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Modified atherogenic lipoproteins induce expression of pentraxin-3 by human vascular smooth muscle cells
Atherosclerosis 175 (2004) 221–228
Modified atherogenic lipoproteins induce expression of pentraxin-3 by human vascular smooth muscle cells Mariam Klouche a,∗ , Giuseppe Peri b,c , Cornelius Knabbe d , Hanns-Henning Eckstein e , Franz-Xaver Schmid f , Gerd Schmitz a , Alberto Mantovani b,c a
Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauß Allee 11 93053 Regensburg, Germany b Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy c Istituto di Patologia Generale, Università di Milano, Milan, Italy d Robert-Bosch-Hospital, Stuttgart, Germany e Department of Vascular Surgery, Ludwigsburg, Germany f Department of Vascular and Thoracic Surgery, University of Regensburg, Regensburg, Germany Received 19 November 2003; received in revised form 1 March 2004; accepted 26 March 2004 Available online 10 June 2004
Abstract Inflammation is a critical contributing factor to the development and the progression of atherosclerosis. Recently, the acute-phase protein pentraxin-3 (PTX3), which has C-terminal sequence homology with the classic pentraxin C-reactive protein (CRP), was described to be increased in patients with myocardial infarction. In this study, we have investigated the capacity of human primary vascular smooth muscle cells (VSMC), derived from arterial specimens of ten different patients, to express PTX3 after incubation with atherogenic lipoproteins. Enzymatically degraded LDL (E-LDL), which is present in human early lesions, mediated a rapid cholesterol loading and foam cell transformation of primary VSMC, which was paralleled by a marked dose- and time-dependent expression of PTX3 mRNA and release of the acute-phase protein. Expression of PTX3 mRNA was delayed and remained almost undetectable for up to 6 h of incubation with E-LDL. However, during extended exposure to E-LDL for more than 24 h, PTX3 mRNA expression increased by more than 15-fold in VSMC foam cells, which was reflected by a concomitant release of up to 211 ng/ml PTX3 protein. We provide evidence for marked expression of PTX3 by VSMC induced by degraded lipoproteins, which may lead to an in situ vascular acute-phase reaction, contributing to the inflammatory pathogenesis of atherosclerosis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Smooth muscle; E-LDL; Acute-phase protein; PTX3
1. Introduction Inflammation plays a key role in the development of atherosclerosis and the related clinical manifestations. The progression of atherosclerosis is characterized by a continuing local inflammatory process, which is initiated by atherogenic lipoproteins and sustained by subendothelial acute-phase proteins and in situ activation of complement. A large body of literature supports oxidized LDL as the classic atherogenic lipoprotein [1]. Oxidized LDL is recognized by macrophage scavenger receptors and induced ∗ Corresponding author. Tel.: +49 941 944 6205; fax: +49 941 280 3210. E-mail address: [email protected] (M. Klouche).
foam cell formation [2,3], cytokine production [4], VSMC proliferation [5], and has been detected in atherosclerotic lesions [6]. We have demonstrated the presence of extensive extracellular deposits of different, enzymatically degraded forms of LDL in human fatty streaks, which appear well before the selective infiltration of monocytes or T-lymphocytes [7]. E-LDL was endowed with crucial properties of an atherogenic lipoprotein, including the induction of foam cell formation [8–10], proinflammatory actions, such as stimulation of chemokine and cytokine expression [9–11] and enhancement of the selective transmigration of monocytes and T-lymphocytes [12], as well as induction of VSMC interleukin (IL)-6 expression and mitogenesis [10]. Of note, lesional E-LDL colocalised with both the terminal complement complex C5b-9 [7] and with C-reactive protein (CRP) [13], and in contrast to Ox-LDL is endowed with the
0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.03.020
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capacity to activate complement [8], indicating an intimate interaction of the modified atherogenic lipoproteins with mediators of the innate immune system. The association of increased serum levels of acute-phase proteins with the progression of atherosclerosis and with the occurrence of atherosclerosis-related adverse events, such as coronary heart disease and myocardial infarction, has been well documented in several epidemiological studies [14,15]. In particular, elevated concentrations of the classic acute-phase protein CRP, as well as of serum amyloid A (SAA), have been implicated as an independent prognostic factor in atherogenesis [14,15]. CRP is the prototype of the family of a pentameric, evolutionary conserved classic pentraxins [16,17]. Members of this family play an important role in innate host defence against microorganisms and altered autologous substances. Classic pentraxins are predominantly expressed in the liver upon activation by IL-6 and other members of the IL-6 family [18], by interleukin (IL)-1 and tumor-necrosis-factor (TNF) ␣, or by foreign substances, such as LPS [16,17]. Thus, subendothelial deposits of CRP in developing atherosclerotic lesions result chiefly from passive transudation of elevated serum levels of this pentraxin. A decade ago, pentraxin 3 (PTX3) or TNF-stimulated gene (TSG)-14 was discovered as a cytokine-inducible gene in vascular endothelial cells [19] and in fibroblasts [20]. PTX3 is the prototype of the novel family of long pentraxins, which primarily encompasses members with unique tissue-restricted expression, such as the neuronal pentraxins NPTX2, NARP and NP, as well as apexin and Xenopus laevis XL-PXN1. Unlike CRP, the liver is not a major site of synthesis of the acute-phase protein PTX3 [21]. PTX3 exhibits C-terminal sequence homology with the classic acute-phase protein C-reactive protein, but displays a unique N-terminal sequence [22,23]. Similar to the classic pentraxins, PTX3 activates complement, and binds to microorganisms and to cellular debris. Moreover, PTX3 has been shown to bind apoptotic cells and to regulate their clearance by dendritic cells, marking its role as a novel scavenger molecule [24]. Recently, the presence of PTX3 protein was demonstrated in human advanced atherosclerotic plaques by immunohistochemistry [25]. In this study, we addressed the capacity of human vascular smooth muscle cells (VSMC) to express pentraxins and to excite a local acute-phase response. Vascular smooth muscle cells actively participate in the atherogenic process, and promote local inflammatory reactions. VSMC secrete proinflammatory cytokines and up-regulate adhesion molecules, migrate, and can be induced to proliferate after incubation with enzymatically degraded LDL [10]. We obtained primary human VSMC cultures from aortic specimens of eight different patients with early or advanced lesions. We then analysed the capacity of degraded lipoproteins to mount an acute-phase response in human primary VSMC, which could generate an in situ vascular inflammation independent from transsudated hepatic CRP. We demonstrate that athero-
genic lipoproteins activated human VSMC foam cells to express PTX3 mRNA and to release marked amounts of the acute-phase protein.
