Published online before print December 27, 2007, doi: 10.1161/ATVBAHA.107.158642
© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Upregulation of Pentraxin-3 in Human Endothelial Cells After Lysophosphatidic Acid Exposure
From the Laboratory of Biochemistry and Cellular Biology, University of Namur (FUNDP), Belgium.
Correspondence to Cindy Gustin, Department of Biochemistry and Cellular Biology, University of Namur (FUNDP), 61, rue de Bruxelles, 5000 Namur, Belgium. E-mail cindy.gustin{at}fundp.ac.be
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Abstract |
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Objective— The earliest event in atherogenesis appears to be endothelium dysfunction. Lysophosphatidic acid (LPA), one of the major bioactive lipid components of oxidized low-density lipoproteins (oxLDL), can cause the activation of endothelial cells (ECs), which start to secrete multiple proinflammatory polypeptides/proteins. The purpose of this study was to better document the proatherogenic properties of LPA using a subproteomic approach focused on the secretome of LPA-treated ECs.
Methods and Results— The secretome of LPA-treated ECs was analyzed using the 2D-DIGE approach. Among the 20 spots displaying significant variations of abundance compared with the control cells, we identified pentraxin-3 by mass spectrometry. Pentraxin-3 upregulation was confirmed at the mRNA and protein level, both on immortalized and primary ECs. LPA- but also oxLDL-induced pentraxin-3 upregulation was reduced in the presence of an antagonist of the LPA-receptors and largely dependent on NF
B activation. Finally, we demonstrated, for the first time, the chemotactic activity of pentraxin-3 on human THP-1 monocytes by using a chemotaxis assay.
Conclusions— Our findings favor the proatherogenic role of LPA, a bioactive lipid produced by activated platelets and present in oxLDL, because it enhances pentraxin-3 secretion that could contribute to the accumulation of monocytes in the atherosclerotic lesion.
Starting from a 2D-gel analytical approach, we demonstrated a LPA-induced pentraxin-3 overexpression in endothelial cells. LPA- but also oxLDL-induced pentraxin-3 upregulation was reduced in the presence of an antagonist of the LPA-receptors and was largely dependent on NFB activation. Finally, we demonstrated the chemotactic activity of pentraxin-3 on monocytes THP-1.
Key Words: lysophosphatidic acid • endothelial cell • atherosclerosis • Pentraxin-3 • chemoattractant
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Introduction |
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Cardiovascular diseases (CVDs), such as atherosclerosis, are the most common causes of death in developed countries. Atherosclerosis is a dynamic process that involves inflammation at all stages, from the early to the complex lesions.1 Inflammation is enhanced by all the CVD risk factors identified in epidemiologic studies, and particularly by elevated levels of low-density lipoprotein (LDL)-associated cholesterol.2 Oxidation of LDL is generally considered as one of the key events in atherogenesis, because oxidized LDL (oxLDL) is a major source of various bioactive modified (lyso)phospholipids.3 Among those oxLDL-derived lipids, lysophosphatidic acid (LPA) has been identified and largely studied to elucidate its role in atherosclerosis.4
LPA is a normal constituent of serum (2 to 20 µmol/L) and plasma (80 nmol/L to 0.7 µmol/L), with palmitoyl- and oleoyl-LPA being predominant.5,6 LPA was primarily described as a growth factor, but it can provoke a large variety of different biological responses in many cell types (for a review see7). LPA exerts its major biological effects by binding to specific transmembrane G protein–coupled LPA1-5 receptors,8 but LPA also binds with high affinity to the nuclear receptor peroxisome proliferator-activator receptor 
(PPAR).9 Vascular wall and blood cells express several types of LPA receptors, and there are data from many experiments indicating that LPA is a potentially athero- and thrombogenic molecule, because it stimulates platelet aggregation,10 promotes proliferation of vascular smooth muscle cells (VSMCs),6 and activates monocytes,11 macrophages,12 as well as vascular endothelial cells (ECs).13 Activated ECs, well known to interact with other cells such as monocytes, macrophages, or platelets, play an important role in the context of atherogenesis by secreting proteins, such as proinflammatory interleukins (interleukin [IL]-1, IL-6) and chemokines (monocyte chemotactic protein-1, IL-8). We therefore investigated the effects of LPA on the proteins secreted by ECs, using the 2D-DIGE approach to analyze the secretome. Significant variations of abundance were observed for 20 spots. One protein, found in 2 spots showing increased abundance, was identified by mass spectrometry and attracted our attention—pentraxin-3.
