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(Circulation Research. 1995;76:958-962.)
© 1995 American Heart Association, Inc.

Articles

Low Concentration of Oxidized Low-Density Lipoprotein and Lysophosphatidylcholine Upregulate Constitutive Nitric Oxide Synthase mRNA Expression in Bovine Aortic Endothelial Cells

Ken-ichi Hirata, Nobuhiko Miki, Yuichi Kuroda, Tsuyoshi Sakoda, Seinosuke Kawashima, Mitsuhiro Yokoyama

From The First Department of Internal Medicine, Kobe (Japan) University School of Medicine.

Correspondence to Ken-ichi Hirata, MD, The First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Abstract Endothelium-dependent relaxation is markedly reduced in atherosclerotic arteries. Recently, the endothelium-dependent relaxing factor has been identified as nitric oxide (NO). We used RNase protection assay and immunoblotting to elucidate the effect of atherogenic lipoprotein on the expression of constitutive NO synthase (cNOS) mRNA and protein levels in bovine aortic endothelial cells. Twenty-four-hour exposure to a low concentration of oxidized low-density lipoprotein (10 µg protein/mL) upregulated cNOS mRNA levels (2.4±0.4-fold, P<.01). However, native low-density lipoprotein and high-density lipoprotein did not have any effect on cNOS mRNA levels. Furthermore, 5 µg/mL of lysophosphatidylcholine (LPC) also upregulated cNOS mRNA levels (2.6±0.5-fold, P<.01) at 8 hours. This action of LPC was abolished with cycloheximide but not with staurosporine. We concluded that atherogenic lipoproteins upregulate cNOS mRNA and protein levels in bovine aortic endothelial cells. This observation supports the hypothesis that an impairment of endothelium-dependent vasodilatation in atherosclerotic vessels may not be due to a decrease in cNOS expression. Moreover, the LPC action on cNOS mRNA levels requires new protein synthesis.

Key Words: oxidized low-density lipoprotein • nitric oxide synthase mRNA • lysophosphatidylcholine

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The endothelium-derived relaxing factor (EDRF) has been identified as either nitric oxide (NO) or a closely related molecule synthesized from L-arginine.¹ NO produced from vascular endothelial cells plays an important role in the regulation of tissue perfusion. Atherosclerosis impairs endothelium-dependent vasodilatation both in animal models^{2 3 4 5} and in human coronary arteries^{6 7} and thereby may predispose to vasoconstriction and arterial spasm. In the atherosclerotic process, oxidized low-density lipoprotein (ox-LDL) is speculated to be an important factor,^{8 9} and it can cause foam cell formation via scavenger receptor uptake in macrophages.¹⁰ Moreover, ox-LDL is detected in atherosclerotic lesions of rabbits and humans.^{11 12} One of the properties of ox-LDL is its increased lysophosphatidylcholine (LPC) content.¹³ Recently, we¹⁴ and another group¹⁵ have demonstrated that ox-LDL inhibits endothelium-dependent arterial relaxation through increased LPC. However, its precise mechanism has yet to be elucidated.

Recently, the constitutive NO synthase (cNOS) cDNA has been cloned from bovine aortic endothelial cells (BAECs) as well as human ones.^{16 17 18} In the present study, we performed RNase protection assays and immunoblotting to investigate the effect of various lipoproteins on cNOS mRNA and protein levels in BAECs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Culture of Endothelial Cells
BAECs were obtained by scraping with a knife the internal surface of aortas excised from freshly slaughtered cows, as described previously.^{19 20} The cells were maintained in DMEM (Flow Laboratories, Inc) with fetal calf serum (FCS, 15% [vol/vol], Boehringer Mannheim Yamanouchi Co). Cells were seeded to 25-cm² flasks and incubated at 37°C under an atmosphere of 5% CO₂/95% air. The cells were separated with 0.25% trypsin-EDTA (Sigma Chemical Co). Cultures used in the present study were from the 5th to 11th passages. The endothelial cells were identified by their typical cobblestone appearance, indicating positive immunofluorescence for anti–factor VIII antibody.²¹ Before stimulation with various lipoproteins and lipids, culture medium was exchanged with FCS-free 0.02% bovine serum albumin containing DMEM for 12 hours; on stimulation, it was further exchanged with FCS and bovine serum albumin–free DMEM.

