Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of Gi, ammonium pyrrolidinedithiocarbamate and BAY 11–7082, inhibitors of the nuclear factor (NF)-κB pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a Gi-, NF-κB-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes.
- lysophosphatidic acid
- sphingosine 1-phosphate
- intercellular adhesion molecule-1
- nuclear factor-κB
- human umbilical cord vein endothelial cells
lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P), low molecular weight LPLs with diverse biological activities (18, 33, 44), are generated by enzymatic cleavage of membrane phospholipids. Recent evidence suggests that autotoxin, a previously identified exoenzyme, might be responsible for the generation of these two phospholipids (4). In serum, LPA and S-1-P reach micromolar concentrations and account for much of the cellular growth effects of serum (12, 46, 55). LPA and S-1-P are enriched in activated platelets, injured cells, and ovarian cancer cells, suggesting potential roles in inflammatory, wound-healing, and tumor-formation events (18, 43, 47, 52, 54, 55).
The cellular signals of LPA and S-1-P are transduced by two subfamilies of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes (Edg Rs; see Refs. 2, 5, 21, 34, 45). Human Edg1 (S-1-P1), Edg3 (S-1-P3), Edg5 (S-1-P2), Edg6 (S-1-P4), and Edg8 (S-1-P5) transduce signals for S-1-P. Human Edg2 (LPA1), Edg4 (LPA2), and Edg7 (LPA3) transduce signals for LPA. Multiple signaling pathways are activated by Edg receptors, which have been characterized in heterologous expression systems, including ras-dependent activation of Erk 1/2, increases in intracellular Ca2+ concentration, and recruitment of rho GTPase and its downstream targets (2, 5, 21, 34, 45).
Endothelial cells form the inner lining of blood vessels and participate in important physiological processes, including materials exchange, coagulation, and wound healing. Several pathological phenotypes, including atherosclerosis, inflammation, and cancer, are associated with excessive activation or abnormalities of endothelial cells (15). Endothelial cells express at least three types of Edg receptors, including Edg1, Edg 2, and Edg3 (26). LPA and S-1-P regulate several endothelial functions, including proliferation, migration, and secretion of proteases (4, 26, 36, 37). It has also been suggested that these lipids might also play a role in the regulation of angiogenesis and blood vessel integrity (4, 28, 29, 31).
The mechanisms by which vascular endothelial cells capture circulating lymphocytes are well documented, and several endothelial cell receptors responsible for these interactions have been described. Among these molecules, intercellular adhesion molecule 1 (ICAM-1; CD54) is one of the most characterized adhesion molecules expressed on endothelial cells. ICAM-1 interacts with LFA-1 and Mac-1, members of the β2-integrin family, which are expressed on activated lymphocytes. Interactions among LFA-1, Mac-1, and ICAM-1 are responsible for the firm interaction between leukocytes and endothelial cells and therefore are important for subsequent processes.
ICAM-1 is constitutively expressed in the microvasculature but not in large arteries or veins. However, in atherosclerotic arteries, ICAM-1 expression is elevated in the endothelium covering atherosclerotic plaques (9, 48). Abnormal interactions between monocytes and endothelial cells have been suggested to be one of the early events in the generation of atherosclerotic plaque and also are necessary for wound healing (17, 40). Both pretreatment of human saphenous veins with anti-ICAM-1 antibodies and pretreatment of monocytes with anti-β2-integrins significantly reduced monocyte adhesion to these vessels (7). Those observations suggest that abnormal ICAM-1 expression might be an important indicator of atherosclerosis.
Expression of ICAM-1 on endothelial cells is upregulated by inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and also by plasma lipoprotein such as oxidized low-density lipoproteins (LDLs; see Refs. 11, 14, 24, 41). Because LPA and S-1-P stimulate the expression of these inflammatory cytokines from macrophages (27), and LPA is generated along with oxidized LDLs (13, 32, 42), we hypothesized that LPA and S-1-P may affect endothelial cell interactions with leukocytes through modulating the expression of ICAM-1. In this report, we present evidence that LPA and S-1-P enhance ICAM-1 expression in human umbilical cord vein endothelial cells (HUVECs) in a time- and concentration-dependent fashion. Furthermore, by using chemical inhibitors, we show that LPA and S-1-P enhance ICAM-1 expression through a Gi/o-, nuclear factor (NF)-κB- and possibly Rac-dependent and Rho-independent mechanism, which is consistent with the signaling pathways activated by LPLs binding to Edg receptors. In addition, the adhesion between U-937 human mononucleated cells and HUVECs is enhanced by LPL treatment. This enhancement is likely because of an increase in ICAM-1 expression in HUVECs, since the enhancement is prevented by pretreatment with functional blocking antibody against human ICAM-1. These results imply that the inflammatory effects of LPL are likely mediated through the enhancement of ICAM-1 expression.
