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Am J Physiol Heart Circ Physiol 279: H1830-H1838, 2000;
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Vol. 279, Issue 4, H1830-H1838, October 2000

Relationship of molecular structure to the mechanism of lysophospholipid-induced smooth muscle cell proliferation

Yuh-Cherng Chai, David G. Binion, and Guy M. Chisolm

Department of Cell Biology, Lerner Research Institute of the Cleveland Clinic Foundation, Cleveland, Ohio 44195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that oxidized low-density lipoprotein and one of its constituents, lysophosphatidylcholine (lysoPC), caused smooth muscle cell proliferation that was inhibitable by vitamin E and by a neutralizing antibody against basic fibroblast growth factor-2 (FGF-2). We now show that the mitogenic activity of lysolipids is highly dependent on structure. Phospholipids with palmitoyl fatty acid and phosphocholine induced DNA synthesis optimally. Shorter and longer fatty acids were significantly less potent, as were phosphoserine and phosphoethanolamine head groups. Structurally related phospholipids [platelet-activating factor (PAF) and lysoPAF] were also mitogens and acted via an analogous FGF-2-dependent, vitamin E-inhibitable mechanism. The mechanism of lysoPC stimulation was distinct from that of another phospholipid mitogen, lysophosphatidic acid (lysoPA), in that lysoPC stimulation was not pertussis toxin inhibitable. Furthermore, lysoPA stimulation was not inhibitable by vitamin E. Despite its distinct cellular pathway for stimulation, lysoPA also ultimately led to FGF-2 release. Our data show that specific structural attributes of lysoPC, PAF, and lysoPAF enable these agents to mediate smooth muscle cell release of FGF-2, which in turn stimulates proliferation.

basic fibroblast growth factor; lysophosphatidylcholine; lysophosphatidic acid; platelet-activating factor; vitamin E


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OXIDIZED LOW-DENSITY LIPOPROTEIN (oxLDL) resides in arterial lesions and in vitro has been shown to influence multiple functions in vascular cells, including having a growth-promoting effect on smooth muscle cells (SMC) (2, 3, 12, 17). Increased vascular SMC proliferation is a central feature in the development of atherosclerotic lesions and may play a role in restenosis after balloon angioplasty. Vitamin E not only inhibits low-density lipoprotein (LDL) oxidation (19), but it also inhibits the oxLDL stimulation of SMC proliferation (2, 17). Vitamin E and other antioxidants have been shown to inhibit both atherosclerosis (7, 29, 41) and restenosis (6, 34), indirectly suggesting a potential role for oxidized lipoproteins in vascular lesion development.

Lysophosphatidylcholine (lysoPC) is formed during oxidative modification of LDL by a LDL-associated phospholipase A2 activity (24, 28, 33), and it is present in atherosclerotic lesions (25, 44). LysoPC alters various intracellular signaling pathways in a variety of cell systems, including inducing cyclooxygenase-2 mRNA and protein (46), inducing nitric oxide synthase type III mRNA and protein (47), stimulating activator protein 1 DNA binding (9), activating c-Jun NH2-terminal kinase (9), activating adenylyl cyclase via a G protein-dependent pathway (45), and causing the release of arachidonic acid (42). Moreover, lysoPC has been shown to alter a variety of biological functions, including recruitment of monocytes (26), inhibition of endothelium-dependent relaxation (15), induction of adhesion molecules in cultured endothelial cells (16), and inhibition of endothelial cell migration (20). Thus understanding how lysoPC interacts with cells is valuable in discerning its influence on cell behavior. We previously reported that oxLDL induced approximately a seven- to eightfold increase in DNA synthesis in rabbit aortic SMC, which was up to 25% of that induced by serum (17). We (2) have also previously shown that a lysolipid-containing fraction extracted from either oxLDL or phospholipase A2-treated native LDL, as well as synthetic palmitoyl lysoPC (but not phosphatidylcholine), can stimulate an approximately eightfold increase in DNA synthesis in rabbit vascular smooth muscle cells, suggesting that lysoPC is a major contributor to oxLDL-induced SMC proliferation.