Preparation of Lipoproteins and Phospholipids
Low-density lipoprotein (LDL; density, 1.020 to 1.063 g/mL) and high-density lipoprotein (HDL; density, 1.063 to 1.210 g/mL) were isolated by sequential ultracentrifugation from freshly harvested normal human plasma collected in EDTA (1 mg/mL, Sigma).²² The quantity of protein was determined by the method described by Bradford,²³ who used bovine serum albumin as a standard. Native LDL (N-LDL, 1 mg protein/mL) was oxidatively modified by exposure to 5 µmol/L copper in phosphate-buffered saline without calcium and magnesium for 24 hours at 37°C according to the method of Quinn et al.²⁴ Oxidative modification of LDL was assessed by thiobarbituric acid–reactive substances (TBARS) and agarose gel electrophoresis. TBARS in N-LDL and ox-LDL were 4 and 58 nmol malondialdehyde equivalents per milligram of LDL protein, and mobility of ox-LDL was 2.7 times that of native LDL. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (PC) was purchased from Avanti Polar Lipids Inc. 1-Palmitoyl-LPC was purchased from Sigma. Phospholipids were dissolved in a mixture (1:1 [vol/vol]) of methanol and chloroform. Appropriate aliquots of the solution were dried with a stream of N₂ gas, followed by sonication for 3 minutes in distilled water before use, and then they were diluted in each solution.

RNase Protection Assay for Detection of cNOS mRNA
Total RNA was isolated from BAECs by the guanidine method.²⁵ RNase protection assay was carried out as described previously.^{26 27} To detect the cNOS mRNA, the region of BAEC cNOS cDNA (from -15 to 225) was amplified with forward primer Np-1 (5'-ATAGAATTCACCAGCACCTTTGGGAATGGCGAT) and reverse primer Cp-1 (5'-ATAGAATTCGGATTCACTGTCTGTGTTGCTGGACTCCTT), which contained the N-terminal myristoylation site. To prepare labeled RNA probes, the 240-bp EcoRI-digested amplified DNA was subcloned into the EcoRI-digested pGEM-4Z plasmid (Promega). This plasmid was linearized with HindIII and transcribed with SP6 RNA polymerase (Promega). To detect the GAPDH mRNA for estimation of applied total RNA, the 124-bp Apa I–Alu I fragment of GAPDH cDNA²⁸ was subcloned into the Apa I–Sma I–digested pBluescriptII plasmid. This plasmid was linearized with BamHI and transcribed with T7 RNA polymerase (Promega). Total RNA (5 µg) was hybridized with ³²P-labeled RNA probes overnight at 54°C in 80% formamide hybridization buffer, followed by digestion with RNase A and RNase T1 (Sigma) at 37°C for 1 hour. The protected fragments were separated in 6% polyacrylamide-urea denaturing gel by electrophoresis and then were exposed to Imaging Plate (BASIII, FUJI XEROX). Relative intensities of signals were determined by Autoimage Analyzer (BAS2000, FUJI XEROX). Relative intensity of cNOS mRNA expression was adjusted with the intensity of GAPDH mRNA, because expression of GAPDH mRNA was not altered by stimulation of phospholipids and lipoproteins for 24 hours.

Immunoblotting of Endothelial cNOS
Immunoblotting was carried out with murine monoclonal antibody against BAEC cNOS (kindly provided by Jennifer Pollock, Abbott Laboratories).²⁹ Signals were detected by using the ECL detection system (Amersham Corp) on standard X-ray film and quantified by densitometry (Chromatoscanner CS-930, Shimadzu).

