, to test LDL's effect on EC eicosanoid generation, ECs
were exposed for 4 days to LDL in concentrations associated with premature atherosclerosis. Low-density lipoprotein's effect in altering EC eicosanoid generation, to date, is largely associated with increases in prostacyclin.36 Because prostacyclin is noted to protect EC function, it is unclear how LDL eicosanoid alterations could promote atherogenesis. However, ECs appear to generate a series of arachidonic acid epoxides that increase vascular permeability7 and intracellular Ca++ mobilization.a These varied and potent effects of epoxyeicosatrienoic acid (EET) products may impact on vascular homeostasis and promote plaque formation. Investigation concerning LDL's effects on EC EET generation may indicate a mechanism(s) by which LDL adversely affects the vasculature. Atherogenic LDL concentrations alter EC arachidonic acid metabolism without causing EC toxicity.3 Based on these findings, protracted LDL exposure could also alter other EC eicosanoids. We therefore examined these cells for hydroxy-eicosanoids (HETE) and EETs, known to be mediators of inflammation.79-1' To test this notion, EC were incubated in high concentrations of LDL for 4 days. In this study, we report that LDL-exposed EC, after thrombin stimulation, generate a series of EET products in substantially greater quantities than do control ECs.
Materials and Methods Materials Human plasma was obtained from Hudson Valley Blood Services (Valhalla, NY). The materials used in this study were purchased as follows: Primeria T-75cm2 flasks from Falcon (Becton Dickinson and Co., Lincoln Park, NJ); reverse-phase purification columns (C18-ODS) from Supleco, Inc. (Bellefonte, PA); high-pressure liquid chroSupported by NIH grants HL 33742 and HL 21429. Accepted for publication February 5, 1990. Address reprint requests to Kirkwood A. Pritchard, Jr., PhD, NYMC, Department of Medicine, Vosburg 302, Valhalla, NY 10595.
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matography (HPLC) equipment and supplies from Beckman Altex Div. (Summerset, NJ); Na2EDTA, KBr, NaCI, KCI, CaCl2, 1 N HCI, NH4OH, petroleum ether, HPLC grade methanol from Fisher Scientific (Springfield, NJ); butylated hydroxytoluene (BHT) and methyl formate from Eastman Kodak Chemical Co. (Rochester, NY); arachidonic acid from NuChek Prep (Elsian, MN); eicosanoid standards, 12HHT, 14,15-EET, 11,12-EET, 8,9-EET, 5,6EET, PGB2, and 8,15-DiHETE from Biomol (Plymouth Meeting, PA); nordihydroguaiaretic acid (NDGA), human thrombin, glucose, and endotoxin assay kit 210 from Sigma Chemical Co. (St. Louis, MO); Spectrapor dialysis tubing, 30,000 MW cutoff (29 mm) from Thomas Scientific (Swedesboro, NJ); eicosatetrayenoic acid (ETYA) from Hoffman La Roche (Nutley, NJ); and 1 -[14C]-arachidonic acid (59.7 mCi/mmol) from Amersham (Rockville, IL). Metapyrone (SKF-525A) was provided by M. Schwartzman. BW755c was provided by M. Gerritsen. Arachidonic acid metabolites were separated on an Ultrasphere 4.6X 250-mm reverse-phase analytical column (C18-ODS) Altex, Beckman Instruments, Inc. (Fullerton, CA). The following tissue culture items were obtained from Gibco (Grand Island, NY); M199, HEPES, penicillin, streptomycin, amphotercin, and heparin.
Preparation of LDL Low-density lipoprotein (1.019 < d < 1.063) was isolated from human plasma containing 0.01% Na2 ethylenediaminetetraacetic acid (EDTA) and 20 ,mol/l (micromolar) BHT, as previously described, with the following modifications.12 Lipoprotein-deficient serum (LPDS, d > 1.25 g/ ml) was prepared by adjusting the density of human serum to 1.25 g/ml with solid KBr, followed by ultracentrifugation at 200,000g for 20 hours.'2 All isolated fractions were sterile filtered (0.22 ;i) into sterile dialysis tubing13 and dialyzed under sterile conditions (four changes, 2000 ml each, of saline-EDTA-BHT [150 mmol/l NaCL, 0.01% EDTA, and 20 ,tmol/I BHT], and one change of Ml 99 containing 18 mmol/l HEPES [2000 ml]). Low-density lipoprotein and LPDS fractions were removed from dialysis tubing, immediately sterile filtered through a 0.22-is syringe filter (Nalge Co., Rochester, NY) and stored at 40C until added to Ml 99 media to obtain the desired LDL concentration. Low-density lipoprotein was routinely tested for oxidation by the thiobarbituric acid-reactive substances test12 and endotoxin by the amebocyte lysate assay.13
Cell Culture Human umbilical vein endothelial cells (EC) were obtained as previously described.3 Endothelial cells were cultured
in M199 with Earl's salts, 25 mmol/l (millimolar) HEPES, 25 mmol/l NaHCO3 (Gibco, Grand Island, NY), supplemented with 20% human serum, 250 ,ug endothelial cell growth factor (ECGF) per milliliter,14 and 90 ltg heparin per milliliter (Gibco, Grand Island, NY), pH 7.45. Experiments were performed on third-passage EC in T75 cm2 flasks coated with 250 mg/ml plasma concentrate from human plasma. Endothelial cells were maintained at confluence for 2 to 3 days before changing to experimental media.