MATERIALS AND METHODS
Reagents and antibodies.
S-1-P was purchased from Biomol (Plymouth, PA). LPA, fatty acid-free BSA, and Clostridium difficile toxin B (Toxin B) were purchased from Sigma (St. Louis, MO). Pertussis toxin (PTx) and BAY 11–7082 (BAY) were from CalBiochem (La Jolla, CA). The C3 exoenzyme was purchased from List Biological Laboratory (Campbell, CA).
Human ICAM-1 antibody (clone M19), FITC-conjugated mouse anti-human ICAM-1 monoclonal antibody (clone 6.5B5), and functional blocking anti-human ICAM-1 monoclonal antibody (clone 15.2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse IgG was obtained from Boehringer Mannheim (Indianapolis, IN). FBS and M199 were purchased from Hyclone (Logan, UT). RPMI-1640 medium and trypsin were purchased from GIBCO-BRL (Grand Island, NY). Endothelial cell growth medium (EGM) was purchased from Cell Applications (San Diego, CA). Penicillin, streptomycin, and l-glutamine were purchased from Invitrogen (Carlsbad, CA).
HUVECs were isolated from fresh umbilical cords by treatment with 1% (vol/vol) collagenase in PBS at 37°C for 10 min. After elution with M199 containing 20% FCS, HUVECs were cultured on 0.04% gelatin-coated (Sigma) 10-cm plates in M199 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine (Invitrogen), 10% (vol/vol) FBS, and 25% (vol/vol) EGM, and cells underwent one passage weekly. Cells were subcultured after trypsinization [0.5% (vol/vol) trypsin solution, supplemented with 0.2% (vol/vol) EDTA] and used throughout passages 2 to 4. The human leukemic monoblast cell line, U-937 (CRL1593), was purchased from ATCC (American Type Culture Collection, Manassas, VA) and cultured in RPMI-1640 medium [supplemented with 0.2% (wt/vol) NaHCO3 and 0.03% (wt/vol) l-glutamine] containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% (vol/vol) FBS. Unless otherwise indicated, all cultures were grown at 37°C in a humidified atmosphere containing 5% CO2 in 175-cm2 culture flasks. U-937 cell stocks were maintained by subculturing cells every 4th or 5th day in fresh medium at a seeding density of 0.5 × 105 cells/ml (subculturing was performed earlier if the cell density exceeded 1.5 × 105 cells/ml).
RT-PCR for ICAM-1.
Total cellular RNA was extracted from HUVECs by the Tri-Zol reagent (GIBCO-BRL), and a Superscript kit (GIBCO-BRL) was used for the RT synthesis of cDNA. PCR amplification was performed with 35 cycles of 30 s at 94°C, 30 s at 62°C, and 2 min at 72°C. Oligonucleotides pairs were 5′-dGCAAGCTCCCAGTGAAATGCAAAC and 5′-dTGTCTACTGACCCCAACCCTTGATG for human ICAM-1. A 498-bp product was expected in the reaction. The primers used to amplify GAPDH were 5′-dACCACAGTTCATGCCATCAC and 5′-dTCCACCACCCTGTTGCTGTA. A 450-bp product was expected in the reaction. PCR products were resolved on 2% agarose gels, stained with ethidium bromide, and photographed.
To assess the levels of ICAM-1 mRNA in HUVECs, RT-PCR results were scanned using a PhosphoreImager and analyzed by ImageQuaNT software (Amersham Biosciences, Piscataway, NJ). Human GAPDH from the same sample in the RT-PCR was used as an internal control to correct the loading.