Determinations
Results are expressed as mean±SEM of four independent experiments. Statistical evaluation of the data was performed by Student's t test for unpaired observations. When more than two groups were compared, the significance of the difference between group means was analyzed by one-way ANOVA and the Bonferroni test for samples. Values were considered to be statistically different at P<.01 and P<.05.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
BAECs were stimulated with N-LDL and ox-LDL for various periods, and cNOS mRNA levels were assessed by RNase protection assay. The RNase protection assay demonstrated that a low concentration of ox-LDL (10 µg protein/mL) increased cNOS mRNA levels (2.4±0.4-fold, P<.01) at 24 hours, whereas a high concentration of ox-LDL (100 µg protein/mL) decreased cNOS mRNA levels at 24 hours. N-LDL (10 µg protein/mL) did not affect cNOS mRNA levels (Fig 1). Copper, used for oxidative modification of LDL, did not affect cNOS mRNA levels (data not shown). Fig 2 illustrates the changes in cNOS mRNA levels at 24 hours with various concentrations of N-LDL, ox-LDL, and HDL. The lower concentration of ox-LDL (1 µg protein/mL) slightly upregulated cNOS mRNA levels, whereas 10 µg protein/mL of ox-LDL had a more marked effect. A higher concentration (>25 µg protein/mL) of ox-LDL induced cytoplasmic vacuolation and a decrease in GAPDH mRNA levels, probably because of the cytotoxic effect. However, N-LDL and HDL did not affect cNOS mRNA levels in any concentrations tested. A recent study demonstrated that diet-induced hypercholesterolemia resulted in upregulation of the synthesis of NO from vascular endothelium and that impaired vasodilatation activity of EDRF by cholesterol feeding may result from loss of an incorporation of NO into a more potent parent compound or from accelerated degradation of EDRF.³⁰ Furthermore, in situ hybridization revealed that the cNOS mRNA expression was normally observed in the endothelial cells overlying human aortic fatty streaks.³¹ These findings suggest that loss of EDRF activity associated with atherosclerosis is not due to an alteration of endothelial cNOS expression.

View larger version (46K): [in this window] [in a new window]   Figure 1. Time course of constitutive nitric oxide synthase (cNOS) mRNA levels by native low-density lipoprotein (N-LDL) and oxidized low-density lipoprotein (ox-LDL). A, Autoradiographs showing bovine aortic endothelial cells exposed to 10 µg protein/mL of N-LDL ( in panel B), 10 µg protein/mL ( in panel B), and 100 µg protein/mL ( in panel B) of ox-LDL. Total RNA (5 µg) was analyzed with RNase protection as described in "Materials and Methods." tRNA extracted from yeast was used as a negative control. B, Graph showing the corrected density for each time point divided by that of the nonstimulated control and plotted as a fold increase (mean±SEM, n=4, **P<.01 compared with the control value).

View larger version (11K): [in this window] [in a new window]   Figure 2. Graph showing dose-response relation of constitutive nitric oxide synthase (cNOS) mRNA levels by native low-density lipoprotein (N-LDL), oxidized low-density lipoprotein (ox-LDL), and high-density lipoprotein (HDL). Bovine aortic endothelial cells were stimulated with various concentrations of N-LDL (), ox-LDL (), and HDL (). Total RNA (5 µg) was analyzed with RNase protection as described in "Materials and Methods." The corrected density for each point was divided by that of the nonstimulated control and was plotted as a fold increase of nonstimulated control (mean±SEM, n=3).

We have demonstrated that LPC extracted from ox-LDL inhibits endothelium-dependent relaxation and that inhibition of endothelium-dependent relaxation by ox-LDL is due to its increased LPC content.¹⁴ Fig 3 shows the time course of changes in cNOS mRNA levels in BAECs with 5 µg/mL concentration of LPC and PC. LPC upregulated cNOS mRNA levels at 8 hours (2.6±0.5-fold, P<.01), whereas PC had no effect. Dose-dependent changes in the expression of cNOS mRNA in BAECs induced by LPC indicated that upregulation of cNOS mRNA levels by LPC peaked at 5 µg/mL. A higher concentration (>25 µg/mL) of LPC induced cytotoxic changes, such as cytoplasmic vacuolization and a decrease of GAPDH mRNA expression, similar to those found with higher concentrations of ox-LDL (data not shown). Immunoblotting revealed that LPC (5 µg/mL) and ox-LDL (10 µg protein/mL) increased cNOS protein associated with cNOS mRNA upregulation in BAECs (Fig 4), whereas N-LDL and PC had no effect. Ox-LDL and LPC dose-dependently increased cNOS protein levels (Fig 5). Higher concentrations of ox-LDL (>25 µg protein/mL) and LPC (>25 µg/mL) decreased cNOS protein because of the cytotoxic effect. The concentrations of LPC used in the present study corresponded to those that produced inhibitory effects on endothelium-dependent relaxations in rabbit aorta, on bradykinin-induced phosphoinositide hydrolysis, and on calcium transients.²⁰ Approximately 40% of PC was converted to LPC during oxidative modification of LDL. The estimated contents of LPC in 1 mg lipoprotein were 20 µg in N-LDL and 220 µg in ox-LDL.¹⁴ However, the concentration of ox-LDL in experiments that produced inhibition of cNOS mRNA was lower than the concentration of ox-LDL in experiments that produced inhibitory effects on endothelium-dependent relaxation in isolated rabbit aorta because of its cytotoxic effect on cultured BAECs. This difference in concentrations is likely to show the difference of the time course of changes in cNOS mRNA levels between ox-LDL and LPC actions.