Pentraxin-3 is a secreted glycoprotein belonging to the pentraxin family of acute-phase proteins, such as C-reactive protein (CRP) and serum amyloid P component (SAP).14 Pentraxin-3, also named TSG-14 (tumor necrosis factor [TNF]
-stimulated gene-14), was originally described as a gene inducible by TNF- in human fibroblasts and, soon after, it was also identified as being induced by IL-1β in ECs.15,16 Pentraxin-3 expression is increased in several cell types including fibroblasts,15 ECs,16 chondrocytes,17 monocytes- macrophages,18 stimulated with TNF, IL-1β, or lipopolysaccharide (LPS). The function currently assigned to pentraxin-3 is to regulate innate immunity at a local level, through its binding to the C1q component.19 The involvement of pentraxin-3 in atherosclerosis remains largely unknown, but a few recent observations suggest a possible role of pentraxin-3 in this context. First, pentraxin-3 is strongly expressed in atherosclerotic lesions compared with normal arteries.20 It has also been reported that pentraxin-3 is induced by oxLDL in VSMCs.21 Finally, Latini et al22 have demonstrated the higher prognostic value of pentraxin-3 in acute myocardial infarction compared with the best-known relevant biological markers. Given the inflammatory nature of atherosclerosis and our results on LPA-induced overexpression of pentraxin-3 in ECs, the data suggest that LPA as well as pentraxin-3 may participate in the early stages of atherosclerosis. The upregulation of pentraxin-3 detected by 2D-DIGE analysis was confirmed by ELISA and Western blot in both immortalized and primary ECs. Pentraxin-3 upregulation was also observed at the transcriptional level, and was largely dependent on NFB activation. In addition, LPA- as well as oxLDL-enhanced transcription of pentraxin-3 was reduced in the presence of Ki16425, an antagonist of the LPA-1 and LPA-3 receptors. Finally, we showed that pentraxin-3 has a clear chemotactic activity on human THP-1 monocytes, which favors a proatherogenic role for pentraxin-3.
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Materials and Methods |
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Please see the supplement data, available online at http://atvb. ahajournals.org for all details.
Cell Culture
The EAhy926 cell line is a hybridoma produced by fusing human umbilical vein endothelial cells (HUVECs) with cells of the epithelial cell line A549. Primary HUVECs were purchased from Clonetics (Cambrex). Human monocytic THP-1 cells were purchased from the American Type Culture Collection (ATCC).
Statistical Analysis
Statistical analysis was achieved by the "Sigmastat" software using Student t test and 1- or 2-way ANOVA followed with post-hoc corrections (Holm-Sidak or Tukey test).
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Results |
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LPA Induces Pentraxin-3 Secretion in Human Endothelial Cells
Before starting the 2D-DIGE experiments, we had to optimize the LPA concentration. Therefore, we analyzed the effects of LPA over an extended range of concentrations on MCP-1 protein secretion on EAhy926 cells (unpublished data, 2007). These data revealed a concentration-dependent effect of LPA from 1 µmol/L to 50 µmol/L, with a marked effect at 25 µmol/L, followed by a plateau for the higher concentrations tested. Hence, EAhy926 cells were treated with 25 µmol/L LPA during 4 hours and the secretome of the LPA-treated cells (=LPA) was compared with nontreated cells (=CTL), using the 2D-DIGE approach and analyzed with the DeCyder software. Among the 20 spots displaying significant variations in abundance, 2 were identified by mass spectrometry as pentraxin-3 (spots 1 and 2, supplemental Figure I; NCBI entry number=4506333). Both spots showed a significant increase of abundance in LPA-treated cells (P<0.0076 and P<0.0032, respectively) with a 1.54- and 1.51-fold induction, respectively.
