Phenotypic Modulation of Vascular Smooth Muscle Cells Induced by Unsaturated Lysophosphatidic Acids
The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA–induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal–regulated kinase and p38 mitogen–activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors.
- vascular smooth muscle cells
- phenotypic modulation
- lysophosphatidic acids
- extracellular signal–regulated kinase
- p38 mitogen-activated protein kinase
The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one, which results in cell proliferation and migration, is a hallmark of the development and progression of atherosclerosis. Numerous studies have reported the possible involvement of cytokines and growth factors in the phenotypic modulation of VSMCs.1,2 The critical factors affecting the VSMC phenotype, however, remain unclear because proper in vitro assay systems have not yet been available. Under conventional culture conditions, VSMCs in primary culture rapidly display a phenotypic change.3,4 Dedifferentiated VSMCs obtained after passaging cannot revert to a differentiated phenotype, even under quiescent culture conditions. Therefore, studies using passaged VSMCs have not been able to investigate adequately the molecular mechanisms underlying the phenotypic modulation of VSMCs. To overcome this obstacle, we recently established primary culture systems of visceral SMCs and VSMCs in which both types of SMCs can maintain a differentiated phenotype for a long time, when cultured on laminin with insulin-like growth factor-I (IGF-I).5,6 Using our culture system, we investigated the signaling pathways affecting the visceral SMC phenotype and found that the IGF-I–stimulated phosphoinositide 3-kinase (PI3-K)/protein kinase B (PKB[Akt]) pathway plays a vital role in maintaining a differentiated phenotype. We also found that the coordinated activation of the extracellular signal–regulated kinase (ERK) and p38 mitogen–activated protein kinase (p38 MAPK) pathways induces SMC dedifferentiation when stimulated by platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), and basic fibroblast growth factor.5,6 Furthermore, we partially characterized the signaling pathways involved in regulating the VSMC phenotype using specific inhibitors for the PI3-K, ERK, and p38 MAPK pathways. The results suggested that identical pathways regulate the vascular and visceral SMC phenotypes.6 Taking these observations together, we hypothesized that changes in the balance between the strengths of the PI3-K/PKB(Akt) pathway and the ERK and p38 MAPK pathways would determine the phenotype of both visceral SMCs and VSMCs.6
It has been well documented that serum potently induces phenotypic modulation of VSMCs.3,4 Here, we further characterized the signaling pathways involved in regulating the VSMC phenotype and used our culture system to search for critical dedifferentiation factors for VSMCs. We thus identified unsaturated lysophosphatidic acids (LPAs) as specific dedifferentiation factors in the polar lipid fraction extracted from human serum and demonstrated that the unsaturated LPA–stimulated VSMC dedifferentiation was mediated through the coordinated activation of ERK and p38 MAPK. This is the first report to identify unsaturated LPAs as critical factors in the phenotypic modulation of VSMCs, suggesting that naturally occurring unsaturated LPAs may be pathogenic in atherosclerosis.
Materials and Methods
Lipids and Reagents
Authentic lysophosphatidylcholine (LPC), phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), sphingosine 1-phosphate (S1P), phosphatidic acid (PA), and 18:1 LPA (oleoyl-LPA) were purchased from Sigma. Other LPA species with various acyl chains were prepared enzymatically from the corresponding PC as previously described.7 LY294002 and wortmannin (PI3-K inhibitors), PD98059 (an MEK inhibitor), SB203580 and SB220025 (p38 MAPK inhibitors), and pertussis toxin (PTX) (Gαi-protein inhibitor) were purchased from Calbiochem. C3 exoenzyme (Rho inhibitor) was kindly provided by Dr S. Narumiya (Graduate School of Medicine, Kyoto University, Japan).