View larger version (31K): [in this window] [in a new window]   Figure 3. Time course of constitutive nitric oxide synthase (cNOS) mRNA levels by lysophosphatidylcholine (LPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (PC), and 12-O-tetradecanoylphorbol 13-acetate (TPA). A, Autoradiographs showing bovine aortic endothelial cells exposed to 5 µg/mL of LPC ( in panel B), 5 µg/mL of PC ( in panel B), and 25 nmol/L of TPA ( in panel B). Total RNA (5 µg) was analyzed with RNase protection as described in "Materials and Methods." B, Graph showing the corrected density for each time point divided by that of the nonstimulated control and plotted as a fold increase of nonstimulated control (mean±SEM, n=4, *P<.05 and **P<.01 compared with the nonstimulated control value).

View larger version (9K): [in this window] [in a new window]   Figure 4. The effect of lysophosphatidylcholine (LPC) and oxidized low-density lipoprotein (ox-LDL) on expression of constitutive nitric oxide synthase (cNOS) protein. A, Immunoblotting of cNOS protein in bovine aortic endothelial cells (BAECs) exposed to LPC and ox-LDL. Lanes are as follows: 1, 20 ng of endothelial cNOS purified from BAECs; 2, 50 µg of homogenate of BAECs without stimulation (control); 3, 50 µg of homogenate of BAECs stimulated with 5 µg/mL of LPC for 12 hours; and 4, homogenate of BAECs stimulated with 10 µg/mL of ox-LDL for 24 hours. B, Graph showing time course of cNOS protein levels stimulated by 5 µg/mL of LPC (), 5 µg/mL of 1-palmitoyl-2-oleoyl-phosphatidylcholine (PC, ), 10 µg/mL of ox-LDL (), and 10 µg/mL of native low-density lipoprotein (). The corrected density for each time point was divided by that of the nonstimulated control and was plotted as a fold increase of nonstimulated control (mean±SEM, n=4, *P<.01 compared with the nonstimulated control value).

View larger version (18K): [in this window] [in a new window]   Figure 5. Graphs showing dose-response relation of constitutive nitric oxide synthase (cNOS) protein levels by oxidized low-density lipoprotein (ox-LDL) and lysophosphatidylcholine (LPC). Bovine aortic endothelial cells were stimulated with various concentrations of ox-LDL () or native low-density lipoprotein () for 24 hours (A) and LPC () or 1-palmitoyl-2-oleoyl-phosphatidylcholine () for 12 hours (B). The corrected density for each point was divided by that of the nonstimulated control and was plotted as a fold increase of nonstimulated control (mean±SEM, n=4, *P<.01 compared with the nonstimulated control value).

Lysophospholipids influence several enzyme systems, including adenylate cyclase, guanylate cyclase,³² and protein kinase C (PKC).³³ Oishi et al³³ have demonstrated that LPC stimulated PKC purified from porcine brain in vitro. Moreover, prolonged exposure to phorbol 12,13-dibutyrate, an activator of PKC, inhibited endothelium-dependent relaxations evoked by histamine in the pig pulmonary artery and by acetylcholine or substance P in the rabbit aorta.^{34 35} These results suggest that PKC activation suppressed receptor-mediated processes linked to the synthesis of EDRF. To determine whether upregulation of cNOS mRNA levels by LPC depends on PKC activation, BAECs were stimulated with 12-O-tetradecanoylphorbol 13-acetate (TPA). Fig 3 shows that TPA upregulated cNOS mRNA levels in a manner similar to LPC. Upregulation of cNOS mRNA by TPA was completely abolished by staurosporine (25 µg/mL). To clarify the mechanism of LPC action on cNOS mRNA expression, we tried to block LPC action with staurosporine (25 µg/mL) and cycloheximide (CHX, 10 µg/mL) as shown in Fig 6. Although the upregulation of cNOS mRNA levels with LPC was not inhibited by staurosporine, CHX blocked the LPC action completely. LPC-induced intercellular adhesion molecule-1 expression in human umbilical vein endothelial cells was not inhibited with staurosporine treatment.³⁶ These data suggest that the potential mechanisms of endothelial cell activation with LPC appear to be multiple and complex and that the mechanism of cNOS mRNA upregulation with LPC did not depend on PKC activation but required new protein synthesis. Further characterization of cNOS mRNA upregulation should be investigated in future studies.

