High-Density Lipoprotein Stimulates Endothelial Cell Migration and Survival Through Sphingosine 1-Phosphate and Its Receptors
Objective— Plasma high-density lipoprotein (HDL) level is inversely correlated with the risk of atherosclerosis. However, the cellular mechanism by which HDL exerts antiatherogenic actions is not well understood. In this study, we focus on the lipid components of HDL as mediators of the lipoprotein-induced antiatherogenic actions.
Methods and Results— HDL and sphingosine 1-phosphate (S1P) stimulated the migration and survival of human umbilical vein endothelial cells. These responses to HDL and S1P were almost completely inhibited by pertussis toxin and other specific inhibitors for intracellular signaling pathways, although the inhibition profiles of migration and survival were different. The HDL-stimulated migration and survival of the cells were markedly inhibited by antisense oligonucleotides against the S1P receptors EDG-1/S1P1 and EDG-3/S1P3. Cell migration was sensitive to both receptors, but cell survival was exclusively sensitive to S1P1. The S1P-rich fraction and chromatographically purified S1P from HDL stimulated cell migration, but the rest of the fraction did not, as was the case of the cell survival.
Conclusions— HDL-induced endothelial cell migration and survival may be mediated by the lipoprotein component S1P and the lipid receptors S1P1 and S1P3.
Plasma lipoproteins are responsible for the transport of cholesterol to cells and the control of cholesterol synthesis.1–3⇓⇓ Low-density lipoprotein (LDL) provides cholesterol to cells through LDL receptors, whereas high-density lipoprotein (HDL) has been shown to remove excess cholesterol from the cells. The so-called reverse transport of cholesterol is thought to be an important mechanism for the antiatherogenic actions of HDL.1,2⇓ Recent studies have shown that HDL induces cytoprotective actions,3,4⇓ proliferation,5 and migration in endothelial cells,2,6⇓ activities presumably independent of cholesterol metabolism,2,3⇓ although the mechanism by which HDL induces these antiatherogenic actions has not been well characterized. It has been reported recently that HDL activates endothelial nitric oxide (NO) production through the scavenger receptor-BI (SR-BI).7 NO production has been shown to be involved in the cytoprotective action of endothelial cells.8
In endothelial cells, sphingosine 1-phosphate (S1P) has been shown to regulate a wide range of cellular activities associated with angiogenesis, wound healing, apoptosis, and atherosclerosis.3,4,8–18⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ S1P promotes cell migration,10–13,16–18⇓⇓⇓⇓⇓⇓ DNA synthesis,10 cell survival,4,9⇓ cell barrier integrity,15 NO production,8,14,16,17⇓⇓⇓ and the expression of several cell adhesion molecules.3 We recently reported that S1P accumulates in the lipoprotein fraction, especially the HDL fraction, and that HDL-associated S1P mediates the cytoprotective actions of HDL in human umbilical vein endothelial cells (HUVECs).4,19⇓ Nofer et al20 reported that sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF) were major components of HDL responsible for these cytoprotective actions. Thus, lipoprotein-associated lipids may also be involved in some HDL-induced actions independent of cholesterol metabolism; however, the species of lipid components are controversial. In the present study, an extension of the previous study,4 we examined the role of the lipid components of HDL in the stimulation of endothelial cell migration, which may be an important antiatherogenic action of lipoproteins.2,6⇓ We also reexamined the role of S1P in the cytoprotective activity of HDL by comparing it with the roles of SPC, LSF, and lysophosphatidic acid (LPA), which are reported to be present in HDL and other lipoproteins.19–21⇓⇓ We found that S1P may mediate HDL-induced migration as well as the cytoprotection of HUVECs through the G-protein–coupled lipid receptors EDG-1/S1P1 and EDG-3/S1P3.
Please see the online data supplement (which can be accessed at http://atvb.ahajournals.org) for materials, preparation of reconstituted HDL, cell culture and transfection, cholesterol efflux, extraction of active components of HDL, cell survival assay, migration assay, measurement of S1P content in lipoproteins, evaluation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase activity, RNA extraction and Northern blot analysis, and data presentation.
