J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tokumura, A.
Right arrow Articles by Fukuzawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tokumura, A.
Right arrow Articles by Fukuzawa, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Journal of Lipid Research, Vol. 43, 307-315, February 2002
Copyright © 2002 by Lipid Research, Inc.

Increased formation of lysophosphatidic acids by lysophospholipase D in serum of hypercholesterolemic rabbits

Akira Tokumuraa, Yumi Kanayaa, Masaki Kitaharab, Maki Miyakea, Yasuko Yoshiokaa, and Kenji Fukuzawaa
a Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
b Shiraoka Research Station of Biological Science, Nissan Chemical Industries, Shiraoka, Minamisaitama-gun, Saitama 349-0294, Japan

Correspondence to: Akira Tokumura, To whom correspondence should be addressed., tokumura{at}ph.tokushima-u.ac.jp (E-mail)


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Lysophosphatidic acid (LPA) is a biologically active phospholipid that has been identified as a vasoactive principle in incubated plasma and serum of mammals. Previously, we found that mammalian plasma and serum contain a lysophospholipase D, which hydrolyzes lysophosphatidylcholines (LPCs) with different fatty acyl groups to the corresponding LPAs during its incubation at 37°C. In this study, we examined whether lysophospholipase D activity and levels of LPCs in rabbit serum were modulated by feeding rabbits a high cholesterol diet. Results showed that the serum levels of LPCs increased gradually in animals fed a high cholesterol diet for 12 weeks. We found that the levels of individual LPAs formed on incubation of serum for 24 h increased with an increase in the period of feeding of rabbits a high cholesterol diet. LPA with a linoleate residue was the most abundant LPA, followed in order by 16:0-, 18:1- and 18:0-LPAs. LPA was found to increase attachment of the monocytic cell line THP-1 to vascular endothelial cells pre-stimulated with tumor necrosis factor-{alpha}.

These results indicated that increases in the levels of LPAs generated by lysophospholipase D in the blood of hypercholesterolemic rabbits may be relevant to attachment of monocytes to vascular walls, a key phenomenon observed at an early stage of atherosclerosis. — Tokumura, A., Y. Kanaya, M. Kitahara, M. Miyake, Y. Yoshioka, and K. Fukuzawa. Increased formation of lysophosphatidic acids by lysophospholipase D in serum of hypercholesterolemic rabbits. J. Lipid Res. 2002. 43: 307–315.

Supplementary key words: lysophosphatidylcholine, phosphatidylcholine, vascular endothelial cell, atherosclerosis, gas chromatography-mass spectrometry


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Patients suffering from chronic diseases relevant to atherosclerosis, as a basic vascular dysfunction, are still increasing in many countries. The formation of fatty streaks in atheromatic lesions has been thought to be related to increased levels of lipids including cholesterol in the blood circulation. The migration of blood monocytes into subendothelial spaces is known to be closely associated with the formation of fatty streaks (1). For evaluation of the relationship of hypercholesterolemia with atherosclerosis or vascular endothelial function, rabbits were fed a high cholesterol diet for several weeks, since atheroma formation rapidly advances in this animal model (2). In this model, high dietary cholesterol was found to promote vascular lesions such as the formation of fatty streaks (2) (3), and to impair dilatory responses of the blood vasculature due to nitric oxide generation (2) (4). Active identities inducing the vascular lesions such as fatty plaques and accompanying vascular dysfunction are still in debate. Oxidative modification of LDL penetrating through vascular endothelial cells has been recognized as a key phenomenon for induction of atherogenesis (1). Two bioactive phospholipids in the oxidized lipoproteins have been suggested as critical for the pathogenesis of atheroma formation. One is a family of phosphatidylcholines (PCs) having an oxidatively shortened fatty acyl chain, the structures of which resemble that of platelet-activating factor (5) (6). The other is lysophosphatidylcholines (LPCs) formed from platelet-activating factor-like PCs by the action of the platelet-activating factor acetylhydrolase in plasma (5). Both components in the oxidized LDL are known to activate blood monocytes (7) (8), which participate in the impairment of vascular cells at an early stage of atherosclerosis (1). However, altered levels of LPC in the blood and vascular tissues were reported in various pathophysiological situations without increased oxidative stress (9). Thus, unrecognized mechanisms are likely to be relevant to early events inducing atherogenesis.

Lysophosphatidic acid (LPA) is an important phospholipid mediator with diverse receptor-mediated biological activities (6) (10) (11). Three different genes encoding G protein-coupled receptors specific for LPA have been characterized so far: Edg-2 (12), Edg-4 (13), and Edg-7 (14). Recently, this bioactive lysophospholipid has been postulated to be responsible for the pathogenesis of atherosclerosis (15) (16), since it aggregates platelets (17) (18), promotes the de-differentiation (16) and proliferation (19) of vascular smooth muscle cells, and activates vascular endothelial cells (15), monocytes (20), and macrophages (21). Second, LPA was identified as a major platelet-aggregating substance in oxidized LDL, a key component for induction of atherogenesis (22).