Analysis of SMC Differentiation Marker Gene Expression
The expression levels of the mRNAs for SMC differentiation marker genes such as caldesmon (CaD)8,9 and calponin (CN)10 were quantified by reverse transcriptase–polymerase chain reaction (RT-PCR) normalized to the expression of GAPDH mRNA. Oligo(dT)19-primed single-stranded cDNAs were synthesized from the total RNA of cultured VSMCs using Super Script II (Life Technologies). First, heat-denatured single-stranded cDNAs were subjected to PCR using Ex Taq DNA polymerase (Takara) and sets of primers specific for rat GAPDH (GenBank accession No. D14437). The intensities of the GAPDH cDNA bands stained by SYBR Green I (FMC Bioproducts) were relatively quantified using a Fluor Imager (Amersham Pharmacia Biotech) at cycle numbers where the intensities increased linearly. Next, defined amounts of heat-denatured single-stranded cDNAs were subjected to PCR to estimate the relative expression levels of SMC differentiation markers in each sample. PCR products were sampled at intervals of three cycles between 21 and 36 cycles and were separated on 1.2% agarose gels. The intensities of the respective cDNA bands were normalized on the basis of the GAPDH cDNA levels. The specific primer sets for h-CaD (AB049626), a common region of the CaD isoforms (U18419), rat CN (X02231), and rat GAPDH were as follows: GAPDH sense primer, 5′-GTGACAAAGTGGACATTGTTG-3′, and GADPH antisense primer, 5′-CATGAGCCCTCCACGATGC-3′; CN sense primer, 5′-ATGTCTTCCGCACACTTTAAC-3′, and CN antisense primer, 5′-GCTCAAATCTCCGCTCTT-3′; h-CaD sense primer, 5′-TGGCGGAGGAACAGGCAAGAAT-3′, and h-CaD antisense primer, 5′-CGGGCTTGTCATCTTGGGATGT-3′; and CaD common sense primer, 5′-TCCCCTACCTCAGTCACTCCT-3′, and CaD antisense primer, 5′-TTCCCTCCCTTCAGCTTCTCTT-3′.
Lipid Extraction and Analyses
Human plasma was prepared from the anticoagulated blood of healthy volunteers with acid/citrate/dextrose by centrifugation at 1500g for 30 minutes at 4°C. Human serum was prepared from the blood of healthy volunteers by spinning clotted blood at 1500g for 30 minutes at 4°C. We tried several extraction methods to recover the dedifferentiation activity. Of these methods, which included extractions with acetone and ether,11 chloroform/methanol under neutralized12 or acidic13 conditions, and n-butanol under neutralized13 or acidic14 conditions tested, n-butanol extraction under acidic conditions14 was the most effective. The recovery of lipid in the extractions was monitored by adding a tracer, [3H]18:1 LPA (New England Nuclear); the extraction efficiency was >95%.
Two-dimensional thin-layer chromatography (TLC) analysis was carried out as follows: chloroform/methanol/water (65:35:5) as the first solvent system and chloroform/methanol/25% ammonium hydroxide (60:40:10) as the second.15 The separated lipids were visualized using iodine vapor and identified by comigration with authentic standards as indicated. To evaluate the dedifferentiation activity in separated polar lipids, the lipid spots were visualized under a UV lamp and scraped.16 LPAs in the polar lipid fractions from human serum were separated by TLC and scraped from silica gels as described above. To estimate the amounts of LPA species, isolated LPAs were incubated with 5% HCl in methanol at 90°C, and the resultant fatty acyl methyl esters were separated and quantified by gas chromatography (model HP-5890A, Hewlett-Packard).
Primary Culture of VSMCs
VSMCs were isolated from the rat aortic media by the enzyme-dispersed method and were primarily cultured on laminin-coated plates in the basal medium (DMEM supplemented with 0.2% fatty acid–free BSA) containing IGF-I (2 ng/mL) for 1 day as described elsewhere.5,6 The medium was then changed to basal medium containing the supplements indicated. Ligand-induced contractility was monitored by a previously reported procedure.5,6 Cell proliferation was determined by bromodeoxyuridine (BrdU) (20 μmol/L) incorporation for 6 hours. Cells were fixed by cold ethanol, stained by monoclonal anti-BrdU antibody (DAKO), and then counted.