HDL Contains Adequate Amount of S1P to Duplicate Lipoprotein-Induced Cell Migration
Consistent with the previous report,6 HDL stimulated the migration of HUVECs in a dose-dependent manner. S1P also stimulated the cell migration (Figure 1). In these experiments, HDL or S1P was placed in the lower chamber and then cells were loaded on the upper chamber of the Boyden apparatus. We also used Transwell chemotaxis chambers, where the cells were first attached on the filers, and then the migration activity in response to HDL and S1P was determined. In this system as well, we observed a significant migration activity by HDL and S1P; the migration activity was increased 305±35% by 100 μg/mL HDL and 345±38% by 100 nmol/L S1P. In the separate experiments, we observed no significant effect of HDL and S1P on the adhesion of the cells on the dishes; the degree of the cells attached on the dishes 1 hour after plating was 80±9% by control, 83±8% by 100 μg/mL HDL, and 81±11% by 100 nmol/L S1P. These results clearly indicate that HDL and S1P enhance migratory activity of the cells but not their adhesion activity to the filter or extracellular matrix.
LDL also stimulated the migration by Boyden chamber method, but to a lesser extent. Oxidation of these lipoproteins markedly modulated their migration activity: oxidized HDL (oxHDL) reduced its activity to the level of LDL and oxLDL failed to exert any significant effect. Reconstituted HDL (rHDL), which was prepared by delipidation of HDL and subsequent reconstitution with phosphatidylcholine and cholesterol, was ineffective (Figure 1, inset), suggesting that lipid components rather than apolipoprotein components may be important for the induction of migration. The S1P content in these lipoproteins was 118±6 pmol/mg protein in HDL, 41±5 pmol/mg protein in oxHDL, and 48±12 pmol/mg protein in LDL (3 observations). The lipid content in oxLDL and rHDL was out of the range of determination (less than 8 pmol/mg protein). Thus, the migration activity induced by lipoproteins was roughly parallel to their S1P content. Based on the S1P content in HDL, the dose-response curve of S1P was overlaid on the same figure (Figure 1). The 2 dose-response curves of HDL and S1P almost completely overlapped, suggesting that the amount of S1P in HDL is high enough to account for the migration activity of HDL. The reconstitution of the inactive rHDL with the corresponding amount of S1P recovered its activity to the level of the native HDL (Figure 1, inset).
HDL and S1P Stimulate Migration Through Gi-Proteins and Intracellular Signaling Pathways Involving Phosphatidyl Inositol 3-Kinase and Rho Kinase
The migration response to HDL and S1P was completely inhibited by PTX, suggesting an involvement of toxin-sensitive Gi-proteins (Figure 2). Whereas the cytoprotective response to HDL and S1P was markedly inhibited by PD98059,4 the extracellular signal-regulated kinase (ERK) kinase inhibitor failed to inhibit the migration response. In contrast, the p38 MAPK inhibitor SB203580 greatly inhibited it (Figure 2). In accordance with these observations, HDL and S1P activated p38 MAPK in a PTX-sensitive manner, as shown in online Figure IA (please see http://atvb.ahajournals.org). These results suggest that p38 MAPK is critical for HDL-induced cell migration. Phosphatidyl inositol 3-kinase (PI3-K) and Rho kinase have also been shown to be involved in endothelial cell migration.11,12,16,17⇓⇓⇓ The migration response to HDL and S1P was markedly inhibited by wortmannin, a PI3-K inhibitor, and Y-27632, a Rho kinase inhibitor, which supports the above findings. Wortmannin inhibited the activation of p38 MAPK, but Y-27632 did not (see online Figure IA). Thus, p38 MAPK seems to be located downstream of Gi-proteins and PI3-K but seems to be independent of Rho kinase.
In addition to p38 MAPK, Akt is located downstream of PI3-K and is involved in S1P-induced migration.11,16⇓ We examined the relationship of p38 MAPK and Akt in the signaling pathways. Both HDL and S1P stimulated the formation of the active phosphorylated Akt in a manner sensitive to PTX and wortmannin (online Figure IB). However, the phosphorylation of Akt was insensitive to either SB203580 or Y-27632. These results suggest that Akt activation is dependent on Gi-proteins and PI3-K but not on Rho kinase, as was the case for p38 MAPK activation, but is independent of p38 MAPK itself. Thus, p38 MAPK and Akt are regulated by the same upstream Gi and PI3-K signaling pathways, but they seem to independently control downstream migration activity. In addition to the PI3-K pathways, Rho kinase may participate in the cell migration response to HDL and S1P.