LPA is known to be produced by sequential intracellular reactions of phospholipid remodeling, and released, in part, into an extracellular medium (6) (23). However, a more specialized mechanism, extracellular production of LPA, is also known. The latter mechanism involves a novel metal-requiring lysophospholipase D activity that was originally found in heparinized rat plasma (24); it hydrolyses different molecular species of LPC to the corresponding LPA (24) (25) (26) (27) (28). In order to gain information about the possible contribution of LPAs generated by lysophospholipase D to the induction of atherogenesis, in this study, we examined whether serum lysophospholipase D activity increased on feeding a high cholesterol diet for several weeks to rabbits, an animal model of atherosclerosis.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Phospholipase D from cabbage, 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17:0/17:02 Fatty acyl moieties of phospholipids are designated in terms of the number of carbon atoms: number of double bonds, e.g., 16:0 for palmitoyl.2-phosphatidylcholine; PC), 1-palmitoyl (16:0)- and heptadecanoyl (17:0)-2-lyso-sn-glycero-3-phosphocholines (16:0- and 17:0-LPCs), 1-oleoyl (18:1)-2-lyso-sn-glycerol-3-phosphate (18:1-LPA), and botulinum toxin C3 were obtained from Sigma Chemical (St. Louis, MO). 1-Linoleoyl (18:2)-2-lyso-sn-glycero-3-phosphocholine (18:2-LPC) was from Doosan Serdary Research Laboratories (London, Canada). 1-Arachidonoyl (20:4)-2-lyso-sn-glycero-3-phosphocholine (20:4-LPC) was prepared by hydrolysis of 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (Doosan Serdary Research Laboratories) as described previously (29). 1-Palmitoyl-, heptadecanoyl-, linoleoyl-, and arachidonoyl-2-lyso-sn-glycerol-3-phosphate (16:0-, 17:0-, 18:2-, and 20:4-LPAs) were prepared by the hydrolyses of 16:0-, 17:0-, 18:2-, and 20:4-LPCs with cabbage phospholipase D (19) (28). Trimethylchlorosilane, N,O-bis(trimethylsilyl)trifluoroacetamide and Sintisol EX-H were from Wako Pure Chemicals (Osaka, Japan). 2,7-Bis(carboxyethyl)-4 or 5-carboxyfluorescein diacetoxymethyl ester (BCECF-AM) was from Dojindo (Kumamoto, Japan).

Male New Zealand white rabbits weighing 2.4–2.9 kg were housed with a cycle of 12 h light and 12 h dark. Rabbits received a normal diet or a diet supplemented with 1% cholesterol (100 g/day) and 250 ml/day of water. Before receiving the diet, blood (5–10 ml) was withdrawn from an ear vein, and serum was prepared by centrifugation of the blood at 3,000 rpm for 15 min after standing it for 3 h at room temperature.

Serum preparations (0.15 ml) were mixed with 230 nmol of 17:0-LPC and 350 nmol of 17:0/17:0-PC per ml of serum, as internal standards. Phospholipids were extracted by the method of Bligh and Dyer (30), and fractionated by TLC on a Whatman Silica gel K6 TLC plate as described above. Both PC and LPC were extracted from silica gel by the method of Bligh and Dyer (30) after their mixing with 1–2 ml of distilled water. The recovered PC and LPC were heated in 0.5 ml of 5% methanolic hydrochloride solution at 100°C for 3 h to convert them to fatty acid methyl esters. The fatty acid methyl esters were extracted with n-hexane three times, and quantified by GLC, as described previously (26).

Aliquots of 0.1 ml of serum preparations from normal and hypercholesterolemic rabbits were incubated with 1-[14C]16:0-LPC (0.15 mCi/mmol, 16.7 nmol/ml of serum) at 37°C for 2, 4, and 6 h. The radioactive LPC and its metabolites were extracted by the method of Bligh and Dyer (30) after the addition of 0.2 g KCl, dilution with distilled water to 1 ml, acidification of the aqueous phase to pH 2.5, and finally separated by TLC with chloroform-methanol-20% ammonium hydroxide (60:35:8, v/v/v) on a Merck Silica gel 60 TLC plate after addition of 18:1-LPA (0.1 µmol/sample) as a carrier. Lipids were localized under an ultraviolet lamp after spraying with 1% p-toluidino-2-naphthalene sulfonic acid in 50 mM Tris/HCl buffer. Silica gel from lipid zones containing PC, LPC, LPA, and free fatty acid were scraped off the plate. The radioactivity of the silica gels was counted in 5 ml of Sintisol EX-H in a liquid scintillation counter to calculate the rate of conversion of [14C]16:0-LPC to LPA (%/h/ml of serum), as described previously (25).

LPAs purified by TLC on a Whatman K6 silica gel TLC plate were treated with 25 µl of pyridine and 25 µl of N,O-bis(trimethylsilyl)trifluoroacetamide containing 2% trimethylchlorosilane at 65°C for 45 min. Aliquots of 1 µl of the reaction mixture were directly injected at 280°C into a gas chromatograph equipped with a DB-1 [J & W Scientific (Foster City, CA); 30 m x 0.25 mm, 0.25 µm thickness] with a program of increase in the column temperature from 200°C to 320°C at a rate of 10°C/min. The temperature of the ion source was 180°C. Electron impact ionization-mass spectra were measured with a JEOL JMS FX102A mass spectrometer coupled to the gas chromatograph under the following conditions: 70 eV of ionizing voltage, 300 µA of ionizing current, 10 kV of accelerating voltage.

To examine whether LPA can impact functions of blood monocytes, we measured adhesion of BCECF-AM-labeled monocytic cell line THP-1 cells to human umbilical venous endothelial cells (HUVEC), essentially as described (31), since use of fluorescent dyes including BCECF-AM in the determination of adherence of human leukocytes to endothelial cells is known to be powerful tool (32). THP-1 cells were stimulated with 18:1-LPA and then co-cultured with HUVEC pre-activated with tumor necrosis factor-{alpha}.