Our (2) previous results demonstrated that oxLDL- and lysoPC-induced vascular smooth muscle cell cycle progression was inhibited not only by the antioxidant vitamin E but also by a neutralizing monoclonal antibody to fibroblast growth factor-2 (FGF-2). In the present study, we used an ELISA to verify more directly that FGF-2 is released by lysoPC and that the conditioned medium containing the released FGF-2 is mitogenic. We also show that the mechanism we describe for rabbit SMC is not species specific but occurs analogously for human SMC. The mitogenic effect was optimal for C16:0 fatty acid and phosphocholine. Our data also show by multiple criteria that the cellular mechanism of lysoPC-induced DNA synthesis is distinct from that of lysophosphatidic acid (lysoPA), a known and well-characterized lysolipid mitogen. Interestingly, however, our data also reveal for the first time a dependency, at least in part, of the proliferative actions of lysoPA, platelet-activating factor (PAF), and lysoPAF on FGF-2 release.


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

Materials. Lysolipids [including lysoPC (1-palmitoyl-2- hydroxy-sn-glycero-3-phosphocholine) and lysoPA (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate)] were obtained from Avanti Polar Lipids (Birmingham, AL). PAF (1-o-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) and lysoPAF (1-o-hexadecyl-sn-glycero-3-phosphocholine) were obtained from Cayman Chemical (Ann Arbor, MI). [Methyl-3H]thymidine (6.7 Ci/mmol) was obtained from ICN (Irvine, CA). A monoclonal neutralizing antibody against FGF-2 and recombinant human FGF-2 were obtained from Upstate Biotechnology (Lake Placid, NY). According to the supplier, the antibody does not cross-react with acidic FGF (FGF-1). Fetal bovine serum (FBS) was obtained from BioWhittaker (Walkersville, MD). A mixture of equal parts of DMEM and Ham's F-12 (DMEM/F-12) cell culture media was purchased from Irvine Scientific (Santa Ana, CA). Mouse IgG and other chemicals were obtained from Sigma Chemical (St. Louis, MO). Pertussis toxin was obtained from Biomol Research Laboratories (Plymouth Meeting, PA).

Cultured rabbit and human vascular SMC. An immortalized rabbit SMC line that overproduces FGF-2 was obtained from Dr. Gene Liau (American Red Cross, Rockville, MD) and was used in most of the experiments shown. Liau and co-workers (40) have characterized this cell line, validating the overproduction of FGF-2 by use of a RNA gel blot hybridization, reverse transcription PCR, and Western blot. We (2) previously showed that these cells are stimulated by oxLDL and lysoPC to increase DNA synthesis by a mechanism identical to that in traditional explant cultures of rabbit aortic SMC, i.e., DNA synthesis was inhibited in both by vitamin E and a neutralizing antibody to FGF-2. Thus the FGF-2-overproducing rabbit SMC offer a tool to examine the same mechanism of the proliferative action of phospholipids in rabbit SMC with an amplified end point. LysoPC induced DNA synthesis by up to 40% of the stimulation by serum in traditional explant-derived rabbit SMC cultures but by ~75-80% that of serum stimulation in the FGF-2-overexpressing cell line. In selected experiments, explant-derived SMC from rabbit aortas were prepared as previously described (2). SMC were grown in DMEM/F-12 medium with 5% FBS and incubated at 37oC and 5% CO2 in air.

Cells were plated into 24-well plates at 20,000-25,000 cells/well (500 µl media/well) and allowed to reach 90% confluence before making the cells quiescent. Quiescence was achieved by replacing the media with serum-free DMEM/F-12 medium for 2-3 days. In all experiments, palmitoyl lysoPC was prepared at 1 mM as a stock solution in PBS and palmitoyl lysoPA was prepared at 1 mM as a stock solution in PBS with sonication and then added to wells at the concentrations indicated. Vitamin E was freshly prepared in ethanol, and the final concentration of ethanol in media was 0.2%. Control cultures had identical levels of ethanol.

Human aortas were obtained from heart transplant donors, and SMC were isolated from explants of excised aortas using procedures analogous to those previously described (1). Use of human tissue was approved by the Institutional Review Board of the Cleveland Clinic Foundation. Human SMC were grown in DMEM/F-12 medium with 10% FBS. Quiescent human SMC were obtained by replacing the media with 0.25% FBS in DMEM/F-12 medium for 2 days.