View larger version (35K): [in this window] [in a new window]   Figure 6. Bar graph showing the effect of staurosporine (Stauro) and cycloheximide (CHX) on lysophosphatidylcholine (LPC) action. Bovine aortic endothelial cells (BAECs) were treated with CHX or Stauro for 1 hour before addition of LPC (5 µg/mL). BAECs were harvested for RNA extraction 8 hours after exposure to LPC. Total RNA (5 µg) was analyzed with RNase protection as described in "Materials and Methods." The corrected density was divided by that of the nonstimulated control and was plotted as a fold increase of nonstimulated control. Each bar represents the mean±SEM for four experiments. **P<.01 compared with each indicated value.

In summary, the present study demonstrates that ox-LDL and LPC upregulate cNOS mRNA and protein expression in BAECs. Furthemore, the mechanism of LPC action on cNOS mRNA levels does not depend on PKC activation but requires new protein synthesis. However, it is necessary to perform further experiments to elucidate the signaling pathway of cNOS mRNA expression by atherogenic lipoprotein and LPC in BAECs.

	Acknowledgments

 
This study was supported by grants-in-aid for scientific research (No. 05670615) from the Ministry of Education, Science, and Culture and for research on cardiovascular disease (1993) from the Ministry of Health and Welfare of Japan, a grant from Japan Cardiovascular Research Foundation, a grant from Research for Molecular Cardiology, and a grant from Kanae Foundation of Research for New Medicine. Bovine aortic endothelial cell NOS cDNA was kindly donated by Prof David G. Harrison (Cardiology Division, Emory University School of Medicine). We thank Dr Jennifer S. Pollock for providing the monoclonal antibody against bovine aortic endothelial cNOS. We are grateful to Seiko Tsutsui for her skillful technical assistance.

Received September 26, 1994; accepted February 16, 1995.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 
1. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;333:664-666. [Medline] [Order article via Infotrieve]

2. Chappell SP, Lewis MJ, Henderson AH. Effect of lipid feeding on endothelial-dependent vascular relaxation in rabbit aorta preparation. Cardiovasc Res. 1987;21:34-38. [Medline] [Order article via Infotrieve]

3. Freiman PC, Mitchell GC, Heistad DD, Armstrong ML, Harrison DG. Atherosclerosis impairs endothelial-dependent vascular relaxation to acetylcholine and thrombin in primates. Circ Res. 1986;58:783-789. [Abstract/Free Full Text]

4. Verbeuren TJ, Jordaens FH, Zonnekeyn LL, CeVan H, Coene MC, Herman AG. Effect of hypercholesterolemia on vascular reactivity in the rabbit, I: endothelial-dependent and endothelial-independent contractions and relaxation in isolated arteries of control and hypercholesterolemic rabbits. Circ Res. 1986;58:552-564. [Abstract/Free Full Text]

5. Hirata K, Akita H, Yokoyama M, Watanabe Y. Impaired vasodilatory response to atrial natriuretic peptide during atherosclerosis progression. Arterioscler Thromb. 1992;12:99-105. [Abstract/Free Full Text]

6. Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelial-dependent relaxation and cyclic guanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest. 1987;79:170-174.

7. Forstermann U, Mugge A, Acheid U, Haverich A, Frolich JC. Selective attenuation of endothelial-mediated vasodilation atherosclerotic human coronary arteries. Circ Res. 1988;62:185-190. [Abstract/Free Full Text]

8. Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1987;84:5928-5931. [Abstract/Free Full Text]

9. Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1987;84:7725-7729. [Abstract/Free Full Text]

10. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]

11. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.

12. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis. 1990;10:336-349. [Abstract/Free Full Text]

13. Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci U S A. 1984;81:3883-3887. [Abstract/Free Full Text]

14. Yokoyama M, Hirata K, Miyake R, Akita H, Ishikawa Y, Fukuzaki H. Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein. Biochem Biophys Res Commun. 1990;16:301-308.

15. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. Impairment of endothelial-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990;344:160-162. [Medline] [Order article via Infotrieve]

16. Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, Murphy TJ. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest. 1992;90:2092-2096.

17. Lamas S, Marsden PA, Li GK, Tempst P, Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci U S A. 1992;89:6348-6352. [Abstract/Free Full Text]

18. Janssens SP, Shimouchi A, Quertermous T, Bloch DB, Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem. 1992;267:14519-14522.[Abstract/Free Full Text]

19. Hirata K, Akita H, Yokoyama M. Oxidized low density lipoprotein inhibits bradykinin-induced phosphoinositide hydrolysis in cultured bovine aortic endothelial cells. FEBS Lett. 1991;287:181-184. [Medline] [Order article via Infotrieve]

20. Inoue N, Hirata K, Yamada M, Hamamori Y, Matsuda Y, Akita H, Yokoyama M. Lysophosphatidylcholine inhibits bradykinin-induced phosphoinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells. Circ Res. 1992;71:1410-1421. [Abstract/Free Full Text]

21. Ryan US, Clements E, Habliston D, Ryan JW. Isolation and culture of pulmonary artery endothelial cells. Tissue Cell. 1978;10:535-554. [Medline] [Order article via Infotrieve]

22. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;43:1345-1353.

23. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]

24. Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proc Natl Acad Sci U S A. 1988;85:2805-2809. [Abstract/Free Full Text]

25. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

26. Miki N, Hatano M, Wakita K, Imoto S, Nishikawa S, Nishikawa S-I, Tokuhisa T. Role of I-A molecules in early stages of B cell maturation. J Immunol. 1992;149:801-807. [Abstract]

27. Miki N, Tokuhisa T. Inhibition of I-A molecule in anti-sense transgenic mice. Biochem Biophys Res Commun. 1992;186:832-837. [Medline] [Order article via Infotrieve]

28. Fort P, Marty L, Piechaczyk M, Sabrouty SE, Dani C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate dehydrogenase multigenic family. Nucleic Acids Res. 1985;13:1431-1442. [Abstract/Free Full Text]

29. Pollock JS, Nakane M, Buttery LDK, Martines A, Springall D, Polak JM, Förstermann U, Murad F. Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies. Am J Physiol. 1993;265:C1379-C1387. [Abstract/Free Full Text]

30. Minor RL, Myers PR, Guerra R, Bates JN, Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest. 1990;86:2109-2116.

31. Sundell CL, Marsden PA, Subramanian RR, Pollock JS, Harrison DG, Wilcox JN. Nitric oxide synthase is expressed by endothelial cells overlying human atherosclerotic plaques. Circulation. 1993;88(suppl I):I-473. Abstract.

32. Shier WT, Baldwin JH, Nilsen-Hamilton M, Hamilton RT, Thanassi NM. Regulation of guanylate and adenylate cyclase activities by lysolecithin. Proc Natl Acad Sci U S A. 1976;73:1586-1590. [Abstract/Free Full Text]

33. Oishi K, Zheng B, Kuo JF. Inhibition of Na, K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid. J Biol Chem. 1990;265:70-75. [Abstract/Free Full Text]

34. Weinheimer G, Wagner B, Osswald H. Interference of phorbol esters with endothelium-dependent vascular smooth muscle relaxation. Eur J Pharmacol. 1986;130:319-322. [Medline] [Order article via Infotrieve]

35. Lewis MJ, Henderson AH. A phorbol ester inhibits the release of endothelium-derived relaxing factor. Eur J Pharmacol. 1987;137:167-171. [Medline] [Order article via Infotrieve]

36. Kume N, Cybulsky MI, Gimbrone MA Jr. Lysophosphatidylcholine, a component of atherogenic lipoprotein, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138-1144.