THP-1 cells were obtained from the American Type Culture Collection (Rockville, MD), and maintained in RPMI-1640 medium supplemented with 20% FBS. HUVEC were obtained from Clontics Corporation (Walkersville, MD), and maintained in endothelial cell basic medium supplemented with 2% FBS, 10 ng/ml epidermal growth factor, 12 µg/ml bovine brain extract, 1 µg/ml hydrocortisone, and 50 µg/ml gentamicin. THP-1 cells were suspended at a cell density of 5 x 105 in 1 ml of the culture medium supplemented with a 0.002 volume of a solution of 18:1-LPA at various concentrations in DMSO. Botulinum toxin C3 (ADP ribosyltransferase C3) was dissolved in the culture medium containing 5 µM digitonin and 50 µM 18:1-LPA. THP-1 cells were incubated in the medium with or without supplementation of 18:1-LPA for 48 h at 37°C under 5% CO2. The cells were then incubated with a medium containing 10 µM BCECF-AM for 30 min. HUVEC cultured at confluence on 96 well culture plates were stimulated with 10 ng/ml tumor necrosis factor-{alpha}. After 8 h, a monolayer of HUVEC was loaded with BCECF-AM-labeled THP-1 cells (8 x 104 cells/well) at 37°C for 2 h under 5% CO2. Nonadherent THP-1 cells were washed out, and fluorescence from the remaining adherent cells was measured at an excitation wavelength of 485 (half bandwidth: 20) nm, and emission wavelength of 530 (half band width: 25) nm. The number of adherent THP-1 cells was calculated from the intensities of fluorescence.

In another experiment, HUVEC were cultured in the presence of 3, 10, or 100 µM 18:1-LPA for 48 h, and then stimulated with 10 ng/ml tumor necrosis factor-{alpha} for 12 h. BCECF-AM-labeled THP-1 cells (1.5 x 105 cells/well) were loaded on the HUVEC culture and incubated for 2 h under 5% CO2. Non-adherent THP-1 cells were washed out, and fluorescence was measured, as described above.

Differences of means were analyzed by Student's t-test, and considered significant at the 5% level.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

Altered serum levels of LPCs by feeding rabbits with a high cholesterol diet affect activity of lysophospholipase D
Rabbits were placed on a diet containing 1% cholesterol for 12 weeks. Blood was collected at intervals, and fresh and incubated sera were prepared. First, we measured the concentration of different LPCs in the serum preparations before incubation. As shown in Table 1, the concentrations of total LPC and individual LPCs in the unincubated serum were greatly increased at an early stage of feeding of rabbits with a cholesterol-rich diet.


 
View this table:
[in this window]
[in a new window]
  		Table 1. Serum levels of different molecular species of LPCs before and after 6 h incubation of sera from normal and hypercholesterolemic rabbits

Previously, we reported that the concentrations of LPC in human serum and rat plasma were significantly increased within several hours after their incubation at 37°C (26) (27) (28). Hence, we examined whether the concentration of LPC was altered significantly during 6 h incubation of serum preparations of rabbits fed a normal or high cholesterol diet. Table 1 shows the levels of various molecular species of LPC in 6 h incubated serum preparations of normal and hypercholesterolemic rabbits. There were increases in the levels of 16:0- and 18:0-LPCs after 6 h incubation of sera from normal rabbits or rabbits fed a high cholesterol diet for 8–12 weeks, whereas the serum levels of other LPCs remain unchanged during the incubation. The changes in 16:0- and 18:0-LPCs were less with 6 h incubated sera from rabbits fed a high cholesterol diet for 1–4 weeks than those in incubated serum from rabbits fed a high cholesterol diet for 8–12 weeks. Fig 1 represents the composition of molecular species of LPC in these serum preparations, expressed as percentages of total LPC. Results with unincubated and 6 h incubated sera showed that the percentage of 18:2-LPC increased at an early stage of high cholesterol-feeding (1–4 weeks), concomitant with decreases in the percentages of 16:0- and 18:0-LPCs. The percentage of 18:2-LPC, however, decreased slowly by prolonging high cholesterol-feeding up to 12 weeks, whereas those of 16:0- and 18:0-LPCs gradually increased to those of normal rabbits. There were no significant changes in the percentages of 18:1- and 20:4-LPCs throughout high cholesterol-feeding.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Fatty acid compositions of lysophosphatidylcholine (LPC) before (A) and after (B) 6 h incubation of serum from normal and hypercholesterolemic rabbits. Values are means ± SEM for 4–5 animals. Symbols used are (open square) (16:0), (open diamond) (18:0), (closed circle) (18:1), (closed triangle) (18:2), and (closed square) (20:4).

By multiplying the level of 16:0-LPC in the 6 h incubated sera from normal and high cholesterol-fed rabbits (Table 1, nmol/ml) by the fractional change of the conversion of [14C]16:0-LPC to LPA (%/h), we estimated the initial production rate of 16:0-LPA (nmol/h) generated by lysophospholipase D activity. Fig 2 shows that the corrected activity of lysophospholipase D was decreased after the high cholesterol-feeding for 1 week followed by a small increase in the activity at early stages of feeding with a high cholesterol diet. However, the corrected activity did not significantly change with further prolonged feeding of a high cholesterol diet.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Lysophospholipase D activity corrected for the level of endogenous substrate, 16:0-LPC, in incubated serum. The amount of 16:0-LPA produced by lysophospholipase D per h was estimated by multiplying the percentage conversion of [14C]16:0-LPC by the level of endogenous 16:0-LPC in 6 h incubated serum (Table 1).