HDL was indistinguishable from S1P with respect to the migration signaling mechanism. In addition, HDL contains a high amount of S1P to duplicate the lipoprotein action (Figure 1). When the cells were pretreated with S1P, they did not respond to S1P that was subsequently applied. Under these conditions, HDL-induced Akt phosphorylation was also completely lost without any reduction of the activity by vascular endothelial growth factor (VEGF) (online Figure IC). These results suggest that S1P or its related lipid mediator may mediate HDL-induced Akt activation and the downstream migration response to the lipoprotein.
SPC, LPA, and S1P Are Potential Lipid Mediators for Endothelial Cell Migration and Survival
As shown in Figure 3A, SPC, LPA, and S1P significantly stimulated migration in a dose-dependent manner, although the potency of SPC was roughly 2 orders lower than that of S1P and the potency of LPA was 4 orders lower. We could not detect any significant stimulation by LSF. S1P and SPC also stimulated cell survival, but neither LPA nor LSF was effective (Figure 3B). We have previously shown that S1P and HDL activate ERK and its activation is critical for their cytoprotective actions.4 Consistent with cell survival activity, SPC, but not LSF or LPA, induced the activation of ERK (data not shown).
Involvement of S1P1 and S1P3 in HDL-Stimulated Cell Migration and Survival
Transfection of antisense oligonucleotides against S1P1 and S1P3 resulted in a dramatic decrease in the expression of the respective S1P receptor mRNA in a specific manner (Figure 4A and online Figure IIA). The migration response to HDL and S1P was approximately 50% inhibited by the respective antisense oligonucleotide and 90% inhibited by their combination (Figure 4B). Under these conditions, the VEGF-induced activity was unchanged, indicating that the antisense strategy is efficacious and selective. Neither S1P1 nor S1P3 antisense oligonucleotide hardly affected the LPA-induced migration (Figure 4B), whereas they were effective for the SPC action (online Figure IIB). These results suggest that both S1P1 and S1P3 are necessary for the full migration response to HDL, S1P, and SPC but that LPA is not a ligand for these S1P receptors and is not a major mediator of lipoprotein-induced migration.
For cell survival activity, S1P1 may be a major receptor, as evidenced by the marked inhibition by S1P1 but the insignificant inhibition by S1P3 antisense oligonucleotides of HDL- and S1P-induced cell survival activity (Figure 4C). Likewise, S1P1 may be crucial to SPC-induced survival (online Figure IIC).
S1P Is a Major Component of HDL Responsible for Endothelial Cell Migration
Although SPC is a potential mediator of HDL-induced actions, its participation may be ruled out (Figure 5). In this experiment, components of HDL were separated into 3 fractions: fraction a, lipid fractions containing the majority of lipids, including SPC; fraction b, lipids soluble in an alkaline aqueous solution, such as S1P and LSF; and fraction c, substances soluble in an aqueous solution. In the previous study, we showed that cell survival activity was recovered in fraction b.4 Migration activity of HDL was also recovered in the S1P-rich fraction b but not in the potentially SPC-rich fraction a or c (Figure 5A). The lipid fraction b was additionally separated by high-performance thin-layer chromatography (Figure 5B), in which authentic S1P, SPC, and LSF migrated to apparently different positions. Although the S1P-rich fraction 5 clearly stimulated cell migration and cell survival,4 neither the potentially SPC-rich fraction 1 nor the potentially LSF-rich fraction 6 induced migration (Figure 5B). These results strongly suggest that the HDL-associated component responsible for cell migration and survival may be S1P but not SPC or LSF.