Analysis of FGF-2 by ELISA. Conditioned media were collected from SMC cultures after treatment with lysoPC for various times. After the conditioned media were removed from the cells, fatty acid-free bovine serum albumin (1 mg/ml) was added to block the lysoPC proliferative effect on the SMC receiving the conditioned media (the effectiveness of albumin to block the proliferative effect of lysoPC was verified in control experiments). The amount of FGF-2 in the media was quantified in duplicate wells by an ELISA kit from R&D Systems (Minneapolis, MN).

Determination of DNA synthesis in SMC. Proliferative stimulation by the conditioned media was assayed by adding the conditioned media plus albumin to quiescent, untreated SMC and quantifying DNA synthesis between 20 and 26 h after addition of the conditioned media. In some experiments, two wells of conditioned media were combined and then added to quiescent SMC. [3H]thymidine incorporation into DNA was determined by adding 0.5-1 µCi/ml [methyl-3H]thymidine to SMC culture medium for the last 6 h of a 26-h exposure to agents or conditioned media being tested for stimulation. After the radioactive medium was removed, cells were washed twice with ice-cold 10% trichloroacetic acid (TCA). TCA-insoluble material was hydrolyzed by 0.25 N NaOH, and radioactivity was assayed in a liquid scintillation counter. To examine the effects of vitamin E or various antibodies on DNA synthesis, cells were treated with these agents for 2 h before, as well as after, adding stimuli. Cells were pretreated with pertussis toxin for 4 h before adding stimuli. Data presented are the means ± SD of three replicate wells.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We (2) have shown that palmitoyl lysoPC was able to stimulate vascular SMC DNA synthesis and that this effect was inhibited by a neutralizing antibody to FGF-2. In this report, we verified more directly the release of FGF-2 into the conditioned media from lysoPC-treated SMC using an ELISA for FGF-2. We measured the concentration of the accumulated FGF-2 in the conditioned media at the time indicated. As shown in Fig. 1A, lysoPC caused rapid release of FGF-2 from SMC. The conditioned medium was able to stimulate SMC DNA synthesis in quiescent recipient SMC (Fig. 1B). To further determine the role of FGF-2 in conditioned media-induced DNA synthesis, the conditioned medium was incubated with a neutralizing antibody against FGF-2 or mouse IgG as a control antibody. As shown in Fig. 2, the neutralizing antibody nearly completely blocked conditioned media-induced DNA synthesis but the control antibody did not. We then tested whether the range of FGF-2 concentration measured in conditioned media was capable of inducing DNA synthesis in quiescent SMC. The addition of exogenous recombinant human FGF-2 at 30 and 100 pg/ml was able to stimulate SMC DNA synthesis to an extent similar to the conditioned media (compare Fig. 3 with Fig. 1).


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  		Fig. 1.   Elevated concentration of active fibroblast growth factor-2 (FGF-2) in the conditioned media from lysophosphatidylcholine (lysoPC)-treated smooth muscle cells (SMC). Quiescent rabbit SMC were treated with 10 µM lysoPC for various times. A: FGF-2 in the conditioned media was measured by an ELISA. Data are averages of duplicates; the experiment shown is representative of the results from 3 experiments. B: mitogenic activity of the conditioned media was assessed as the enhancement of DNA synthesis (measured through incorporation of [3H]thymidine) after transferring the media to quiescent SMC. Albumin was added during the transfer to block the lysoPC remaining in the conditioned media from inducing DNA synthesis in the recipient cells; the effectiveness of this blockage was verified in control experiments. Data are expressed as means ± SD.



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  		Fig. 2.   LysoPC-induced release of FGF-2 is responsible for the mitogenic activity of FGF-2. Conditioned media were collected from quiescent rabbit SMC treated with lysoPC for 30 min. During the collection, 1 mg/ml of fat acid-free BSA was added to the conditioned media to bind the free lysoPC. Conditioned media were incubated with 10 µg/ml of neutralizing antibody (Ab) to FGF-2 (Ab-bFGF) or mouse IgG for 1 h at 21oC before adding them to another quiescent recipient SMC for measuring DNA synthesis. Data are expressed as means ± SD.