The kinase activities were determined by immunoprecipitation with anti-ERK, -p38 MAPK, or -PKB(Akt) antibodies (Santa Cruz Biotechnology), followed by kinase assays as described elsewhere.5,6
Analysis of LPA Receptor Expression
The expression of three LPA receptor (Edg-2, Edg-4, and Edg-7) mRNAs in VSMCs was analyzed by RT-PCR. Single-stranded cDNAs from rat testis, in which the three receptor mRNAs are expressed,7,17 were used as a positive control. The expression levels of three receptor mRNAs in VSMCs and testis were semiquantified by normalizing their respective mRNA levels to the GAPDH mRNA levels as described above. The specific primer sets for rat Edg-2 (AF014418), mouse Edg-4 (NM 020028), and mouse Edg-7 (AF293845) were as follows: Edg-2 sense primer, 5′-ATGGCAGCTGCCTCTACTTCC-3′, and Edg-2 antisense primer, 5′-CTAAACCACAGAGTGGTCATTGC-3′; Edg-4 sense primer, 5′-ATGGGCCAGTGCTACTACAAC-3′, and Edg-4 antisense primer, 5′-GCATTGACCAGTGAGTTGGC-3′; and Edg-7 sense primer, 5′-AAGACACATGTCAATCATGAGG-3′, and Edg-7 antisense primer, 5′-GCCGTTTTTATTGCACACCGGG-3′.
Characterization of the VSMC Differentiated Phenotype
The phenotypes of VSMCs in primary culture were characterized by the following three criteria: cell morphology, ligand-induced contractility, and expression of SMC differentiation marker genes (Figure 1A). VSMCs cultured on laminin under IGF-I–stimulated conditions showed spindle-like cell shapes, carbachol-induced contractility, and high levels of h-CaD and CN expression, indicating a fully differentiated phenotype. In these cells, IGF-I activated the PKB(Akt) activity, and this activation was suppressed by treatment with LY294002 (30 μmol/L) or wortmannin (30 nmol/L), suggesting that the activation of PKB(Akt) depends on the PI3-K pathway (Figure 1D). However, IGF-I did not activate the ERK or p38 MAPK (Figures 1B and 1C). VSMCs stimulated with PDGF-BB and EGF showed a typical dedifferentiated phenotype, which comprises fibroblast-like shape change, loss of contractility, and downregulation of h-CaD and h-CaD+l-CaD and of CN expression (Figure 1A). The signaling pathways of PDGF-BB and EGF were different from those of IGF-I; they coordinately activated ERK and p38 MAPK (Figures 1B and 1C). PDGF-BB also activated PKB(Akt), but EGF did not (Figure 1D). Consistent with our previous partial characterization of the VSMC phenotype,6 PDGF-BB–induced dedifferentiation was eliminated by simultaneous blocking of the ERK and p38 MAPK pathways by PD98059 and SB203580 (Figures 1A through 1D). Thus, these results indicate that PDGF-BB activates the dual pathways, in which the PI3-K/PKB(Akt) pathway is involved in maintenance of the differentiated phenotype and the coordinated activation of the ERK and p38 MAPK pathways induces dedifferentiation.
VSMC Dedifferentiation Is Induced by the Human Serum LPA Fraction
Human plasma had no effect on the differentiated phenotype of VSMCs. In contrast, human serum potently induced dedifferentiation as determined by the three criteria described above (Figure 2A). The serum was then separated into the lipid and aqueous fractions. Of the extraction methods tested (described in Materials and Methods), n-butanol extraction under acidic conditions13 was the most effective for extracting the dedifferentiation activity (Figure 2A); >75% of the total dedifferentiation activity in the human serum was recovered in the n-butanol fraction, and <25% in the aqueous fraction. Neither the lipid nor the aqueous fractions of plasma showed dedifferentiation activity (data not shown). Two-dimensional TLC of the plasma and serum lipid fractions revealed the following four major polar lipids that were common components in the lipid fractions of both: LPC, PC, SM, and PE. LPA was detected only in the serum lipid fraction (Figure 2B). Of the five polar lipid fractions extracted from silica gel spots, only the LPA fraction exhibited the dedifferentiation activity (Figure 2C). None of the other polar lipid fractions affected the VSMC phenotype, even at concentrations of up to 30 μmol/L. We also confirmed that the other fractions of human serum lipids had no effect on the SMC phenotype (data not shown). We estimated that the dedifferentiation activity of VSMCs in the LPA fraction was >90% of the total activity in the serum lipid fraction, suggesting that the serum lipid–induced dedifferentiation activity is mostly dependent on LPAs.