The present study showed that HDL stimulated endothelial cell migration and survival through S1P1 and S1P3, S1P-specific receptors. First, both HDL and S1P stimulated early intracellular signaling pathways in association with cell migration and survival. The involvement of intracellular signaling pathways in these antiatherogenic actions was supported by their inhibition by several specific inhibitors for Gi-proteins, PI3-K, Rho kinase, ERK kinase, and p38 MAPK, although the inhibition profiles of migration and survival differed. Second, desensitization of the S1P receptors led to the disappearance of the HDL- and S1P-induced phosphorylation of ERK4 and Akt (online Figure IC); the activation of these enzymes seems to be essential to survival and migration, respectively (see below). Third, the inhibition of the expression of S1P1 and S1P3 by the antisense oligonucleotide against the respective receptor resulted in the inhibition of HDL- and S1P-induced survival and migration (Figure 4). Fourth, HDL contains an adequately high amount of S1P to account for lipoprotein action. Finally, the S1P-rich fraction and chromatographically purified S1P from HDL stimulated cell migration, but the other fractions did not (Figure 5), which was also true for cytoprotective action.4
Nofer and colleagues20,22⇓ recently reported that SPC and LSF were concentrated in HDL and that these lipids mediate HDL-induced cytoprotective actions. In the present study, we confirmed that exogenous SPC mimics antiatherogenic migration and cytoprotective responses to HDL, and we found that these actions are mediated through S1P1 and S1P3. However, the fraction of HDL in which endogenous SPC is supposed to be recovered did not exert any significant action. In the case of LSF, we could not detect any significant response to either exogenous LSF or the potentially LSF-rich fraction of HDL. LPA was recently shown to be present in lipoproteins, especially oxLDL, and to stimulate stress fiber formation, gap formation, and cell permeability in endothelial cells.21 In the S1P-rich fraction of the HPTLC used in the present study, LPA was also partially recovered. However, HDL contains at most 100 pmol/mg protein LPA, as estimated based on its ability to inhibit cAMP accumulation in LPA1-expressing RH7777 cells (data not shown). We usually used 100 μg protein/mL HDL, which corresponds to 10 nmol/L LPA. However, the induction of significant migration activity required ≈100 to 1000 nmol/L LPA (Figure 3A), and no significant effect was detected at up to 10 μmol/L LPA on survival (Figure 3B). Furthermore, antisense oligonucleotides against S1P1 and S1P3 did not exert any significant effect on exogenous LPA-induced migration (Figure 4). These results may rule out the possibility that SPC, LSF, and LPA mediate HDL-induced actions.
The reason for the discrepancy between the present results and those of Nofer et al20,22⇓ remains unclear. However, it should be noted that the content of LSF and SPC in HDL estimated by Nofer et al22 is extremely high, exceeding the sum of major phospholipid components, ie, phosphatidylcholine and sphingomyelin. The chemical composition of HDL (by weight) is roughly 20% in cholesterol, 10% in triglyceride, 20% in phospholipids, and 50% in protein.23 Phosphatidylcholine and sphingomyelin account for approximately 80% of the total phospholipids.23 Based on these values, the content of these phospholipids in HDL is roughly 0.32 mg/mg protein. In accordance with these observations, we noticed that phosphatidylcholine and sphingomyelin are major phospholipids in HDL by HPTLC (data not shown). On the other hand, Nofer et al22 stated that LSF plus SPC reached approximately 10 μmol/mg of protein, corresponding to approximately 5 mg of LSF plus SPC/mg of protein in HDL based on their molecular weight of approximately 500. Mysteriously, in Nofer et al’s procedure by extraction with acetonitrile and separation by high-performance liquid chromatography of lipid components of HDL, they did not seem to recognize major phospholipid components phosphatidylcholine and sphingomyelin in HDL; instead, they mentioned that LSF and SPC were major lipid components of HDL.
S1P is concentrated in LDL, although to a lesser degree than in HDL. During oxidation, the S1P content in LDL markedly decreases.4,19⇓ This fact may in part explain why LDL significantly induced cell migration but oxLDL did not (Figure 1). In addition, lysophosphatidylcholine accumulates during the oxidation of LDL,4 and this lipid is known to inhibit the migration of endothelial cells.24 Lysophosphatidylcholine accumulation might also be partially involved in the loss of migration activity by oxLDL.