Analysis of LPA generated by lysophospholipase D during incubation of serum
Because the effect of dietary high cholesterol on the serum lysophospholipase D activity toward the single molecular species of LPC was rather inhibitory, and somewhat complicated, we attempted to examine this issue by adapting other approaches that can monitor the conversions of different LPCs to corresponding LPAs. For this purpose, we decided to utilize direct analysis of TLC-purified LPAs by GC-MS after their conversion to volatile derivatives having three trimethylsilyl (TMS) groups, without any hydrolytic pretreatment. Fig 3B shows typical results with LPAs purified from 24 h incubated serum of a rabbit fed a high cholesterol diet for 4 weeks, indicating the ion-current profiles of various ions at m/z 639, 637, 635, 625, 611 corresponding to [M - CH3]+ of TMS derivatives of 18:0-, 18:1-, 18:2-, 17:0-, and 16:0-LPA, respectively, with various magnifications for the 24 h incubated serum. These ion peaks were normalized to have the same peak height by magnifying them to different degrees, as shown in Fig 3. Little signals of [M - CH3]+ of TMS derivatives of endogenous LPAs were observed for unincubated serum of a rabbit fed with a high cholesterol diet for 4 weeks (Fig 3A). The amounts of LPAs in these serum preparations were determined on the basis of peak areas of [M - CH3]+ of TMS derivatives of endogenous LPAs relative to that of TMS-17:0-LPA, an internal standard, after corrections on the basis of variable heat-stability of various TMS derivatives of LPAs in the gas phase and their efficiencies of ionization upon electron impact. For this purpose, we measured ratios of [M - CH3]+ of TMS derivatives of standard LPAs to that of TMS derivative of 17:0-LPA with different molar ratios. Because the retention times of TMS derivatives of 18:1- and 18:2-LPAs were the same under our conditions, the [M - CH3]+ of TMS derivative of 18:1-LPA could not be distinguished from [M - CH3 + 2]+ of TMS-18:2-LPA. This was also corrected for quantification of 18:1-LPA as follows: the peak area of [M - CH3 + 2]+ due to 18:2-LPA-TMS was subtracted from the observed peak area of [M - CH3]+ of 18:1-LPA-TMS (m/z 637). The former was calculated by multiplying the observed peak area due to [M - CH3]+ of 18:2-LPA-TMS (m/z 635) by the ratio of [M - CH3 + 2]+ to [M - CH3]+ obtained by GC-MS of standard 18:2-LPA-TMS. Fig 4 shows results of such quantitative experiments for LPAs in the 24 h incubated serum preparations of rabbits fed a normal diet or high cholesterol diet. The total amount of LPA accumulated during the 24 h incubation of serum was found to be remarkably elevated with increase in the feeding period for up to 8 weeks, and thereafter declined slightly. The most predominant molecular species of LPA was 18:2-LPA followed by 16:0-, 18:1-, and 18:0-LPAs in order. At a late stage of feeding of the high cholesterol diet, the production of 18:2-LPA declined, whereas the generation of 16:0-LPA increased slightly for up to 12 weeks. The levels of other LPAs reached maxima after 8–10 weeks feeding of the high cholesterol diet. However, the molecular species compositions of fresh serum LPA from hypercholesterolemic rabbits were not similar to those from normal animals; 18:2-LPA was the predominant species for the hypercholesterolemic groups, whereas 16:0-LPA was abundant in the serum of normal rabbits ( Fig 5).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Gas chromatography-mass spectrometry (GC-MS) of trimetylsilyl (TMS) derivatives of lysophosphatidic acids (LPAs) purified from lipid extracts of rabbit serum before and after incubation for 24 h. Serum from a rabbit fed a high cholesterol diet for 4 weeks was incubated at 37°C for 24 h. Lipids were extracted from the serum before (A) and after (B) incubation, and fractionated by TLC. Purified LPAs were recovered from the silica gel, and converted to volatile derivatives with three TMS groups. Ion-current profiles of five selected ions (m/z 639, 637, 635, 625, 611 for [M - CH3]+ of 18:0-, 18:1-, 18:2, 17:0-, and 16:0-LPAs, respectively) are shown at various magnifications where the major ions has the same peak height.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. Levels of various LPAs in 24 h incubated sera from rabbits fed a normal or high cholesterol diet. Serum was prepared from rabbits fed a normal diet or a high cholesterol diet for various periods. Levels of four major species of LPA were measured in these serum preparations after incubation at 37°C for 24 h (A). Data for 16:0-, 18:0-, and 18:1-LPAs were enlarged (B). Values are means ± S.E.M. for 4–5 animals. Symbols used are (open square) (16:0), (open diamond) (18:0), (closed circle) (18:1), (open triangle) (18:2), and total (open circle).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Levels of various LPAs in unincubated sera from rabbits fed a normal or high cholesterol diet for various periods. Serum preparations were prepared from rabbits fed a normal diet or a high cholesterol diet for various periods. Levels of four major species of LPA were measured on these serum preparations. Values are means ± SEM for two or three animals. White, hatched, and black bars show results on rabbits fed a normal diet, high cholesterol diet for 1–4 weeks, and high cholesterol diet for 8–12 weeks, respectively. ND, not detected.