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  		Fig. 3.   Recombinant human FGF-2-induced SMC DNA synthesis (measured through incorporation of [3H]thymidine) at concentrations similar to those found in conditioned media. Quiescent rabbit SMC were treated with FGF-2 at the indicated concentrations, and DNA synthesis was analyzed according to MATERIALS AND METHODS. Data are expressed as means ± SD.

We (2) had previously shown that lysoPC stimulated proliferation of both explant-derived cultures of rabbit aortic SMC and an FGF-2-overexpressing rabbit SMC line (2, 40) via a vitamin E-inhibitable, FGF-2-dependent mechanism. We tested whether the proliferative response was relevant to human SMC and whether the mechanism was similar. LysoPC induced human SMC DNA synthesis at concentrations similar to those that stimulated rabbit SMC (Fig. 4). The response was also inhibited by vitamin E and the monoclonal neutralizing antibody against FGF-2.


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  		Fig. 4.   LysoPC-induced DNA synthesis (measured through incorporation of [3H]thymidine) in human SMC was inhibited by vitamin E and by neutralizing Ab-FGF-2. Quiescent human SMC were treated with lysoPC, with or without vitamin E (Vit. E) and neutralizing Ab-FGF-2, using the same protocol as that used for rabbit SMC. [3H]thymidine incorporation into DNA was measured between 20 and 26 h. Data are expressed as means ± SD. PDGF, platelet-derived growth factor.

We then sought the importance of the molecular species of lysoPC on SMC DNA synthesis. Using the rabbit SMC line that overproduces FGF-2, we exposed cells to lysoPC with fatty acid chain lengths of C12:0, C14:0, C15:0, C16:0, C17:0, and C18:0 at the sn-1 position. Among them, palmitoyl (C16:0) lysoPC exhibited the most potent stimulation of DNA synthesis on an equimolar basis among the lysolipids tested (Fig. 5). A dramatic decrease in DNA synthesis occurred at 30 µM palmitoyl lysoPC, reflecting the well-known cytotoxicity of lysoPC at high concentrations (39). Interestingly, the lysolipids pentadecanoyl (C15:0) lysoPC and heptadecanoyl (C17:0) lysoPC, nonphysiological forms of lysoPC, were as effective as palmitoyl lysoPC in inducing vascular SMC proliferation. Lauroyl lysoPC (C12:0) at concentrations up to 30 µM failed to stimulate DNA synthesis to an appreciable extent. Myristoyl lysoPC (C14:0) and stearoyl lysoPC (C18:0) showed a concentration-dependent stimulation of DNA synthesis but to a significantly lower level than C16:0 lysoPC. We also tested oleoyl (C18:1) and linoleoyl (C18:2) lysoPC; neither was more effective than C18:0 lysoPC in stimulating DNA synthesis (data not shown). In a comparison among selected classes of lysophospholipids, we found that palmitoyl lysophosphatidylethanolamine and palmitoyl lysophosphatidylserine did not induce DNA synthesis at concentrations up to 30 µM (data not shown).


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  		Fig. 5.   The effect of varying fatty acid chain length (C12:0, C14:0, C15:0, C16:0, C17:0, and C18:0) of lysoPC on DNA synthesis (measured through incorporation of [3H]thymidine) in vascular SMC. Quiescent rabbit SMC were treated with lysoPC for 26 h, and [3H]thymidine incorporation into DNA was measured between 20 and 26 h. Data are expressed as means ± SD.

The significance of the chain length and head group of lysophospholipids to influence SMC DNA synthesis was substantiated by analyzing the release of FGF-2 in response to selected lysolipids. In independent experiments, the ELISA for FGF-2 showed that C15:0 and C16:0 lysoPC, but not C12:0 and C14:0 lysoPC or C16:0 lysophosphatidylethanolamine, caused a twofold increase of FGF-2 in the conditioned media after treating SMC with even submaximal concentrations of the lysolipids (data not shown). Our data thus suggest that both the chain length and head group determine the effectiveness of lysophospholipids to increase DNA synthesis in vascular SMC and reveal specificity for C16:0 and phosphocholine, respectively.