Incubation of EC with Atherogenic Levels of LDL Endothelial cells were cultured for 4 days with media changes every 48 hours. Control EC (C-EC) were cultured in M199 media, pH 7.45, supplemented with 2.5% human serum, 17.5% LPDS, 100 U/ml penicillin, 100 ,ug/ml streptomycin, 0.25 Ag/ml amphotericin, and 18 mmol/l HEPES (EC-Media). Experimental EC (LDL-EC) were cultured in EC-media containing LDL at a final concentration of 1500 ,ug protein per milliliter (240 mg LDL cholesterol per deciliter). The LDL content in control media was determined to be less than 20 Ag protein per milliliter (
Atherogenic Concentrations of Low-density Lipoprotein Enhance Endothelial Cell Generation of Epoxyeicosatrienoic Acid Products
Kirkwood A. Pritchard, Jr.,*t P. Y.-K. Wong,t and M. B. Stemerman* From the Departments ofMedicine* and Physiology,t New York Medical College, Valhalla, New York
To investigate the effects ofprotracted low-density lipoprotein (LDL) exposure on endothelial cell (EC) epoxyeicosatrienoic acid (EET) generation, human umbilical vein ECs were incubated in atherogenic concentrations of LDL (240 mg cholesterolper deciliter) (LDL-EC). After 4 days' incubation with LDL, EC were stimulated with human thrombin in the presence of 1-['4CJ-arachidonic acid. Substantially more EETproducts were generated by LDL-ECs than by cells not exposed to high levels of LDL (C-EC). Thrombin stimulation caused LDL-EC to producefive- to eightfold more in 14,15-EET, 11, 12-EET, 8,9-EET, and 5,6-EET, with 14,15-EET as the major product. This is the first demonstration, to date, that EETs can be induced in EC. Metapyrone (SKF-525A) markedly inhibited EC EET generation, indicating a role for the cytochrome P-450 enzyme system in human EC arachidonic acid metabolism. One EET product, 14,15EET, has been found to be chemotactic and to promote adhesion of U93 7 cells, a human monocytic lymphoma cell line, to EC. Thus, protracted exposure to atherogenic LDL concentrations increases the generation of chemotactic and adhesion factors (ie, 14,15-EET) after thrombin stimulation, possibly through the cytochrome P-450 enzyme system. (AmjPathol 1990, 136:1383-1391)
Elevated plasma low-density lipoprotein (LDL) concentrations are associated with the premature atherosclerosis1; however, the pathophysiology remains unclear. Low-density lipoprotein may affect endothelial cell (EC) function through alteration of eicosanoid production.23 Therefore, to test LDL's effect on EC eicosanoid generation, ECs
were exposed for 4 days to LDL in concentrations associated with premature atherosclerosis. Low-density lipoprotein's effect in altering EC eicosanoid generation, to date, is largely associated with increases in prostacyclin.36 Because prostacyclin is noted to protect EC function, it is unclear how LDL eicosanoid alterations could promote atherogenesis. However, ECs appear to generate a series of arachidonic acid epoxides that increase vascular permeability7 and intracellular Ca++ mobilization.a These varied and potent effects of epoxyeicosatrienoic acid (EET) products may impact on vascular homeostasis and promote plaque formation. Investigation concerning LDL's effects on EC EET generation may indicate a mechanism(s) by which LDL adversely affects the vasculature. Atherogenic LDL concentrations alter EC arachidonic acid metabolism without causing EC toxicity.3 Based on these findings, protracted LDL exposure could also alter other EC eicosanoids. We therefore examined these cells for hydroxy-eicosanoids (HETE) and EETs, known to be mediators of inflammation.79-1' To test this notion, EC were incubated in high concentrations of LDL for 4 days. In this study, we report that LDL-exposed EC, after thrombin stimulation, generate a series of EET products in substantially greater quantities than do control ECs.