To confirm these data, we measured pentraxin-3 released into the supernatant of EAhy926 cells after 4 hours stimulation with LPA at 1, 10, and 25 µmol/L and after 6 and 8 hours incubation with LPA (25 µmol/L) and TNF (10 ng/mL). The latter, known to induce pentraxin-3 expression in several cell lines,16,18 was used as positive control. As measured by ELISA, LPA induces a concentration-dependent pentraxin-3 release with a maximal effect at 25 µmol/L after 6 hours (release of about 600 ng/mL pentraxin-3 compared with 200 ng/mL for the untreated cells; Figure 1). TNF significantly increases pentraxin-3 secretion reaching a maximum of 800 ng/mL after 8 hours incubation. We further studied pentraxin-3 expression by Western blots on LPA-stimulated EAhy926 and HUVECs. As shown in Figure 2, pentraxin-3 secretion increases after 4 and 6 hours stimulation with 25 µmol/L LPA both in EAhy926 (1.5- to 2.5-fold induction) and HUVECs (1.5- to 2-fold induction). The 2 observed bands probably correspond to the glycosylated (G-PTX3) and nonglycosylated (PTX3) forms of pentraxin-3, considering that the latter has an expected molecular mass of 40 kDa. This is also in agreement with the 2 spots identified as being pentraxin-3 in the 2D-gel analysis. Taken together, these data demonstrate an increased secretion of pentraxin-3 both in immortalized and primary ECs after LPA stimulation.
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Kinetics and Concentration-Dependent Effect of LPA-Induced Pentraxin-3 Expression
We further investigated whether the LPA-induced pentraxin-3 overexpression was also controlled at the mRNA level by using real-time RT-PCR. For both EC models, kinetics of LPA (25 µmol/L) stimulation were performed. As shown in Figure 3, we observed a rapid increase in the abundance of pentraxin-3 mRNA, normalized to GAPDH mRNA, which peaked at 2 hours (±240%) for the EAhy926 cells (Figure 3A) and at 3 hours (±220%), for the HUVECs (Figure 3B), followed by a second wave of induction for both cell types.
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The effect of LPA on pentraxin-3 mRNA expression observed in both EC models at lower LPA concentrations (1 µmol/L and 10 µmol/L), was concentration-dependent with a significant effect already observed with 1 µmol/L LPA (supplemental Figure II).
LPA Is One of the Major Bioactive Phospholipids in oxLDL Inducing Pentraxin-3 Overexpression
Because LPA accumulates during LDL oxidation, EAhy926 cells were treated for 2 hours with different concentrations of oxLDL (from 50 to 200 µg/mL) and pentraxin-3 gene expression was monitored by real-time RT-PCR. As shown in Figure 4A, oxLDL induces a concentration-dependent increase of pentraxin-3 expression with approximately a 2.5-fold induction (with 200 µg/mL), almost comparable to the one observed in the presence of LPA (Figure 3A). Moreover, a preincubation with 10 µmol/L Ki16425 (Ki), a specific antagonist of LPA-1 and LPA-3 receptors,23 before the incubation with oxLDL (200 µg/mL) for 2 hours, markedly diminished this overexpression to levels that are not significantly different compared with the negative control (Figure 4B). Native LDL (LDL) (Figure 4B) and Ki16425 alone were ineffective (data not shown). To check whether LPA was the major bioactive lipid responsible for this response, we tested 2 other lipids present within oxLDL, sphingosine-1–phosphate (S1P) and lysophosphatidylcholine (LPC). EAhy926 cells were incubated during 2, 4, 6, and 8 hours with these lipids. After real-time RT-PCR analysis, no overexpression was observed at any time or at any of the concentrations used (1 µmol/L and 20 µmol/L), as represented in Figure 4B for cells stimulated during 2 hours with 1 µmol/L S1P or LPC. We also combined S1P and LPA treatments, but S1P was unable to affect the magnitude of pentraxin-3 upregulation induced by LPA (data not shown). All together, these data demonstrate that oxLDL induces pentraxin-3 expression through LPA-1 and LPA-3 receptors in EAhy926 cells.