LPA activates monocytes to attach to vascular endothelial cells
As described above, the increased formation of LPA by lysophospholipase D in the blood circulation in hypercholesterolemic rabbits may contribute to early events inducing atherosclerosis. In this study, we examined whether LPA was able to activate monocytic THP-1 cells to attach to vascular endothelial cells that is known to be a key phenomenon related to atherogenesis. After THP-1 cells were pre-activated with various concentrations of 18:1-LPA, the monocytic cells were incubated with HUVEC pre-stimulated with tumor necrosis factor-{alpha}. Under these conditions, LPA promoted the adhesion of THP-1 cells to the endothelial cells in a concentration-dependent manner ( Table 2, experiment A). The enhancing effect of 50 µM 18:1-LPA was inhibited by pretreatment of THP-1 cells with 15 or 50 µg/ml ADP ribosyltransferase C3, indicating that LPA augments the monocyte/vascular endothelial cell interaction by a Rho-mediated mechanism (Table 2, experiment B). Fig 6 shows a result of similar adherent experiment where HUVEC, but not THP-1 cells, were incubated with LPA, and then stimulated with tumor necrosis factor-{alpha}. The result showed that LPA failed to enhance the activating effect of tumor necrosis factor-{alpha} on HUVEC to bind THP-1 cells.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 6. No significant enhancing effect of LPA on capability of HUVEC stimulated with tumor necrosis factor-{alpha}-induced to bind THP-1 cells. Basal: experiment on HUVEC unstimulated with tumor necrosis factor-{alpha}. Control: experiment on HUVEC stimulated with tumor necrosis factor-{alpha} alone. LPA: experiments on HUVEC incubated with LPA and then stimulated with tumor necrosis factor-{alpha}.


 
View this table:
[in this window]
[in a new window]
  		Table 2. Stimulatory effect of LPA on adhesion of the THP-1 monocytic cell line to HUVEC activated by tumor necrosis factor-{alpha} and its inhibition by treatment of monocytes with ADP ribosyltransferase C3


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

An animal model is useful in studies on LPA generation under pathophysiological conditions to evaluate the source of serum LPA and diseases to which the altered level of LPA may be relevant. Therefore, we conducted experiments with rabbits fed a high cholesterol diet as a model of atherosclerosis. We found great, gradual increases in PC content in the serum of the animals given 1% cholesterol in their diet for 12 weeks (data not shown), consistent with an earlier report of about nine times higher PC in the serum of rabbits fed a diet with 1% cholesterol than in normal rabbits (33). There are several reports that the level of LPC, the endogenous substrate of lysophospholipase D, is increased in hypercholesterolemic rabbits (34) (35) (36), and our results confirmed these previous findings. These results appear to be closely related to a large increase in lipoprotein concentration in the serum of hypercholesterolemic rabbits. Plasma LPC was proposed as a potential causal agent of atherosclerosis, based on the good correlation between atherosclerotic risk factors and LPC levels (9). In mammals other than rabbits, hypercholesterolemic and hyperlipidemic serum or plasma preparations were shown to contain more LPC than normal preparations (37) (38) (39) (40).

In this study, we found that the total amount of LPA in unincubated serum of hypercholesterolemic (8–12 weeks) was higher than that of normal rabbits. The higher serum level of total LPA in the hypercholesterolemic rabbits may be due to the increased level of lipoproteins that are potential reservoirs for plasma LPA, while a predominant portion of LPA in rat plasma was reported to be bound to albumin, not to lipoprotein (26). The reason for the observed low level of total LPA in fresh serum from hypercholesterolemic rabbits (1–4 weeks) remains obscure. However, the increased level of 18:2-LPA was more likely to be attributed to elevated apparent activities of LPA-producing enzymes such as lysophospholipase D, as discussed below.

At the end of the 1970s, LPA was reported to be a vasoactive principle in incubated plasma and serum of mammals (17), but this finding attracted little attention. There has been more interest in the occurrence of LPA in animal serum since a report by van Corven et al. who identified LPA as an active component in animal serum, causing proliferation of fibroblasts (41). Although the origin of LPA in human serum was shown to be activated platelets (42), the serum preparation contains lysophospholipase D activity that may convert endogenous LPCs in serum to LPAs during its preparation (28). For preparing the serum preparation, the withdrawn blood was often stood for several hours at various temperatures. Therefore, caution would be needed to suppress potential formation of LPA by lysophospholipase D activity during the preparation of serum. For assessment of the LPA level in the blood circulation in vivo, plasma anticoagulated with EDTA would be suitable, since we previously found that lysophospholipase D activity in rat plasma was suppressed by pretreatment of the blood with EDTA, but not citrate or heparin (25). However, the absolute amounts of LPA in unincubated rabbit serum preparations, that seem to reflect in vivo situation, were much less than those in in vitro 24 h incubated serum preparations.

The present study showed that 18:2-LPC was an abundant molecular species of LPC as well as 16:0-LPC (Fig 1), but the generation of 18:2-LPA was preferable to that of 16:0-LPA in rabbit serum incubated at 37°C for 24 h, suggesting that the substrate specificity of rabbit serum lysophospholipase D resembles that of lysophospholipase D in rat (24) (25) (26) (27) and human (28) plasma; they prefer unsaturated LPCs over saturated LPCs. An additional novel observation is that percentage conversion of 18:2-LPC to 18:2-LPA in hypercholesterolemic rabbits after 2 weeks feeding with a high cholesterol diet was about 2-fold that in normal rabbits. Two explanations are likely for this observation: the first is that a lysophospholipase D with a distinct substrate specificity preferring more 18:2-LPC may be secreted into the blood circulation of rabbits after high cholesterol feeding, and cooperate with a constitutive lysophospholipiase D, giving rise to more 16:0-LPA. Alternatively, the relative enrichment of 18:2-LPC over other LPCs in the blood plasma may tend to preferential production of 18:2-LPA over other LPAs. The increased levels of lipoprotein in the blood after cholesterol feeding of rabbits may lead to not only the increases in serum levels of individual LPC, but also the alteration of the pattern of their distribution among proteins in the serum. The altered pattern of LPC distribution in hypercholesterolemic rabbits may be related to utilization of much more 18:2-LPC than other LPCs by lysophospholipase D. Taken together, we conclude that the feeding of rabbits with a high cholesterol diet not only induces elevation of the total amount of LPA in the serum, but also change its molecular species composition.