Identification of Unsaturated LPAs as Potent Factors in Inducing VSMC Dedifferentiation
We then quantified the LPA species in the LPA fraction derived from human serum by gas chromatography. The LPA contents were as follows (in nmol/mL): 14:0 LPA, 1.43±0.35; 16:0 LPA, 11.95±4.04; 18:0 LPA, 7.93±3.51; 18:1 LPA, 3.01±0.90; 18:2 LPA, 2.99±2.47; and 20:4 LPA, 0.37±0.09. Saturated (16:0 and 18:0) LPAs were dominant, but significant amounts of unsaturated (18:1, 18:2, and 20:4) LPAs were also present. We further compared the dedifferentiation activity of the following LPA species, 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, and 18:2 LPAs. At an equimolar (1 μmol/L) concentration of LPA species, unsaturated (16:1, 18:1, and 18:2) LPAs potently induced changes in cell shape and suppressed the expression of SMC differentiation marker genes, but saturated (12:0,14:0,16:0, and 18:0) LPAs did not (Figure 3A). The apparent IC50 values of unsaturated LPAs ranged from 20 to 30 nmol/L, whereas a >200-fold excess of saturated LPAs or their structural analogue and precursor, S1P and PA, had no effect on the SMC phenotype (Figure 3B). Because 18:1 LPA is a naturally occurring unsaturated LPA that induces VSMC dedifferentiation and is the richest unsaturated LPA in human serum, we used 18:1 LPA in the following experiments.
Signaling Pathways Stimulated by Unsaturated LPAs
We investigated the unsaturated LPA–stimulated signaling pathways in VSMCs. The ERK, p38 MAPK, and PKB(Akt) were maximally activated within 10 minutes after 18:1 LPA stimulation (Figure 4). The ERK activation by 18:1 LPA was specifically inhibited by 100 ng/mL PTX or 20 μmol/L PD98059, but not 10 μg/mL C3 exoenzyme or 10 μmol/L SB203580 (Figure 4A). Similar effects were also seen at the following concentrations of inhibitors: 200 to 300 ng/mL PTX, 30 μmol/L PD98059, 20 μmol/L SB203580 or 10 to 20 μmol/L SB220025, and 5 and 20 μg/mL C3 (data not shown). These results suggest that the Gαi-coupled cascade is directly linked to ERK activation. p38 MAPK activation was inhibited by SB203580 (Figure 4B) or SB220025 (data not shown), but not by PTX, C3, or PD98059 (Figure 4B). PKB(Akt) activation by 18:1 LPA was sensitive only to LY294002 (10 to 30 μmol/L) or wortmannin (10 to 30 nmol/L), but not to the other inhibitors examined here (Figure 4C), indicating that the activation is dependent on the PI3-K pathway.
The fully differentiated SMC phenotype was maintained, even under 18:1 LPA–stimulated conditions, when the ERK and p38 MAPK pathways were simultaneously blocked by PTX or PD98059 and SB203580 (Figure 5) or SB220025 (data not shown). In contrast, the 18:1 LPA–induced dedifferentiation was not blocked by PTX, PD98059, SB203580, or SB220025 alone (data not shown). C3 had no effect on the 18:1 LPA–induced loss of contractility or downregulation of the SMC differentiation marker gene expression but significantly blocked the change in cell shape (Figure 5), suggesting that the Rho-mediated pathway is involved only in SMC morphology. LY294002 (30 μmol/L) (Figure 5) or wortmannin (30 nmol/L) (data not shown) did not affect the 18:1 LPA–induced VSMC dedifferentiation. Thus, these results indicate that dedifferentiation of VSMCs by 18:1 LPA is also mediated through the coordinated activation of ERK and p38 MPAK. Consistent with the dedifferentiation activities of LPA species (Figure 3), the ERK and p38 MAPK pathways were activated by 18:2 LPA, but not by 18:0 LPA (data not shown).
Persistent Activation of the MAPK Pathways Evoked by Unsaturated LPA
We monitored the progressive changes in VSMCs stimulated with 1 μmol/L 18:1 LPA (Figure 6). Within 5 minutes, 18:1 LPA rapidly induced VSMC contraction, which was followed within 12 hours by a fibroblast-like shape change. Consistent with this shape change, VSMCs showed the downregulation of SMC marker gene expression within 12 to 24 hours, and then the cell migration activity. By the third day in culture, significant increases in cell proliferation, as detected by BrdU incorporation, were observed, suggesting that the phenotypic modulation of VSMCs precedes cell migration and proliferation.