Subconfluent cultures of HUVECs were serum starved with or without chemical inhibitors for 16 h and incubated with LPA or S-1-P for the indicated durations at 37°C. Suspensions of 106 HUVECs in a 50-μl volume of PBS with 0.1% fatty acid-free BSA received 20 μl FITC-conjugated human anti-ICAM-1 (clone 6.5B5) and were incubated for 30 min at 4°C. Antibody binding of HUVECs with and without stimulation was determined by CyFlow SL (Partec, Germany) and analyzed by WinMDI version 2.7 software.
Western blotting for ICAM-1.
Endothelial cells plated in six-well plates were treated with the indicated concentrations of LPA or S-1-P. Treated cells were then lysed with RIPA buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) containing protease inhibitor cocktail (Sigma) and 2 mM sodium vanadate. Equal amounts of cell lysates were separated by ∼4–10% SDS-PAGE and electrophoretically transferred to Immobilon membranes. The transferred membranes were blocked with blocking buffer (PBS + 0.1% Tween 20 with 1% BSA) at room temperature for 1 h. The membranes were immunoblotted with anti-human ICAM-1 monoclonal antibody for 2 h and then washed in washing buffer (0.1% Tween 20, without 1% BSA) one time for 15 min followed by two washes for 5 min. The membranes were blocked again in new blocking buffer for 1 h at room temperature and then immunoblotted with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h. Membranes were subsequently washed in washing buffer one time for 15 min followed by two washes for 5 min each. Proteins on each immunoblot were visualized with Renaissance Western blot chemiluminescence reagent (NEN Life Science, Boston, MA). Blots were stripped and reprobed with an antibody against human actin to demonstrate uniform loading of proteins.
HUVECs were grown and treated on glass coverslips at 37°C. After being fixed in methanol (−20°C) for 5 min and air-dried, 50 μl of mouse anti-human ICAM-1 monoclonal antibody (4.5 g/ml in 0.5% FBS-0.5% Tween 20-PBS) were applied as the primary antibody solution and incubated overnight at 4°C in a humidified chamber. After extensive rinsing with PBS, the coverslips were incubated with 50 μl of Cy3-conjugated goat anti-mouse IgG (DAKO, Carpinteria, CA) as the secondary antibody (1:20 in 0.5% FBS-0.5% Tween 20-PBS) for 2 h at 37°C. After three washes with PBS, cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (1:5,000 in 0.5% FBS-0.5% Tween 20-PBS) purchased from Sigma. Glass coverslips were subsequently washed three times with double-distilled water, mounted with Fluoromount G (Electron Microscopy Sciences, Washington, PA), and examined using a confocal microscope.
To examine the distribution of ICAM-1, immunostained cells were mounted with Immunomount G. Cells were observed using a laser-scanning confocal microscope (Leica model TCS SP2) with a Leica Mellis-Griot ×63 numerical aperture oil immersion objective, with a pinhole of 1.5 and an electronic zoom of 1.5 or 2. Cy3 was excited using a 543-nm argon/krypton laser and detected with an ∼550- to 620-nm band-pass filter. Images were manipulated with a Leica TCS SP2 scanner.
Assay for U-937 adhesion to treated endothelium.
HUVECs cultured in six-well plates were stimulated for 8 h with 5 μM LPA or S-1-P. After being gently washed three times with M199, U-937 monocytes (1 ml, 5 × 106 cells/ml) were added to the cultures and then incubated at 37°C for 1 h. In the inhibitory experiments, anti-human ICAM-1 functional blocking antibodies (10 μg/ml) or normal mouse IgG was added before the addition of U-937 monocytes. Cultures were then washed by placing 1 ml of M199 gently on the cultures three to five times until no visible suspension of U-937 cells was observed. After being washed, cell cultures were imaged by microscopy with a Kodak digital camera, and the number of monocytes per culture was counted.
Significant differences between treatment groups were tested using ANOVA followed by Duncan's new multiple range test (StatView; Abacus Concept, Berkeley, CA). Each experiment was repeated at least three times. A value of P < 0.05 was considered statistically significant.
LPA and S-1-P increase ICAM-1 mRNA in HUVECs.