A biologically important, structurally related, subclass of phospholipids includes PAF (which has an ether-linked fatty acid at the sn-1 position, an acetyl group at sn-2, and phosphocholine at sn-3) and lysoPAF (which does not have the sn-2 acetyl group). We tested whether PAF and lysoPAF (with C16:0 at sn-1) could increase DNA synthesis in vascular SMC and whether they act by mechanisms analogous to lysoPC. In Fig. 6, we show that palmitoyl PAF induced vascular SMC DNA synthesis at concentrations similar to effective levels of lysoPC. LysoPAF also stimulated DNA synthesis at comparable concentrations and to an extent comparable to lysoPC. Like lysoPC, at higher concentrations (30 µM) both PAF and lysoPAF were cytotoxic. Because both PAF and lysoPAF induced SMC DNA synthesis at concentrations similar to lysoPC, we investigated whether these acted by a similar cellular mechanism. The mitogenic activity of lysoPAF was readily inhibited by both vitamin E (Fig. 7A) and the neutralizing monoclonal antibody against FGF-2 (Fig. 7B), whereas a control antibody failed to inhibit lysoPAF-induced SMC DNA synthesis. The release of FGF-2 by PAF or lysoPAF was confirmed by ELISA (data not shown). Vitamin E pretreatment and the neutralizing monoclonal antibody against FGF-2 were also capable of inhibiting PAF-induced SMC DNA synthesis (data not shown). The proliferative effects of PAF and lysoPC were not inhibited by either of two PAF receptor antagonists, WEB-2086 (10 and 50 µM) and L-659,989 (100 µM) (12), in these rabbit SMC (data not shown). These inhibitors were ineffective at concentrations previously shown by others (12) to block the PAF receptor.


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  		Fig. 6.   The effect of palmitoyl platelet-activating factor (PAF) and lysoPAF on DNA synthesis (measured through incorporation of [3H]thymidine) in vascular SMC. Quiescent rabbit SMC were treated with or without PAF and lysoPAF for 26 h, and [3H]thymidine incorporation into DNA was measured between 20 and 26 h. Data are expressed as means ± SD.



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  		Fig. 7.   LysoPAF-induced DNA synthesis (measured through incorporation of [3H]thymidine) was inhibited by vitamin E and by neutralizing Ab-FGF-2. A: vitamin E inhibited palmitoyl lysoPAF-induced DNA synthesis in rabbit SMC. Quiescent SMC were pretreated for 2 h with or without 50 µM vitamin E before treatment with or without lysoPAF for 26 h; vitamin E treatment was continued during lysoPAF exposure. Data are expressed as means ± SD. B: lysoPAF stimulation of DNA synthesis was inhibited by Ab-FGF-2. In a separate experiment, quiescent rabbit SMC were pretreated with or without 10 µg/ml of monoclonal neutralizing Ab-FGF-2 or a control antibody (mouse IgG) for 2 h before treatment without or with lysoPAF for 26 h. [3H]thymidine incorporation into DNA was measured between 20 and 26 h. Data are expressed as means ± SD.

We then examined whether the mechanism we observed, i.e., the pathway inhibited by both vitamin E and the neutralizing monoclonal antibody against FGF-2, was pertinent to the previously reported mitogenic effects of another phospholipid, lysoPA. We used multiple approaches to determine whether the mechanism of lysoPC stimulation was similar to that of lysoPA. Our results confirmed the expected lysoPA stimulation of DNA synthesis in our vascular SMC system in a concentration-dependent manner; however, vitamin E did not inhibit the lysoPA effect (data not shown). In further contrast to lysoPC, lysoPA was not toxic to cells at higher concentrations (e.g., 50 µM) (data not shown).