Results of the experiments using specific inhibitors for the signaling pathways suggest that S1P-induced migration involves Gi-proteins, PI3-K, small G-proteins such as Rho and Rac, and Akt.10–13,16–18,25⇓⇓⇓⇓⇓⇓⇓ The pathways may be composed of 2 major pathways: the PI3-K/Akt and Rac pathway, which leads to the formation of cortical actin assembly, and the Rho/Rho kinase pathway, which leads to the formation of focal contact and stress fiber.9,11,12⇓⇓ S1P1 and S1P3 may prefer the former pathway and the latter pathway, respectively, although their coupling is not always strict.9,11,12⇓⇓ The formation of cortical actin assembly may also function as an antiatherosclerosis event by enhancing the endothelial barrier integrity and reducing endothelial permeability.15 The present results are consistent with this scheme and indicate that HDL signals migration through its component S1P and the lipid receptors S1P1 and S1P3. In addition to these signaling events, p38 MAPK may play an obligatory role in the migration response to HDL and S1P, as evidenced by complete inhibition by a specific inhibitor to the enzyme. p38 MAPK is suggested to be involved in the migration of endothelial cells10,13,26⇓⇓ and other cell types.27–29⇓⇓ The activation of p38 MAPK was sensitive to PTX and wortmannin but not to Y-27632, suggesting that its activity is regulated by Gi-proteins and PI3-K but is independent of Rho kinase. Rac has recently been shown to be involved in p38 MAPK-dependent migration28,29⇓ and might function as an intermediate signaling molecule to transduce signals from PI3-K to p38 MAPK.
On the other hand, p38 MAPK may play little role in S1P- and HDL-induced cell survival, as suggested by a finding that a p38 MAPK inhibitor failed to inhibit the cell survival response to S1P and HDL. Instead, ERK activation may be essential for this pathway.9,10⇓ In addition, PI3-K/Akt-dependent NO synthase activation might play a critical role in the survival of HUVECs,8,20⇓ although the involvement of NO in cell survival is controversial.17 The ERK pathway and the PI3-K/Akt pathway may be independent. The inhibition of PI3-K by wortmannin14 and dominant-negative Akt11 hardly affected the S1P-induced activation of ERK. We also found that wortmannin did not affect the HDL-induced activation of ERK (data not shown). Although both S1P1 and S1P3 seem to participate in the migration response to HDL and S1P, S1P1 is likely to be a major S1P receptor involved in cytoprotective activity, as suggested by the finding that antisense oligonucleotides against S1P1 but not S1P3 markedly attenuated the cell survival activity of HDL and S1P (Figure 4C). Additional studies are necessary to clarify the signaling pathways and their molecular mechanisms, leading to the migration and survival responses to S1P and HDL.
The proposed mechanisms by which HDL induces antiatherogenic cell migration and survival in addition to the well-known action on cholesterol metabolism in endothelial cells are shown in Figure 6. As for cell survival and migration, the lipid component S1P in HDL may play an important role through the S1P receptors S1P1 and S1P3, which couple to Gi-proteins. The S1P1/ERK pathway is essential for cell survival, and both S1P1 and S1P3 are required for cell migration through the PI3-K/Akt and p38 MAPK pathway and the Rho/Rho kinase pathway. A similar mechanism mediated by S1P may account for other HDL-induced antiatherogenic actions independent of cholesterol metabolism. Cholesterol metabolism, on the other hand, is mediated by apolipoprotein receptors such as SR-BI and ABC1.30 SR-BI also may be involved in NO production in endothelial cells, although the signaling mechanism is not well characterized.7 HDL exerts antiatherogenic actions through multiple mechanisms involving more than 2 kinds of receptors and their intracellular signaling systems.
This work was supported in part by a research grant from the Ministry of Education, Science, and Culture of Japan and by research grants from the Mitsubishi Foundation and Yamanouchi Foundation for Research on Metabolic Disorders. The authors are grateful to Masayo Yanagita, Mayumi Komachi, and Masayuki Tobo for their technical assistance.
- Received December 24, 2002.
- Accepted May 7, 2003.
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