By DNA array analysis, we found that several genes that participate in inflammatory processes are upregulated by both LPA and S-1-P treatments. To confirm these observations, we determined the expression of ICAM-1 mRNA in LPL-treated HUVECs by RT-PCR. As shown in Fig. 1A, middle, and Fig. 2A, middle, ICAM-1 mRNA levels increased in both LPA- and S-1-P-treated samples in a concentration (Fig. 1A)- and time (Fig. 2A)-dependent manner. The expression patterns of GAPDH, which was used as the loading control, did not differ significantly in either LPA- or S-1-P-treated samples (Figs. 1A and 2A, bottom). The RT-PCR results were quantified and are shown in Figs. 1A and 2A, top. The maximum stimulatory effects were first observed with treatment at a concentration of 0.5 μM and peaked at 5 μM for both LPA and S-1-P treatments. Significant increases in ICAM-1 mRNA levels were first observed as early as 1 h after the treatments were added, peaked at 4 h, and decreased thereafter. These results confirmed our previous observations from DNA array experiments that both LPA and S-1-P enhance ICAM-1 mRNA expression in HUVECs.
LPA and S-1-P increase ICAM-1 protein expression.
Because mRNA levels were increased by LPLs, we further investigated whether the elevated mRNA levels of ICAM-1 were also correlated to protein expression levels. The total ICAM-1 protein levels in LPL-treated cells were detected by Western blotting. After 8 h of treatment, both LPA and S-1-P enhanced total ICAM-1 protein expression in a concentration (Fig. 1B)- and time (Fig. 2B)-dependent manner. Consistent with the RT-PCR results, the enhancement effects of LPA and S-1-P on ICAM-1 expression in HUVECs peaked at a concentration of 5 μM. In the time course experiments, the enhancement effects of both LPA (Fig. 2B, top) and S-1-P (Fig. 2B, bottom) peaked at 8 h after treatment and decreased thereafter. By confocal microscopy, we also observed a significant enhancement of ICAM-1 expression in LPA- and S-1-P-treated HUVECs (Fig. 3). By FACscan analysis, which detects the antigenic activities of proteins on cell surfaces, we observed a significant increase in cell-surface ICAM-1 levels in LPL-treated HUVECs (Fig. 4). These results indicated that the enhancement effects of LPLs on ICAM-1 also occur at the surface protein level, which might affect interactions between leukocytes and the endothelium during wound-healing and inflammatory processes.
In the time course experiments, we showed that both LPA (Fig. 5A) and S-1-P (Fig. 5B) enhancement of ICAM-1 protein expression on the surface of HUVECs also occurred in a time-dependent manner. LPL-enhanced HUVEC surface ICAM-1 expression was first observed at 4 h postligand treatment and peaked at 8 h postligand treatment. After 16 h, the effects of LPL on ICAM-1 expression levels of HUVECs returned to basal levels. These results indicate that both LPA and S-1-P are potent stimulators of ICAM-1 expression on HUVECs.
PTx, PDTC, and BAY, but not exoC3, blocked LPA and S-1-P effects on ICAM-1 expression.
Because LPA and S-1-P are ligands for Edg receptors, and at least three different Edg receptors are expressed on HUVECs (26, 30), we further investigated if the effects of LPLs on ICAM-1 expression are mediated through these receptors. We addressed this question by using chemical inhibitors known to impede signaling processes of activated Edg receptors. PTx, a specific inhibitor for Gi/o proteins that has been shown to inhibit Gi/o-dependent LPL effects in different cell systems, including HUVECs (26, 30), was used in our assay. Pretreatment with 15 ng/ml PTx for 16 h had no significant effect on the basal level of ICAM-1 expression in HUVECs (Fig. 8, A and B, top left). However, the stimulatory effects of 5 μM LPA or S-1-P on ICAM-1 expression in HUVECs were totally suppressed by the toxin treatment (Fig. 6, A and B, top left). On the other hand, pretreatment with 200 μM PDTC, an inhibitor of the NF-κB-dependent pathway (8), had no effects on basal ICAM-1 expression but significantly inhibited LPL-enhanced ICAM-1 expression on HUVECs (Fig. 6, A and B, bottom left). Moreover, pretreatment with 10 μM BAY, another specific inhibitor of the NF-κB pathway that selectively inhibits phosphorylation of inhibitory factor-κB-α, had no effects on basal ICAM-1 expression but significantly inhibited LPL-enhanced ICAM-1 expression on HUVECs (Fig. 6, A and B, bottom right). Pretreatment with 1 μg/ml exoC3, a specific inhibitor of Rho (26, 30), had no effects on either basal or LPL-enhanced ICAM-1 expressions on HUVECs (Fig. 6, A and B, top right). These results suggest that the enhancement effects of LPL on ICAM-1 expression are Gi/o- and NF-κB-dependent, but independent of the function of Rho. Similar results were also observed at the RNA level, as detected by RT-PCR (Fig. 7).