To reveal the progressive changes that connect the 18:1 LPA–induced ERK and p38 MAPK activation with VSMC dedifferentiation, changes in the ERK and p38 MAPK activities were analyzed (Figure 7A). The ERK activity was maximally activated at 10 minutes after 18:1 LPA (1 μmol/L) stimulation, and this activation was significantly retained for at least 24 hours. The p38 MAPK activity stimulated by 18:1 LPA was also sustained for 24 hours. To examine the persistent activation of the MAPK pathways, VSMCs stimulated with 18:1 LPA for 10 minutes to 6 hours were cultured for a further 24 hours without LPA. These short exposures sustained the ERK and p38 MAPK activation and induced the VSMC dedifferentiation (Figure 7B), indicating that the 18:1 LPA–induced phenotypic modulation is irreversible. Furthermore, the 18:1 LPA containing cultured medium at indicated time points (3, 6, 12, and 24 hours) also activated the ERK and p38 MAPK activities and induced VSMC dedifferentiation within 24 hours after stimulation (Figure 7C), indicating that 18:1 LPA in culture medium might be stable. On the other hand, 18:1 or 18:2 LPAs (1 μmol/L) in basal medium without BSA had no effect on the induction of dedifferentiation (data not shown). These results suggest that BSA in basal medium might stabilize unsaturated LPAs.
Expression of LPA Receptors in VSMCs
Three LPA receptors, Edg-2, Edg-4, and Edg-7, have been reported.7,17,18 RT-PCR using single-stranded cDNAs from differentiated and 18:1 LPA–induced dedifferentiated VSMCs significantly amplified the Edg-2 and Edg-7 cDNAs, but not the Edg-4 cDNA (Figures 8A and 8B). As a control, cDNAs encoding the three receptors were readily amplified using single-stranded cDNAs from rat testis (Figure 8C). These results indicate that although Edg-2 and Edg-7 are expressed in differentiated and dedifferentiated VSMCs, Edg-4 is either not expressed or is expressed at an undetectable level in these cells.
In this study, we characterized the signaling pathways in differentiated VSMCs and confirmed that IGF-I potently activates the PI3-K/PKB(Akt) pathway, but not the ERK and p38 MAPK pathways (Figure 1). We also showed that the PI3-K/PKB(Akt) pathway plays a vital role in maintaining the differentiated phenotype of VSMCs, because treatment with PI3-K inhibitors induces a fibroblast-like shape change and loss of contractility.5,6 In contrast, PDGF-BB or EGF activated the ERK and p38 MAPK pathways coordinately, which induce VSMC dedifferentiation. Of these, PDGF-BB also activated PI3-K/PKB(Akt). Consequently, simultaneous blocking of the ERK and p38 MAPK pathways by their inhibitors was sufficient to maintain the differentiated phenotype, even under PDGF-BB–stimulated conditions (Figure 1). We further characterized the signaling pathways stimulated by unsaturated LPAs (Figures 4 and 5⇑); like the PDGF-BB stimulation, the ERK, p38 MAPK, and PKB(Akt) pathways were activated by 18:1 LPA, and the simultaneous blocking of both the ERK and p38 MAPK pathways suppressed SMC dedifferentiation. These results are understandable, because the PKB(Akt) pathway, which plays a vital role in maintaining the differentiated phenotype, was activated under conditions in which both the MAPK pathways were blocked. Thus, the unsaturated LPA–stimulated pathways in VSMCs presented here support our hypothesis that the VSMC phenotype is regulated by the balance in strengths between the PI3-K/PKB(Akt) pathway and the ERK and p38 MAPK pathways. Furthermore, ERK activation by 18:1 LPA was specifically suppressed by PTX, but p38 MAPK activation was not (Figure 4), suggesting that in differentiated VSMCs, the Gαi-coupled cascade directly links to ERK. As shown in Figure 7, ERK and p38 MAPK activities were sustained in VSMCs even after short exposure with 18:1 LPA. This persistent activation of two MAPKs would be directly involved in induction of VSMC dedifferentiation. Further study is required to determine whether this persistent activation is regulated upstream or downstream of the MAPK pathways.