The position of the fatty acyl moiety attaching to the glycerol backbone of LPA in rabbit serum was not determined in this study. However, the determining the percentage of the sn-1 and 2-acyl LPA is of potential value for a better understanding of their physiological role, because Edg-7 has been characterized as a gene of an LPA receptor selective for LPAs having an sn-2-unsaturated fatty acyl group (14). We previously reported that unsaturated LPCs in fresh and incubated rat plasma existed as mainly an 1-acyl isomer, indicating that unsaturated LPAs generated from the LPCs by lysophospholipase D activity during the incubation of rat plasma would be predominantly 1-acyl isomers (26). Croset et al. (43) reported similar results on ratios of positional isomer of LPCs in human and rat plasma. They further found that about 50% of the unsaturated LPC was 2-acyl isomer when the blood was directly collected in organic solvent, indicating that the fast isomerization of LPC occurred during preparation of plasma. Perhaps both isomers of unsaturated LPCs circulate in the blood in vivo. These results suggest that both 1- and 2-unsaturated acyl LPAs can be produced from the corresponding LPCs in the blood circulation. In this context, it should be mentioned that a recent paper by Hayashi et al. (16) who found that unsaturated LPA, but not saturated LPAs, in human serum modulated a phenotype of vascular smooth muscle from the differentiated state to the dedifferentiated one, that is involved in the development of atherosclerosis, although they did not determine the position of the unsaturated fatty acyl moiety on these LPAs.

Activation of blood monocytes and their adhesion to injured vascular endothelium has been suggested as a participant in the early processes leading to atherosclerosis (1) (8). In this study, we first focused on the effect of LPA on monocytes, because LPA generated in blood circulation was expected to interact with blood monocytes, and little is known about LPA-induced monocyte activation except for a report showing that high concentrations (0.1–1 mM) of LPA induced directional migration of human monocytes by a haptotactic mechanism after binding to its receptor coupled to a pertussis toxin-sensitive G protein in a polycarbonate filter assay (20). Our results clearly showed that LPA at physiological concentrations activated the monocytic cell line THP-1 to attach to HUVEC pre-stimulated by tumor necrosis factor-{alpha}, and that the effect of 18:1-LPA was prevented by pretreatment of THP-1 cells with ADP ribosyltransferase C3 exoenzyme. These results suggest that LPA stimulates cell surface adhesion molecules via activation of Rho GTPase in THP-1 cells. Rho GTPase is well-known to be involved in the intracellular signaling pathways triggered by LPA in a variety of cells (44) (45). We also found that LPA failed to enhance the adhesion of HUVEC pre-stimulated with tumor necrosis factor-{alpha} to resting THP-1 cells. However, LPA was found to activate human aortic endothelial cells to bind monocytes and neutrophilic HL-60 cells, by the mediating of the pertussis toxin-sensitive induction of vascular cell adhesion molecule-1 and E-selectin on the endothelial cells (15). The reason for the inconsistencies between these studies remains unknown, however, might be due to different cell types and/or distinct experimental conditions with or without stimulation by tumor necrosis factor-{alpha}.

Taken altogether, we suggest that the elevated production of LPAs by lysophospholipase D in the blood plasma of hypercholesterolemic rabbits may play a role in induction of atherosclerosis by enhancing the penetration of blood monocytes into the subendothelial space of vascular tissues. We are now conducting experiments on the structure-activity relationship and mechanism of LPA-induced activation of monocytes to attach vascular endothelial cells.


  ACKNOWLEDGMENTS

This study was in part supported by a Grant-in-Aid (10672043) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from The Japan Health Science Foundation.

Manuscript received July 9, 2001; and in revised form October 25, 2001

Abbreviations: GC-MS, gas chromatography-mass spectrometry; HUVEC, human umbilical venous endothelial cell; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; TMS, trimethylsilyl


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

  1. Steinberg, D. 1997. Low density lipoprotein oxidation and its pathophysiological significance. J. Biol. Chem. 272:20963-20966.[Free Full Text]

  2. Osborne, J. A., Lento, P. H., Siegfried, M. R., Stahl, G. L., Fusman, B., Lefer, A. M. 1989. Cardiovascular effects of acute hypercholesterolemia in rabbits: reversal with the lovastatin treatment. J. Clin. Invest. 83:465-473.