LPA and S-1-P enhanced U-937 cell adherence to treated HUVECs.
Because ICAM-1 expression on endothelial cells is responsible for their firm adhesion to leukocytes, we tested the hypothesis that LPL treatments enhance endothelial cell and monocyte adhesion by an in vitro adhesion assay. Five micromolars of LPA or S-1-P treatment on endothelial cells enhanced cell adhesion by the human monocystic cell line U-937 (Fig. 8). Furthermore, preincubation with the functional blocking antibody against human ICAM-1 significantly inhibited the enhancement effect of LPL on U-937 cell adhesion. On the contrary, a nonspecific antibody had no effects in these assays (Fig. 8, A and B).
Toxin B blocked the enhancement effects of LPA and S-1-P on ICAM-1 expression.
Rac has been shown to play important roles in regulating ICAM-1 expression in human endothelial cells (1). In addition, LPA has also been shown to be able to activate Rac in neuroblastoma cells (49). Therefore, we intended to determine whether Rac also participate in the enhancement effects of LPL on ICAM-1 expression on HUVEC. Toxin B, an inhibitor of the small GTPases Rho and Rac (16), was used in our assay. Pretreatment with 0.5 nM Toxin B for 2 h had no significant effect on the basal level of ICAM-1 protein expression in HUVECs, as detected by FACS. However, the stimulatory effects of 5 μM LPA or S-1-P on ICAM-1 expression in HUVECs were partially suppressed by Toxin B treatment (Fig. 9). Rho has no significant effect in the stimulatory property of LPA and S-1-P on ICAM-1 expression, suggesting that Rac might be involved in the enhancement effects of these LPLs on ICAM-1 expression on HUVEC.
The results of this report show evidence that LPLs generated from activated platelets or other cell regions might enhance ICAM-1 expression by surrounding endothelial cells. Previous work suggested that LPA induces the expression of ICAM-1 on endothelial cells (35). In this report, we show that S-1-P, an LPL that binds receptors belonging to the Edg family, has a similar effect on ICAM-1 expression on HUVECs. The enhancement effects might be mediated through a Gi/o-, NF-κB-, and Rac-dependent mechanism, which is consistent with the fact that Edg receptors activate these pathways. Furthermore, the time- and concentration-dependent activation of ICAM-1 by LPLs is also consistent with a receptor-mediated mechanism. It has been shown that both LPA and S-1-P receptors are expressed on HUVEC (26, 30). Therefore, the effects of LPA and S-1-P on these cells are expected to be mediated through these receptors.
In our previous study, we showed that LPLs are wound-healing factors in the endothelium (26). Another report also suggested that LPA facilitates wound healing in in vivo systems (3). Because ICAM-1 is an important regulator of interactions between leukocytes and endothelial cells, this suggests that the facilitating effects of LPLs on wound healing might partially be mediated through enhancement of ICAM-1 expression. From DNA array analysis and also by results reported by others, we know that LPA also enhances the expression of endothelial-leukocyte adhesion molecule (ELAM) on endothelial cells (28, 29). ELAM is responsible for the initial rolling effect during interactions between leukocytes and endothelial cells. These results suggest that the effects of LPLs on leukocyte-endothelial cell interactions during wound healing occur at multiple levels. This is consistent with the fact that, when tissue encounters a mechanical wound, LPLs are released from activated platelets, which might act as one of the initiating signals for the subsequent wound-healing processes.
With confocal microscopy, we also observed that LPL-enhanced ICAM-1 expression formed significant capping on HUVECs. The aggregation of cell-surface molecules upon antibody cross-linking or association with other cell types has been described in several papers (20, 22). The capping effects are likely because of surface-expressed ICAM-1 associated with the intracellular cytoskeletal structure. However, the physiological roles of this capping by adhesion molecules is currently unclear.
Activation of Rac has been shown to play important roles in regulating ICAM-1 expression in human endothelial cells (1). In addition, binding of LPA to LPA1 also activates Rac (49). In this study, we showed that the enhancement effects of LPA and S-1-P on ICAM-1 expression in HUVECs could be partially blocked by Toxin B, an inhibitor of Rac. These results suggest that the effects of LPLs on ICAM-1 in HUVECs are at least partially mediated through Rac, which is consistent with the previous reports.