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				Y. Rikitake, K.-i. Hirata, T. Yamashita, K. Iwai, S. Kobayashi, H. Itoh, M. Ozaki, J. Ejiri, M. Shiomi, N. Inoue, et al. Expression of G2A, a Receptor for Lysophosphatidylcholine, by Macrophages in Murine, Rabbit, and Human Atherosclerotic Plaques Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2049 - 2053. [Abstract] [Full Text] [PDF]

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				J. L. Mehta and D. Li Identification, regulation and function of a novel lectin-like oxidized low-density lipoprotein receptor J. Am. Coll. Cardiol., May 1, 2002; 39(9): 1429 - 1435. [Abstract] [Full Text] [PDF]

				Y. Rikitake, S. Kawashima, T. Takahashi, T. Ueyama, S. Ishido, N. Inoue, K.-I. Hirata, and M. Yokoyama Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H266 - H274. [Abstract] [Full Text] [PDF]

				M. Morimoto, N. Kume, S. Miyamoto, Y. Ueno, H. Kataoka, M. Minami, K. Hayashida, N. Hashimoto, and T. Kita Lysophosphatidylcholine Induces Early Growth Response Factor-1 Expression and Activates the Core Promoter of PDGF-A Chain in Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 771 - 776. [Abstract] [Full Text] [PDF]

				R. Govers and T. J. Rabelink Cellular regulation of endothelial nitric oxide synthase Am J Physiol Renal Physiol, February 1, 2001; 280(2): F193 - F206. [Abstract] [Full Text] [PDF]

				K. K. Koh Effects of statins on vascular wall: vasomotor function, inflammation, and plaque stability Cardiovasc Res, September 1, 2000; 47(4): 648 - 657. [Abstract] [Full Text] [PDF]

				S. M. Colles and G. M. Chisolm Lysophosphatidylcholine-induced cellular injury in cultured fibroblasts involves oxidative events J. Lipid Res., August 1, 2000; 41(8): 1188 - 1198. [Abstract] [Full Text]

				Q. Jing, S.-M. Xin, W.-B. Zhang, P. Wang, Y.-W. Qin, and G. Pei Lysophosphatidylcholine Activates p38 and p42/44 Mitogen-Activated Protein Kinases in Monocytic THP-1 Cells, but Only p38 Activation Is Involved in Its Stimulated Chemotaxis Circ. Res., July 7, 2000; 87(1): 52 - 59. [Abstract] [Full Text] [PDF]

				K. Yokoyama, F. Shimizu, and M. Setaka Simultaneous separation of lysophospholipids from the total lipid fraction of crude biological samples using two-dimensional thin-layer chromatography J. Lipid Res., January 1, 2000; 41(1): 142 - 147. [Abstract] [Full Text]

				A. Gomez-Munoz, J. S. Martens, and U. P. Steinbrecher Stimulation of Phospholipase D Activity by Oxidized LDL in Mouse Peritoneal Macrophages Arterioscler Thromb Vasc Biol, January 1, 2000; 20(1): 135 - 143. [Abstract] [Full Text] [PDF]

				K. Cieslik, C.-M. Lee, J.-L. Tang, and K. K. Wu Transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Interaction between Casein Kinase 2 and Protein Phosphatase 2A J. Biol. Chem., December 3, 1999; 274(49): 34669 - 34675. [Abstract] [Full Text] [PDF]

				C. A. Hubel Oxidative Stress in the Pathogenesis of Preeclampsia Experimental Biology and Medicine, December 1, 1999; 222(3): 222 - 235. [Abstract] [Full Text]

				C. D. Searles, Y. Miwa, D. G. Harrison, and S. Ramasamy Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase During Cell Growth Circ. Res., October 1, 1999; 85(7): 588 - 595. [Abstract] [Full Text] [PDF]

				A. Papapetropoulos, R. D. Rudic, and W. C Sessa Molecular control of nitric oxide synthases in the cardiovascular system Cardiovasc Res, August 15, 1999; 43(3): 509 - 520. [Abstract] [Full Text] [PDF]

				A. M Dart and J. P.F Chin-Dusting Lipids and the endothelium Cardiovasc Res, August 1, 1999; 43(2): 308 - 322. [Abstract] [Full Text] [PDF]