Materials and Methods Materials Human plasma was obtained from Hudson Valley Blood Services (Valhalla, NY). The materials used in this study were purchased as follows: Primeria T-75cm2 flasks from Falcon (Becton Dickinson and Co., Lincoln Park, NJ); reverse-phase purification columns (C18-ODS) from Supleco, Inc. (Bellefonte, PA); high-pressure liquid chroSupported by NIH grants HL 33742 and HL 21429. Accepted for publication February 5, 1990. Address reprint requests to Kirkwood A. Pritchard, Jr., PhD, NYMC, Department of Medicine, Vosburg 302, Valhalla, NY 10595.
1383
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Pritchard, Wong, and Stemerman
AJPJune 1990, Vol. 136, No. 6
matography (HPLC) equipment and supplies from Beckman Altex Div. (Summerset, NJ); Na2EDTA, KBr, NaCI, KCI, CaCl2, 1 N HCI, NH4OH, petroleum ether, HPLC grade methanol from Fisher Scientific (Springfield, NJ); butylated hydroxytoluene (BHT) and methyl formate from Eastman Kodak Chemical Co. (Rochester, NY); arachidonic acid from NuChek Prep (Elsian, MN); eicosanoid standards, 12HHT, 14,15-EET, 11,12-EET, 8,9-EET, 5,6EET, PGB2, and 8,15-DiHETE from Biomol (Plymouth Meeting, PA); nordihydroguaiaretic acid (NDGA), human thrombin, glucose, and endotoxin assay kit 210 from Sigma Chemical Co. (St. Louis, MO); Spectrapor dialysis tubing, 30,000 MW cutoff (29 mm) from Thomas Scientific (Swedesboro, NJ); eicosatetrayenoic acid (ETYA) from Hoffman La Roche (Nutley, NJ); and 1 -[14C]-arachidonic acid (59.7 mCi/mmol) from Amersham (Rockville, IL). Metapyrone (SKF-525A) was provided by M. Schwartzman. BW755c was provided by M. Gerritsen. Arachidonic acid metabolites were separated on an Ultrasphere 4.6X 250-mm reverse-phase analytical column (C18-ODS) Altex, Beckman Instruments, Inc. (Fullerton, CA). The following tissue culture items were obtained from Gibco (Grand Island, NY); M199, HEPES, penicillin, streptomycin, amphotercin, and heparin.
Preparation of LDL Low-density lipoprotein (1.019 < d < 1.063) was isolated from human plasma containing 0.01% Na2 ethylenediaminetetraacetic acid (EDTA) and 20 ,mol/l (micromolar) BHT, as previously described, with the following modifications.12 Lipoprotein-deficient serum (LPDS, d > 1.25 g/ ml) was prepared by adjusting the density of human serum to 1.25 g/ml with solid KBr, followed by ultracentrifugation at 200,000g for 20 hours.'2 All isolated fractions were sterile filtered (0.22 ;i) into sterile dialysis tubing13 and dialyzed under sterile conditions (four changes, 2000 ml each, of saline-EDTA-BHT [150 mmol/l NaCL, 0.01% EDTA, and 20 ,tmol/I BHT], and one change of Ml 99 containing 18 mmol/l HEPES [2000 ml]). Low-density lipoprotein and LPDS fractions were removed from dialysis tubing, immediately sterile filtered through a 0.22-is syringe filter (Nalge Co., Rochester, NY) and stored at 40C until added to Ml 99 media to obtain the desired LDL concentration. Low-density lipoprotein was routinely tested for oxidation by the thiobarbituric acid-reactive substances test12 and endotoxin by the amebocyte lysate assay.13
Cell Culture Human umbilical vein endothelial cells (EC) were obtained as previously described.3 Endothelial cells were cultured
in M199 with Earl's salts, 25 mmol/l (millimolar) HEPES, 25 mmol/l NaHCO3 (Gibco, Grand Island, NY), supplemented with 20% human serum, 250 ,ug endothelial cell growth factor (ECGF) per milliliter,14 and 90 ltg heparin per milliliter (Gibco, Grand Island, NY), pH 7.45. Experiments were performed on third-passage EC in T75 cm2 flasks coated with 250 mg/ml plasma concentrate from human plasma. Endothelial cells were maintained at confluence for 2 to 3 days before changing to experimental media.
Incubation of EC with Atherogenic Levels of LDL Endothelial cells were cultured for 4 days with media changes every 48 hours. Control EC (C-EC) were cultured in M199 media, pH 7.45, supplemented with 2.5% human serum, 17.5% LPDS, 100 U/ml penicillin, 100 ,ug/ml streptomycin, 0.25 Ag/ml amphotericin, and 18 mmol/l HEPES (EC-Media). Experimental EC (LDL-EC) were cultured in EC-media containing LDL at a final concentration of 1500 ,ug protein per milliliter (240 mg LDL cholesterol per deciliter). The LDL content in control media was determined to be less than 20 Ag protein per milliliter (