  3. Sun, Y. P., Zhu, B. Q., Sievers, R. E., Isenberg, W. M., Parmley, W. W. 1993. Aspirin inhibits platelet activity but does not attenuate experimental atherosclerosis. Am. Heart J. 125:79-86.[CrossRef][Medline]

  4. Laight, D. W., Matz, J., Caesar, B., Carrier, M. J., Anggard, E. E. 1996. Investigation of endogenous nitric oxide vascular function in the carotid artery of cholesterol-fed rabbits. Br. J. Pharmacol. 117:1471-1474.[Medline]

  5. McIntyre, T. M., Zimmerman, G. A., Prescott, S. M. 1999. Biologically active oxidized phospholipids. J. Biol. Chem. 274:25189-25192.[Free Full Text]

  6. Tokumura, A. 1995. A family of phospholipid autacoids: occurrence, metabolism and bioactions. Prog. Lipid Res. 34:151-184.[CrossRef][Medline]

  7. Kern, H., Volk, T., Knaufe-Sciefer, S., Mieth, T., Rustow, B., Kox, W. J., Schlame, M. 1998. Stimulation of monocytes and platelets by short-chain phosphatidylcholines with and without terminal carboxyl group. Biochim. Biophys. Acta. 1394:33-42.[Medline]

  8. Quinn, M. T., Parthasarathy, S., Steinberg, D. 1988. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc. Natl. Acad. Sci. USA. 85:2805-2809.[Abstract/Free Full Text]

  9. Vidaver, G. A., Ting, A., Lee, J. W. 1985. Evidence that lysolecithin is an important causal agent of atherosclerosis. J. Theor. Biol. 115:27-41.[CrossRef][Medline]

  10. Jalink, K., Hordijk, P. L., Moolenaar, W. H. 1994. Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator. Biochim. Biophys. Acta. 1198:185-196.[Medline]

  11. Goetzl, E. J., An, S. 1998. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate. FASEB J. 12:1589-1598.[Abstract/Free Full Text]

  12. Hecht, J. H., Weiner, J. A., Post, S. R., Chun, J. 1996. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex. J. Cell Biol. 135:1071-1083.[Abstract/Free Full Text]

  13. An, S., Bleu, T., Hallmark, O. G., Goetzl, E. G. 1998. Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid. J. Biol. Chem. 273:7906-7910.[Abstract/Free Full Text]

  14. Bandoh, K., Aoki, J., Hosono, H., Kobayashi, S., Kobayashi, T., Murofushi-Murakami, K., Tsujimoto, M., Arai, H., Inoue, K. 1999. Molecular cloning and characterization of a novel human G-protein coupled receptor, EDG7, for lysophosphatidic acid. J. Biol. Chem. 274:27776-27785.[Abstract/Free Full Text]

  15. Rizza, C., Leitinger, N., Yue, J., Fischer, D. J., Wang, D., Shih, P. T., Lee, H., Tigyi, G., Berliner, J. A. 1999. Lysophosphatidic acid as a regulator of endothelial/leukocyte interaction. Lab. Invest. 79:1227-1235.[Medline]

  16. Hayashi, K., Takahashi, M., Nishida, W., Yoshida, K., Ohkawa, Y., Kitabatake, A., Aoki, J., Arai, H., Sobue, K. 2001. Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids. Circ. Res. 89:251-258.[Abstract/Free Full Text]

  17. Schumacher, K. A., Classen, H. G., Späth, M. 1979. Platelet aggregation evoked in vitro and in vivo by phosphatidic acids and lysoderivatives: identity with substances in aged serum (DAS). Thromb. Haemostas. 42:631-640.[Medline]

  18. Gerrard, J. M., Kindom, S. E., Peterson, D. A., Peller, J., Krantz, K. E., White, J. G. 1979. Lysophosphatidic acid: influence on platelet aggregation and intracellular calcium flux. Am. J. Pathol. 96:423-438.[Abstract]

  19. Tokumura, A., Iimori, M., Nishioka, Y., Kitahara, M., Sakashita, M., Tanaka, S. 1994. Lysophosphatidic acids induces proliferation of cultured vascular smooth muscle cells from rat aorta. Am. J. Physiol. 267:C204-C210.[Abstract/Free Full Text]

  20. Zhou, D., Luini, W., Bernasconi, S., Diomede, L., Salmona, M., Mantovani, A., Sozzani, S. 1995. Phosphatidic acid and lysophosphatidic acid induce haptotactic migration of human monocytes. J. Biol. Chem. 270:25549-25556.[Abstract/Free Full Text]

  21. Koh, J. S., Lieberthal, W., Heydrick, S., Levine, J. S. 1998. Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway. J. Clin. Invest. 102:716-727.[Medline]

  22. Siess, W., Zangl, K. J., Essler, M., Bauer, M., Brandl, R., Corrinth, C., Bittman, R., Tigyi, G., Aepfelbacher, M. 1999. Lysophosphatidic acid mediated the rapid activation of platelets and endothelial cells by mildly oxidized low density lipoprotein and accumulates in human atherosclerotic lesions. Proc. Natl. Acad. Sci. USA. 96:6931-6936.[Abstract/Free Full Text]

  23. Gaits, F., Fourcade, O., Le Balle, F., Gueguen, G., Gaigé, B., Gassama-Diagne, A., Fauvel, J., Salles, J., Mauco, G., Simon, M., Chap, H. 1997. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis. FEBS Lett. 410:54-58.[CrossRef][Medline]

  24. Tokumura, A., Harada, K., Fukuzawa, K., Tsukatani, H. 1986. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma. Biochim. Biophys. Acta. 875:31-38.[Medline]

  25. Tokumura, A., Miyake, M., Yoshimoto, O., Shimizu, M., Fukuzawa, K. 1998. Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma. Lipids. 33:1009-1015.[Medline]

  26. Tokumura, A., Nishioka, Y., Yoshimoto, O., Shinomiya, J., Fukuzawa, K. 1999. Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma. Biochim. Biophys. Acta. 1437:235-245.[Medline]

  27. Tokumura, A., Fujimoto, H., Yoshimoto, O., Nishioka, Y., Miyake, M., Fukuzawa, K. 1999. Production of lysophosphatidic acid by lysophospholipase D in incubated plasma of spontaneously hypertensive rats and Wistar Kyoto rats. Life Sci. 65:245-253.[CrossRef][Medline]