				T. Ishida, K.-i. Hirata, T. Sakoda, S. Kawashima, H. Akita, and M. Yokoyama Identification of mRNA for 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries Cardiovasc Res, January 1, 1999; 41(1): 267 - 274. [Abstract] [Full Text] [PDF]

				E. Nishi, N. Kume, Y. Ueno, H. Ochi, H. Moriwaki, and T. Kita Lysophosphatidylcholine Enhances Cytokine-Induced Interferon Gamma Expression in Human T Lymphocytes Circ. Res., September 7, 1998; 83(5): 508 - 515. [Abstract] [Full Text] [PDF]

				N. Kume, T. Murase, H. Moriwaki, T. Aoyama, T. Sawamura, T. Masaki, and T. Kita Inducible Expression of Lectin-like Oxidized LDL Receptor-1 in Vascular Endothelial Cells Circ. Res., August 10, 1998; 83(3): 322 - 327. [Abstract] [Full Text] [PDF]

				R. O. Cannon III Role of nitric oxide in cardiovascular disease: focus on the endothelium Clin. Chem., August 1, 1998; 44(8): 1809 - 1819. [Abstract] [Full Text] [PDF]

				U. Förstermann, J.-p. Boissel, and H. Kleinert Expressional control of the `constitutive' isoforms of nitric oxide synthase (NOS I and NOS III) FASEB J, July 1, 1998; 12(10): 773 - 790. [Abstract] [Full Text]

				K. Cieslik, A. Zembowicz, J.-L. Tang, and K. K. Wu Transcriptional Regulation of Endothelial Nitric-oxide Synthase by Lysophosphatidylcholine J. Biol. Chem., June 12, 1998; 273(24): 14885 - 14890. [Abstract] [Full Text] [PDF]

				J. T. Wong, K. Tran, G. N. Pierce, A. C. Chan, K. O, and P. C. Choy Lysophosphatidylcholine Stimulates the Release of Arachidonic Acid in Human Endothelial Cells J. Biol. Chem., March 20, 1998; 273(12): 6830 - 6836. [Abstract] [Full Text] [PDF]

				S. Ramasamy, S. Parthasarathy, and D. G. Harrison Regulation of endothelial nitric oxide synthase gene expression by oxidized linoleic acid J. Lipid Res., February 1, 1998; 39(2): 268 - 276. [Abstract] [Full Text]

				L. Zhao and R. L. Tackett Oxidized Low-Density Lipoprotein Inhibits Acetylcholine-Induced Vasorelaxation and Increases 5-HT-Induced Vasoconstriction in Isolated Human Saphenous Vein J. Pharmacol. Exp. Ther., February 1, 1998; 284(2): 637 - 643. [Abstract] [Full Text]

				F. M. FARACI and D. D. HEISTAD Regulation of the Cerebral Circulation: Role of Endothelium and Potassium Channels Physiol Rev, January 1, 1998; 78(1): 53 - 97. [Abstract] [Full Text] [PDF]

				J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]

				Y. Miwa, K.-i. Hirata, S. Kawashima, H. Akita, and M. Yokoyama Lysophosphatidylcholine Inhibits Receptor-Mediated Ca2+ Mobilization in Intact Endothelial Cells of Rabbit Aorta Arterioscler Thromb Vasc Biol, August 1, 1997; 17(8): 1561 - 1567. [Abstract] [Full Text]

				S. Hara, T. Shike, N. Takasu, and T. Mizui Lysophosphatidylcholine Promotes Cholesterol Efflux From Mouse Macrophage Foam Cells Arterioscler Thromb Vasc Biol, July 1, 1997; 17(7): 1258 - 1266. [Abstract] [Full Text]

				X. Fang, S. Gibson, M. Flowers, T. Furui, R. C. Bast Jr., and G. B. Mills Lysophosphatidylcholine Stimulates Activator Protein 1 and the c-Jun N-terminal Kinase Activity J. Biol. Chem., May 23, 1997; 272(21): 13683 - 13689. [Abstract] [Full Text] [PDF]

				S. T. Davidge, A. P. Signorella, C. A. Hubel, D. L. Lykins, and J. M. Roberts Distinct Factors in Plasma of Preeclamptic Women Increase Endothelial Nitric Oxide or Prostacyclin Hypertension, November 1, 1996; 28(5): 758 - 764. [Abstract] [Full Text]

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