The mitogenic activity of lysoPA on fibroblasts has been shown to be via a pertussis toxin-sensitive, Gi protein-dependent signaling pathway (37). Consistent with this finding, pertussis toxin inhibited ~85% and 70% of the DNA synthesis induced by 20 and 50 µM lysoPA, respectively, in our SMC system; however, pertussis toxin failed to inhibit lysoPC-induced DNA synthesis (Fig. 8) or PAF- or lysoPAF-induced DNA synthesis (data not shown). In a control experiment, pertussis toxin did not inhibit FGF-2-induced DNA synthesis (data not shown). Both lysoPC and lysoPA have similar binding affinities to albumin (35), and bovine serum albumin has been shown to decrease the cytotoxicity of lysoPC in vascular endothelial cells (21). In our vascular SMC system, albumin inhibited the DNA synthesis induced by lysoPC but not that induced by lysoPA (data not shown). Taken together, these data indicate that the mechanism of lysoPA-induced DNA synthesis in vascular SMC is distinct from that of lysoPC, PAF, and lysoPAF. Interestingly, however, the neutralizing antibody against FGF-2 also significantly inhibited a portion of the lysoPA-induced vascular SMC proliferation (Fig. 9). The release of FGF-2 by lysoPA was confirmed by ELISA (data not shown). We then asked whether the release of FGF-2 by lysoPA was particular to the FGF-2-overproducing SMC. We assessed lysoPA stimulation in the more conventional, explant-derived rabbit SMC cultures, prepared as previously described (2). Both pertussis toxin and the neutralizing antibody against FGF-2 significantly inhibited the DNA synthesis induced by 50 and 100 µM lysoPA. For example, the increase in DNA synthesis by 100 µM lysoPA in the explant-derived SMC was inhibited ~80% by pertussis toxin and ~70% by the neutralizing antibody to FGF-2. The release of FGF-2 from lysoPA-treated SMC was not due to mortal cell injury, because lysoPA did not increase the specific release of radioactivity from [14C]adenine-preloaded SMC (data not shown).


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  		Fig. 8.   The effect of pertussis toxin on phospholipid-induced DNA synthesis (measured through incorporation of [3H]thymidine) in vascular SMC. Quiescent rabbit SMC were pretreated with or without 100 ng/ml pertussis toxin for 4 h before treatment with or without lysophosphatidic acid (lysoPA) or lysoPC for 26 h; pertussis toxin treatment was continued during lysoPA or lysoPC exposure. [3H]thymidine incorporation into DNA was measured between 20 and 26 h. Data are expressed as means ± SD.



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  		Fig. 9.   Neutralizing Ab-FGF-2 inhibited lysoPA-induced DNA synthesis (measured through incorporation of [3H]thymidine). Quiescent rabbit SMC were pretreated without or with 10 µg/ml monoclonal neutralizing antibody against FGF-2 (Ab-FGF-2) or a control antibody (mouse IgG) for 2 h before treatment with or without lysoPA for 26 h. [3H]thymidine incorporation into DNA was measured between 20 and 26 h. Data are expressed as means ± SD.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, our data reveal novel findings relating to lysoPC-induced stimulation of SMC proliferation. Specific structures of lysophospholipids were required for optimal stimulation; a fatty acid chain length of C16:0 at the sn-1 position and the presence of phosphocholine as the head group at the sn-3 position were both crucial determinants. Two structurally related molecules, PAF and lysoPAF, were also mitogens to vascular SMC, and they acted analogously to lysoPC, i.e., via a mechanism inhibitable by vitamin E and a neutralizing antibody against FGF-2. The fact that PC did not stimulate DNA synthesis (2), but PAF and lysoPC did, suggests that either the absence of a fatty acid or a short chain moiety in the sn-2 position is another essential structural feature to attain optimal proliferative activity. We have also shown by multiple criteria that the common cellular mechanism of lysoPC, PAF, and lysoPAF stimulation of DNA synthesis is distinct from that of another well-characterized lysolipid mitogen, lysoPA. Unlike lysoPC, the effect of lysoPA was inhibitable by pertussis toxin but was not blocked by vitamin E. However, lysoPA-induced DNA synthesis shared one trait with lysoPC, lysoPAF, and PAF. All acted, at least in part, by a FGF-2-mediated pathway.