  28. Tokumura, A., Miyake, M., Nishioka, Y., Yamano, S., Aono, T., Fukuzawa, K. 1999. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids in vitro fertilization patients. Biol. Reprod. 61:195-199.[Abstract/Free Full Text]

  29. Satouchi, K., Oda, M., Yasunaga, K., Saito, K. 1985. Evidence for production of 1-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine concomitantly with platelet-activating factor. Biochem. Biophys. Res. Commun. 128:1409-1417.[Medline]

  30. Bligh, E. G., Dyer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.

  31. Theilmeier, G., Lenaerts, T., Remacle, C., Collen, D., Vermylen, J., Hoylaerts, M. F. 1999. Circulating activated platelets assist THP-1 monocytoid/endothelial cell interaction under shear stress. Blood. 94:2725-2734.[Abstract/Free Full Text]

  32. De Clerck, L. S., Bridts, C. H., Mertens, A. M., Moens, M. M., Stevens, W. J. 1994. Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function. J. Immunol. Methods. 172:115-124.[CrossRef][Medline]

  33. McCandless, E. L., Zilversmit, D. B. 1956. The effect of cholesterol on the turnover of lecithin, cephalin and sphingomyelin in the rabbit. Arch. Biochem. Biophys. 62:402-410.[CrossRef][Medline]

  34. Rogausch, H., Distler, E. 1986. Erythrocyte rheology in cholesterol-fed rabbits. Int. J. Microcirc. Clin. Exp. 5:27-36.[Medline]

  35. Taketomi, T., Hara, A., Uemura, K., Kyogashima, N. 1989. Effect of long-term treatment with sulfatide on hyperlipidemia and progression of atherosclerosis in WHHL rabbits. Jpn. J. Exp. Med. 59:221-231.[Medline]

  36. Keaney, J. F., Jr., Xu, A., Cunningham, D., Jackson, T., Frei, B., Vita, J. A. 1995. Dietary probucol preserves endothelial function in cholesterol-fed rabbits by limiting vascular oxidative stress and superoxide generation. J. Clin. Invest. 95:2520-2529.

  37. Portman, O. W. 1969. Atherosclerosis in nonhuman primates: sequences and possible mechanisms of change in phospholipid composition and metabolism. Ann. N.Y. Acad. Sci. 162:120-136.[CrossRef][Medline]

  38. Wells, I. C., Peitzmeier, G., Vincent, J. K. 1986. Lecithin:cholesterol acyltransferase and lysolecithin in coronary atherosclerosis. Exp. Mol. Pathol. 45:303-310.[CrossRef][Medline]

  39. Engelmann, B., Streich, S., Schöntheir, U. M., Richter, W. O., Duhm, J. 1992. Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. I. Increased phosphatidylcholine and reduced sphingomyeline in patients with elevated levels of triacylglycerol-rich lipoproteins. Biochim. Biophys. Acta. 1165:32-37.[Medline]

  40. Kulschar, R., Engelmann, B., Bräutigam, C., Duhm, J., Thiery, J., Richter, W. O. 1995. Fast transmission of alterations in plasma phosphatidylcholine/sphingomyelin ratio and lyso-phosphatidylcholine levels into changes of red blood cell membrane phospholipid composition after low density lipoprotein apheresis. Europ. J. Clin. Invest. 25:258-265.[Medline]

  41. van Corven, E. J., Groenink, A., Jalink, K., Eichholz, T., Moolenaar, W. H. 1989. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell. 59:45-54.[CrossRef][Medline]

  42. Eichholtz, T., Jalink, K., Fahrenfort, I., Moolenaar, W. H. 1993. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem. J. 291:677-680.

  43. Croset, M., Brossard, N., Polette, A., Lagarde, M. 2000. Characterization of plasma unsaturated lysophosphatidylcholines in human and rat. Biochem. J. 348:61-67.

  44. Machesky, L. M., Hall, A. 1997. Rho: a connection between membrane receptor signalling and the cytoskeleton. Trend. Cell Biol. 6:304-310.

  45. Moolenaar, W. H., Kranenburg, O., Postoma, F. R., Zondag, C. M. 1997. Lysophosphatidic acid: G-protein signalling and cellular response. Curr. Biol. 9:168-173.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
D. L. Baker, Y. Fujiwara, K. R. Pigg, R. Tsukahara, S. Kobayashi, H. Murofushi, A. Uchiyama, K. Murakami-Murofushi, E. Koh, R. W. Bandle, et al.
Carba Analogs of Cyclic Phosphatidic Acid Are Selective Inhibitors of Autotaxin and Cancer Cell Invasion and Metastasis
J. Biol. Chem., August 11, 2006; 281(32): 22786 - 22793.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Tokumura, E. Majima, Y. Kariya, K. Tominaga, K. Kogure, K. Yasuda, and K. Fukuzawa
Identification of Human Plasma Lysophospholipase D, a Lysophosphatidic Acid-producing Enzyme, as Autotaxin, a Multifunctional Phosphodiesterase
J. Biol. Chem., October 11, 2002; 277(42): 39436 - 39442.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
M. Umezu-Goto, Y. Kishi, A. Taira, K. Hama, N. Dohmae, K. Takio, T. Yamori, G. B. Mills, K. Inoue, J. Aoki, et al.
Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production
J. Cell Biol., July 22, 2002; 158(2): 227 - 233.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tokumura, A.
Right arrow Articles by Fukuzawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tokumura, A.
Right arrow Articles by Fukuzawa, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today