It is well known that LPA plays a variety of biological roles.19,20 Several studies of LPA-induced SMC proliferation have also been published.16,21–23 However, all of these studies were performed using passaged (dedifferentiated) VSMCs and extremely high concentrations of LPA (10 to 100 μmol/L). Furthermore, these studies did not address LPA species. Of these, two studies reported the stimulation of MAPK in LPA-induced SMC proliferation.21,23 Here, we used our culture system to screen dedifferentiation factors for VSMCs and identified unsaturated LPAs in the serum lipid fraction as potent factors (Figures 2 and 3⇑).
The total concentration of unsaturated (18:1, 18:2, and 20:4) LPA species in human serum was calculated to be 6.37±2.74 μmol/L. As shown in Figure 2, the differentiated phenotype of VSMCs was completely abolished by the lipid fraction that corresponded to 2% human serum, which we calculated to be 73 to 180 nmol/L unsaturated LPAs. This result agrees well with the dose dependency of unsaturated LPA species (Figure 3). We also found that 18:1 LPA rapidly and irreversibly converted the VSMC phenotype followed by cell migration and proliferation (Figures 4 and 7⇑). Considering the potency of unsaturated LPAs in human serum in inducing VSMC dedifferentiation at low concentrations in vitro, naturally occurring unsaturated LPAs may be potent atherogenic factors. Although serum LPAs are generated in large part as the products of LPC by hydrolysis of lysophospholipase D,15 the amount of LPAs released from activated platelets is far smaller than that of serum LPAs.14 Platelet activation is, however, known to be involved in atherogenesis.24 LPA is generated through phospholipase A2–mediated deacylation of newly generated PA in activated platelets.25 Because the local concentration of LPA released from locally activated platelets is thought to be much higher,14 platelet-derived LPAs may be a source of unsaturated LPAs that induce atherogenesis. It has been well documented that oxidation of LDL is critically involved in atherogenesis.26 Indeed, oxidized LDL has been detected in the plasma of atherosclerotic patients and in atherosclerotic lesions.27,28 Most, if not all, of the atherogenic effects of oxidized LDL are derived from specific oxidized lipids. Siess et al29 recently demonstrated the formation of LPA from mildly oxidized LDL and the accumulation of LPA in atherosclerotic lesions, and suggested a possible role for LPA in thrombotic complications. However, they did not determine LPA species. We detected significant amounts of unsaturated LPA species in mildly oxidized LDL (Hayashi K, Nishida W, Yoshida K, Aoki J, Arai H, Ogawa A, Sobue K, unpublished data, 2001). These observations support our present insight that unsaturated LPA species might function as potent atherogenic factors.
Three LPA receptors (Edg-2, Edg-4, and Edg-7) have been identified.7,17,18 Egd-7 shows affinity for saturated and unsaturated LPAs, but the affinity for unsaturated LPAs is 2.5-fold higher than that for saturated ones.30 Although Edg-2 and Edg-4 show broad ligand specificities,30,31 the affinity of Edg-2 for unsaturated LPA is weak compared with that for Edg-4.17,30 As shown in Figure 8, VSMCs expressed two types of LPA receptor transcripts (Edg-2 and Edg-7). However, our present results (Figure 3) showed that the induction of VSMC dedifferentiation was specifically dependent on unsaturated LPA species. On the basis of these findings, we thought that Edg-7 may be partially involved in this unsaturated LPA–stimulated VSMC dedifferentiation, and we suspect the presence of as-yet-unidentified LPA receptors. Further studies are necessary to identify such receptor(s).
In this report, we demonstrated the identification of naturally occurring unsaturated LPAs as potent factors for inducing VSMC dedifferentiation and revealed unsaturated LPA–stimulated signaling pathways. Our present results provide clues for understanding the molecular mechanisms underlying phenotypic modulation of VSMCs in vitro and the development and progression of atherosclerosis in vivo.
This work was supported by Grants-in-Aid for Research on Brain Science from the Ministry of Health and Welfare of Japan (to K.S.) and in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to K.S.). We thank Dr S. Narumiya (Graduate School of Medicine, Kyoto University) for providing C3 exoenzyme.
Original received March 20, 2001; revision received June 8, 2001; accepted June 8, 2001.
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