By DNA array analysis, we also observed that LPLs enhanced both IL-1β and TNF-α expression in macrophages (27). Both IL-1β and TNF-α are cytokines that regulate the blood vessel environment, specifically endothelial cell physiology. Expression of ICAM-1 on endothelial cells is also upregulated by these cytokines (11, 14). IL-1β and TNF-α also participate in a self-augmentation induction mechanism (10), which allows a positive-feedback mechanism to amplify the effects of these cytokines within a local milieu. Furthermore, TNF-α directly and potently stimulates sphingosine kinase activity in HUVECs, inducing the generation of S-1-P, which further enhances endothelial and macrophage functions (53). These results suggest that platelet-derived LPLs might facilitate wound-healing processes through complex cell-cell interactions in the local environment.
It has been reported that concentrations of LPA and S-1-P in serum can reach micromolar levels (19). Therefore, it is likely that, at the wounded area, local concentration of LPLs may easily reach micromolar concentrations. The high concentration of LPLs at these wounded areas might generate a gradient of ICAM-1 expression, which is expected to peak at the spot of platelet activation. This might result in maximized leukocyte-endothelial interaction at these hot spots, thereby facilitating the wound-healing process.
Hypercholesterolemia is a major risk factor for atherosclerosis. Delivery of cholesterol to HUVECs results in an increase in ICAM-1 levels (56). Upregulation of vascular cell adhesion molecule-1, ICAM-1, and E-selectin in endothelial cells by inflammatory cytokines such as vascular endothelial growth factor has also been implicated in facilitating the formation and progression of atherosclerotic plaque (6, 25). Abnormal expression of adhesion molecules on endothelial cells and the large amount of trans-endothelial macrophage accumulation are two of the early events in the process of atherosclerosis generation. As mentioned earlier, ICAM-1 plays a crucial role in leukocyte-endothelial cell interactions. Previous studies also indicated that ICAM-1 is an important molecule involved in atherosclerosis (51). These results strongly suggest that LPLs may play multiple roles in the process of atherosclerosis through regulating the expression of ICAM-1 and inflammatory cytokines.
Several groups have reported the effects of LPA and S-1-P on leukocyte interactions with the endothelium. In one study, S-1-P inhibited neutrophil-endothelial cell interactions and subsequent neutrophil invasion (23), whereas in another report (39), LPA enhanced HL-60 cell adherence to human aortic endothelial cells. Apparent diversity of the effects of LPA and S-1-P on leukocyte-endothelial cell interaction may be attributed to the use of different leukocytes and endothelial cell types. In our report, we clearly showed that both LPA and S-1-P are potent enhancers of U-937 cell adhesion to HUVECs, and this is likely because of the enhancement of ICAM-1 expression. Our results suggest that platelet-derived LPLs enhanced leukocyte-endothelial cell interactions, consistent with the fact that they might be important regulators of the wound-healing process.
In summary, our results clearly indicate that LPLs increase ICAM-1 mRNA and protein levels in a time- and concentration-dependent manner. This induction is inhibited by specific inhibitors for NF-κB, Gi/o, and Rac. Furthermore, the enhancement effects of LPLs on ICAM-1 expression on HUVECs are responsible for augmentation of the adherence of U-937 cells to treated endothelium. Our results suggest that LPLs might be important physiological regulators of interactions between endothelial cells and mononuclear phagocytes. Therefore, these lipids might play an important role in the regulation of wound healing and possibly the generation of atherosclerosis.
This work was supported by National Science Council (NSC) Grants 90-2316-B-002-019, 91-2311-B-002-071, and 92-2311-B-002-095 (to H. Lee) and partly supported by NSC Grants 92-2314-B-010-039 and 92-2314-B-010-040, National Health Research Institutes Grant NHRI-EX93-9211SI (to H.-Y. Hsu), and Ministry of Education Program for Promoting Academic Excellence of Universities Grant 91-B-FA09-2-4 (to H.-Y. Hsu and H. L. Wu).
We thank Dr. J. H. Su Pang at Chang Gung University for technical assistance in endothelial cell culture.
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.
- Copyright © 2004 the American Physiological Society