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Regulation of Pentraxin-3 Expression in LPA-Stimulated EAhy926 Cells
First, we highlighted the LPA-induced NFB transactivating activity by using a specific luciferase reporter plasmid (supplemental Figure IIIA). Then, using a colorimetric assay,24 we observed a significant increase in the DNA binding activity of NFB (using an anti-p65 antibody) after LPA (25 µmol/L) treatment during 45 minutes (supplemental Figure IIIB). However, this increase in DNA binding activity was markedly inhibited in the presence of 10 µmol/L Ki16425 (79% inhibition) and of 500 nmol/L evodiamine (90% inhibition), a recently described NFB inhibitor. Interestingly, evodiamine (EVO) by itself inhibits the constitutive DNA binding activity of NFB, as mentioned by Takada et al25 on myeloma cells. Finally, we tested these inhibitors on the LPA-induced pentraxin-3 upregulation by real-time PCR. As shown in supplemental Figure IIIC, the increase in the abundance of pentraxin-3 mRNA induced by 25 µmol/L LPA (after 2 hours stimulation) was inhibited by approximately 77% and 68% after preincubation with Ki16425 (10 µmol/L) and evodiamine (500 nmol/L), respectively. We also demonstrated that palmitoyl-LPA (a LPA isoform with a saturated fatty acyl chain unable to activate LPA-3 receptor) induces a dose-dependent effect on pentraxin-3 mRNA expression with inductions similar to the ones obtained with oleoyl-LPA (supplemental Figure IV). This increase in pentraxin-3 expression was inhibited by approximately 80% to 100%, after preincubation with Ki16425. All together, these data suggest that LPA-1 receptor is mainly involved in the LPA-induced pentraxin-3 overexpression. Ki16425, but also evodiamine alone, had no effect on the abundance of pentraxin-3 mRNA (data not shown). Experiments performed with BAY 11-7082 (another well-described NFB inhibitor), despite a higher cytotoxicity of BAY 11-7082 compared with evodiamine, confirmed the involvement of NFB.
Pentraxin-3 Induces the Transmigration of Monocytes In Vitro
Because the functions of pentraxin-3 are not yet completely defined, we wondered whether pentraxin-3 could be chemotactic for monocytes. To test this hypothesis, we used recombinant human pentraxin-3 (rhPentraxin-3) at concentrations ranging from 200 ng/mL to 800 ng/mL, which is comparable to the concentrations of pentraxin-3 achieved in the supernatants of ECs after LPA stimulation (Figure 1). As shown in Figure 5A, rhPentraxin-3 used in a modified Boyden’s chamber for 4 hours induces a significant monocyte transmigration with a weak concentration-dependent effect. We then evaluated the supernatants of LPA (1 µmol/L)-treated cells (4 hours) using the same assay and as shown in Figure 5B, we clearly demonstrate an effect of these supernatants on monocyte transmigration (LPA compared with CTL). This effect is even more pronounced than with rhPentraxin-3 probably because of the presence of additional chemotactic factors such as MCP-1 and IL-8 (unpublished data, 2007). Moreover, as demonstrated in supplemental Figure V, by adapting the assay, we could exclude that residual LPA present in the supernatant of conditioned media (CM) was required. We thus went on to evaluate the role of secreted pentraxin-3 in this chemotactic activity by abrogating pentraxin-3 expression with specific double-stranded siRNA (small interfering RNA). Using an ELISA, we showed that anti–pentraxin-3 siRNA attenuated pentraxin-3 expression by 80% (at the protein level) after 24 hours transfection (supplemental Figure VI). So, CM of EAhy926 cells stimulated with LPA (1 µmol/L) and transfected or not for 24 hours with siRNA against pentraxin-3 were tested on the THP-1 transmigration. First, CM from LPA-treated cells transfected with anti–pentraxin-3 siRNA (LPA+siRNA) displayed a significantly lower chemotactic activity (Figure 5B). Moreover, this inhibition was counteracted when 300 ng/mL of exogenous rhPentraxin-3 (concentration achieved in the supernatant of ECs after LPA treatment) were added (LPA+siRNA+rhPTX3) during the transmigration assay. Secondly, we also observed a significant effect of pentraxin-3 silencing in control cells (CTL+siRNA; Figure 5B). This is not surprising because nonstimulated EAhy926 cells secrete detectable amounts of pentraxin-3 (Figure 1), sufficient to almost double THP-1 chemotaxis (Figure 5A). CM of cells stimulated or not with LPA and treated with control siRNA did not alter monocyte migration (CTL and LPA RF).