The mechanism of release of FGF-2 from cells is not well understood. Lysis of a portion of the cell population and release of FGF-2 from lysed cells might have explained our observations; however, the release of FGF-2 by PAF, lysoPAF, and lysoPA did not require mortal cell injury because there were proliferation-inducing concentrations of these lysolipids that did not cause detectable increases in specific 14C release from SMC loaded with [14C]adenine (data not shown). This is consistent with our previous result with lysoPC (2). In addition, mitogenic concentrations of lysoPC were observed without injury using lactate dehydrogenase release (Sigma diagnostic kit) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays (Sigma procedure) of cell death (Y.-C. Chai and G. M. Chisolm, unpublished data). These data are consistent with lysolipid-induced cell permeabilization playing a role in the release of FGF-2 from SMC. Interestingly, the amount of FGF-2 in SMC-conditioned media after lysoPC treatment was in a range similar to that found after transient mechanical stimulation of human SMC (5).

We were interested to know whether the mechanism we identified in rabbit SMC was pertinent to human cells. LysoPC indeed exerted a proliferative effect on human SMC by a mechanism analogous to that observed in rabbit SMC, i.e., the lysoPC-stimulated DNA synthesis in these two cell types was inhibitable by both vitamin E and a neutralizing antibody to FGF-2. Despite the fact that this occurs in human SMC, it does not resolve the unknown issue of the role of lysoPC influencing cells in vivo. LysoPC has been shown to be present in human lesions (25, 44). The lysoPC concentration in healthy human plasma is reported to be ~130-150 µM (23) but up to 1.7 mM in hyperlipidemic patients (27). However, because lysoPC exists in equilibrium between bound and free forms, it is not clear what levels of free lysoPC are reached in an extravascular, extracellular inflammation site.

Our results show that FGF-2 is an essential component in conditioned media responsible for the optimum mitogenic activity stimulated by lysoPC, because the neutralizing antibody to FGF-2 nearly completely blocked the proliferative effect of the conditioned media but a control antibody, mouse IgG, did not. Moreover, the concentration of FGF-2 in conditioned media (typically, tens of pg/ml) was capable of stimulating SMC DNA synthesis to an extent similar to that stimulated by exogenously added recombinant human FGF-2 at 30 pg/ml. The proliferative response of cells to conditioned media taken from SMC treated with lysoPC was less than that of cells treated directly with lysoPC. This may have been due to the binding of FGF-2 to cells or the matrix of the donor cultures or the dilution of FGF-2 from mixing with the bulk media upon collection of the media, which resulted in the dispersion of the higher concentration of FGF-2 in the unstirred layer adjacent to the SMC plasma membranes where FGF-2 is secreted. The secretion of FGF-2 from lysoPC-treated SMC was protein synthesis independent, because cycloheximide treatment did not inhibit the lysoPC-induced release of FGF-2 in the conditioned media (Y.-C. Chai and G. M. Chisolm, unpublished data). This result suggests that the release of FGF-2 is from preexisting cellular pools of FGF-2.

LysoPC has also been reported by others (4, 30) to induce vascular SMC proliferation. These studies showed that lysoPC induced Ca2+ influx (4), stimulated p44/42 mitogen-activated protein kinase (43), and stimulated cationic amino acid uptake and the metabolism of growth-stimulatory polyamines (8). Our results suggest that these studies should be extended to determine which of these effects is due to a more direct effect of lysoPC and which to the effects of FGF-2 (or other growth factors) released secondary to lysoPC exposure.

LysoPC has also been shown to exert multiple effects on cell function other than the proliferative stimulus and, for some of these effects, the relationship of the structure of related bioactive lysolipids has been correlated with their cellular effects. These include chemoattraction of human monocytes (26), upregulation of adhesion molecules in endothelial cells (16), upregulation of heparin-binding epidermal growth factor-like mRNA in human monocytes (22), inhibition of endothelial cell migration (20), and release of arachidonic acid from endothelial cells (42). Interestingly, the studies of the lysolipid molecular structures that are necessary to elicit these particular cellular responses reveal that different, specific lysolipid structures are important in eliciting the different cell functions.

