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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.

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