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These data point out for the first time a new function of pentraxin-3 as a chemoattractant for monocytes.
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Discussion |
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The endothelium plays a major role in atherosclerosis given its function as secretory tissue, releasing several bioactive molecules.26 Therefore, analyzing the secretome of activated ECs should further contribute to a better understanding of the role of these cells in the initiation of atherogenesis. To activate ECs, we chose LPA, a bioactive lipid present within oxLDL and well known because sometime as a growth factor,27 but still a controversial molecule in the context of atherosclerosis, considered as either pro- or antiatherogenic.28
We demonstrated, by a subproteomic approach, that LPA clearly modulates the secretome of EAhy926 cells and significantly increases the abundance of pentraxin-3.
The 2D-gel data on pentraxin-3 were confirmed by ELISA and Western blot analysis on EAhy926 cells. Although the EAhy926 cell line is now considered as a well characterized vascular EC line,29 displaying various characteristics typical of human ECs,30,31 we confirmed the LPA-induced pentraxin-3 overexpression in HUVECs. We next performed a time-course analysis of pentraxin-3 mRNA expression. In both EC models, the abundance of pentraxin-3 transcripts rapidly increases, with a first peak at 2 hours and 3 hours, for the EAhy926 and HUVECs, respectively, followed by a second wave of induction. These pentraxin-3 protein and mRNA patterns of expression are comparable to those described in the literature with proinflammatory agents. For example, Alles et al18 demonstrated that LPS induces a peak of pentraxin-3 mRNA expression between 4 hours and 8 hours in peripheral blood mononuclear cells. They also showed that the regulation of pentraxin-3 expression requires an active protein synthesis in monocytes, which supposes the production of "intermediate proteins" not yet identified. We could propose a similar hypothesis in the induction of the second wave of pentraxin-3 expression after LPA treatment. Indeed, Lin et al32 have demonstrated that the induction of MCP-1 and IL-8 by LPA after 12 hours is regulated by IL-1, produced by the HUVECs in response to the primary stimulus (LPA). Therefore, based on our data obtained by the 2D-DIGE analysis, the "intermediate protein" could be one of the proteins overexpressed after LPA treatment, but not yet identified, because of the very limited amounts of material available for mass spectrometric analysis. Interestingly, the levels of induction of pentraxin-3 secretion induced by LPA and TNF are very similar. So, our data suggests that LPA may act as a true proinflammatory molecule. Secondly, we confirmed that LPA is able to modulate pentraxin-3 expression at concentrations as low as 1 µmol/L to 10 µmol/L, on both EC models. These concentrations might seem extremely high as nanomolar concentrations of LPA are sufficient to activate the LPA membrane receptors LPA1-5.8 But, as described in the literature, micromolar concentrations of LPA are required to induce DNA synthesis in cultured cells, and more specifically to induce transcriptional effects.33,34 Moreover, micromolar concentrations of LPA are not at all uncommon in vivo. LPA is known to be present in serum in concentrations of up to 2 to 20 µmol/L6 and is present in atherosclerotic lesions in concentrations 13 fold higher compared with normal arteries,4 although the absolute concentrations are not known. One might speculate that during the onset of inflammatory reactions, recruitment of LPA-producing cells such as monocytes-macrophages and ECs and multiplication of other potential sources for LPA production (activated platelets, higher concentrations of oxLDL, secretion of lyso-phospholipase D... ), could contribute to the delivery of high levels of LPA in a localized environment. Moreover, we