A proliferative response to PAF in various cells, including vascular SMC (12, 31, 32), has been previously reported (14, 18). Heery et al. (12) reported that PAF induction of DNA synthesis in bovine SMC occurred at 100 nM and was inhibited by two PAF receptor antagonists, WEB-2086 and L-659,989. In our system (i.e., in rabbit SMC), the stimulatory concentrations of PAF were optimal at much higher concentrations, in the 10-15 µM range, and the same two receptor antagonists did not inhibit PAF-induced DNA synthesis. Furthermore, lysoPAF, which is not generally considered to be biologically active, induced DNA synthesis to a degree similar to that by PAF. Therefore, our data in rabbit SMC suggest that the DNA synthesis enhancement by PAF is via a PAF receptor-independent pathway compared with DNA synthesis reported in bovine SMC, which appeared to be via a receptor-dependent pathway (12). The response to PAF and lysoPAF thus appears to vary among species of SMC. Stoll and Spector (31) reported that PAF can induce porcine vascular SMC proliferation by a PAF receptor-independent mechanism.

Our data show that lysoPC, PAF, and lysoPAF induction of DNA synthesis in vascular SMC is via a mechanism distinct from that by lysoPA. LysoPA is one of the major components in fetal calf serum responsible for the mitogenic effect of serum (36). The mitogenic action of lysoPA is via a pertussis toxin-sensitive, Gi protein-signaling pathway (37). The cDNA encoding lysoPA receptors have been cloned (10, 11). Most of the reported effects of lysoPA are mediated by receptor-dependent actions. The effective concentrations for lysoPA-induced cell proliferation have been reported to be up to 100 µM for fibroblasts (38) and vascular SMC (36). It has been suggested by Jalink et al. (13) that the relatively high concentration of lysoPA required may be due to vigorous uptake by cells or conversion in culture medium. In our system, lysoPA stimulated SMC DNA synthesis at both higher and lower concentrations than lysoPC, although the maximal response to lysoPA was lower than the maximal response to lysoPC. In contrast to lysoPC, vitamin E did not inhibit lysoPA-induced DNA synthesis. Furthermore, pertussis toxin inhibited lysoPA stimulation but did not inhibit stimulation by lysoPC, PAF, and lysoPAF. Interestingly, however, despite the differences in cellular pathways between lysoPC and lysoPA, lysoPA appeared to exert its proliferative effects in part by evoking FGF-2 release. The inhibition of the proliferative effects of high concentrations (e.g., 50 µM) of lysoPA treatment by either pertussis toxin or the neutralizing monoclonal antibody to FGF-2 was incomplete. This suggests that the effect of lysoPA may be by dual mechanisms, only one of which is receptor dependent. The release of FGF-2 by lysoPA is not particular to the FGF-2-overexpressing SMC used in most of our experiments, because we also observed that pertussis toxin and the neutralizing monoclonal antibody to FGF-2 inhibited 50 and 100 µM lysoPA-induced DNA synthesis in the more conventional explant-derived rabbit SMC (data not shown).

The present work provides insights into the cellular mechanisms for phospholipid stimulation of vascular SMC growth and shows very specific structural requirements for phospholipid-induced release of FGF-2 and increased DNA synthesis, although the mechanism for FGF-2 release remains unknown. Our work implies potentially important pathways by which lysoPC, PAF, lysoPAF, and lysoPA can mediate the release of growth factors and subsequent vascular SMC proliferation. The results may have implications for vascular lesion growth, because lysoPC is a major component of oxLDL and a known component of atherosclerotic lesions.


    ACKNOWLEDGEMENTS

WEB-2086 was generously provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT), and L-659,989 was generously provided by Merck (Rahway, NJ).


    FOOTNOTES

This study was supported in part by the National Heart, Lung, and Blood Institute Grant HL-29582 (to G. M. Chisolm) and a postdoctoral fellowship from the American Heart Association, Northeast Ohio Affiliate (to Y.-C. Chai).

Present address of D. G. Binion: Div. of Gastroenterology and Hepatology, Medical College of Wisconsin, Milwaukee, WI 53226.

Address for reprint requests and other correspondence: G. M. Chisolm, Dept. of Cell Biology, Cleveland Clinic Foundation (NC-10), 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: chisolg{at}ccf.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 September 1998; accepted in final form 24 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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