demonstrated that oxLDL enhances pentraxin-3 expression through the activation of LPA-1 or LPA-3 receptors. To further study the specificity of the LPA-induced pentraxin-3, we also tested other bioactive lipids present within oxLDL, such as S1P and LPC, but they were unable to modulate pentraxin-3 gene expression. S1P has also be claimed to be able to counteract LPA, for instance, in human platelets.35 Combined with LPA, S1P (preincubated or together with LPA) was unable to affect the magnitude of pentraxin-3 upregulation induced by LPA (data not shown). We next started a preliminary study to unravel some of the mechanisms involved in the regulation of pentraxin-3, using a inhibitor of NFB (evodiamine) and an antagonist of LPA-receptors (Ki16425). We showed that LPA is able to induce pentraxin-3 through a mechanism involving mainly the LPA-1 receptor (because similar effects were obtained with both palmitoy- and oleoyl-LPA), as well as NFB activation, that is in agreement with the identification of 2 B elements in the promoter of the human pentraxin-3 gene.36
Finally, we investigated what could be the pathophysiological significance of pentraxin-3 secretion by ECs. Previous studies have suggested that CRP may favor the monocyte chemotactic response to MCP-1.37 We thus wanted to look for a possible chemotactic activity of pentraxin-3 on monocytes, an activity not assigned up to now to pentraxin-3. Using an in vitro chemotaxis assay, we demonstrated for the first time that rhPentraxin-3 (200 to 800 ng/mL) was able to induce a significant and concentration-dependent chemotaxis of THP-1 monocytes. We next abrogated pentraxin-3 gene expression by using siRNA. Anti–pentraxin-3 siRNA reduces the chemotactic activity of the supernatants of LPA-treated cells, but this activity was recovered when using the same CM supplemented with 300 ng/mL of rhPentraxin-3. The effect of anti–pentraxin-3 siRNA on monocyte migration was only partial (about 30% inhibition), probably because the expression of pentraxin-3 was not completely abrogated. Parenthetically, LPA induces other chemotactic agents such as MCP-1 and IL-8 (unpublished data, 2007).
In this manuscript, we show for the first time that LPA enhances pentraxin-3 expression at the mRNA as well as the protein level, in immortalized and primary ECs. Interestingly, LPA by so doing, is able to mimic oxLDL, on the contrary to S1P and LPC. We showed that this LPA-induced pentraxin-3 upregulation was reduced in the presence of an antagonist of LPA-1 and LPA-3 and was largely dependent on NFB activation. Finally, our findings suggest, at least in vitro, a chemotactic effect of pentraxin-3, which strengthens the proatherogenic role of LPA contributing to the pathogenesis of atherosclerosis, through the secretion of pentraxin-3. Consequently, our results suggest that pentraxin-3 and LPA may be new relevant therapeutic targets to consider for the treatment of inflammatory vascular lesions.
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Acknowledgments |
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We thank Doctor Karim Zouaoui Boudjeltia from the Experimental Medicine Laboratory (ULB-U222, CHU-Charleroi, Belgium) for providing the LDL and John Burns for editorial assistance.
Sources of Funding
This manuscript presents research results of the Belgian Programme on Interuniversity Poles of Attraction (PAI 6/30) initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. Cindy Gustin is a recipient of the FRIA (Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture) doctoral fellowship. We thank the Fonds de la Recherche Fondamentale Collective and the Facultés Universitaires Notre Dame de la Paix for financial support.
Disclosures
None.
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Footnotes |
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Original received September 3, 2007; final version accepted December 13, 2007.
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