2. Materials and methods 2.1. Isolation and culture of human primary vascular smooth muscle cells Human primary VSMC were derived from arterial specimens from 10 different patients (mean age 68 years, seven male, three female), obtained during vascular operations after informed consent. Primary VSMC cultures were prepared as described [10]. Briefly, VSMC were obtained by outgrowth from pieces of media after 2–6 weeks in medium (PromoCell, Heidelberg, Germany) supplemented with 1% FCS, recombinant human FGF (1 ng/ml) and EGF (25 g/ml), and 25 g/ml Gentamicin. The typical morphology and positive staining for smooth muscle cell-specific ␣-actin (clone 1A4, IgG2␣, Cy3; Sigma, Deisenhofen, Germany) was confirmed. Prior to experiments, VSMC were kept in medium without supplements for 16 h, and cells up to the fifth passage were used. 2.2. Lipoprotein isolation and generation of E-LDL and Ox-LDL Native LDL (d = 1.020–1.062 g/ml) was obtained by preparative ultracentrifugation from healthy blood donor plasma [9]. Enzymatically degraded LDL was generated by subjecting native human LDL to treatment with trypsin, cholesterol esterase and neuraminidase, resulting in extensively degraded apolipoprotein B, as described in [8]. Dialysed E-LDL was generated after dialysis against Tris-buffer (5 mmol/L Tris; 150 mmol/L NaCl; pH 7,4) using a 10 kDa exclusion membrane (Perbio Science, Bonn, Germany). Oxidized LDL was obtained by incubating native LDL with 50 mol/L CuSO4 for 20 h at 37 ◦ C, and used after dialysis [9]. 2.3. Intracellular lipid labelling and quantification of cholesterol uptake VSMC foam cell formation was visualized using lipoproteins labelled with a fluorescent lipid dye 1,1-dilinoleyl-3,3, 3,3-tetramethylindocarbocyanine perchlorate (FAST DiI, Molecular Probes, Leiden, The Netherlands). After incubation with DiI (20 g/ml) for 12 h at 37 ◦ C, unbound chromophore was removed by ultracentrifugation in KBr gradients (d < 1.063), and labelled lipoproteins were dialyzed against Tris-buffer. 2.4. RNA extraction and RT-PCR Total RNA was extracted from two confluent wells (8 cm2 ) (Qiagen), and conventional RT-PCR for PTX3
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was performed using 1 g total RNA, as described [26]. PTX3 (Accession number NM 002852 and X63053) specific primers were selected: 5 -tggctgccggcaggt-3 and 5 -tccacccaccacaaacactat-3 , and absence of binding to the classic acute-phase protein CRP (Accession number NM 000567) was ascertained. Cyclophilin B (CPB) was consistently expressed by human VSMC and was used as a housekeeping gene (Seach-LC, Heidelberg, Germany). The PTX3 amplificate was cloned into the pCR3.1 TA vector (Invitrogen, Groningen, Netherlands), the standard was used in the same RT-PCR reaction, allowing quantification of PTX3 mRNA from 5 × 100 to 5 × 105 fM by real-time one tube RT-PCR with the Light Cycler technology (Roche, Mannheim, Germany), as described [26]. 2.5. Quantification of PTX3 protein PTX3 protein was measured using an enzyme-linked immunosorbent assay method based on PTX3-specific monoclonal antibody MNB4 and on biotinylated rabbit PTX3-specific polyclonal IgG, as described [27]. The VSMC culture supernatants were collected from two confluent wells after stimulation with E-LDL, Ox-LDL, LDL or in the presence of medium without additives (control). The amount of PTX3 released was normalized to the cellular protein content (mean 99 ± 3.7 g), corresponding to a mean VSMC number of 3 × 106 in two confluent wells, as determined in triplicate (n = 9). 2.6. Cell viability testing Viability of VSMC after incubation with E-LDL was assessed by Trypan Blue (0.1% in NaCl, Sigma) exclusion and by determination of unaltered intracellular ATP content, as described [9,10].
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Fig. 1. VSMC foam cell formation induced by degraded lipoproteins. Foam cell formation of VSMC after incubation with degraded lipoproteins was visualized by intracellular accumulation of fluorescent dye labelled (DiL-I) lipids. While E-LDL (upper right) and Ox-LDL (lower right) induced marked foam cell formation, native LDL had no effect on cholesterol-loading of VSMC (lower left). (Upper left) VSMC kept in medium alone served as controls. Lipoproteins were added at 50 g/ml based on cholesterol content for 24 h.
ter 30 min of E-LDL-induced cholesterol-loading. Similarly, Ox-LDL induced foam cell formation, while native LDL had no effect on intracellular lipid accumulation. VSMC kept in medium alone served as controls. E-LDL and Ox-LDL (each at 50 g/ml) mediated a pronounced upregulation of PTX3 mRNA in VSMC foam cells after 24 h of incubation, as shown for two different VSMC cultures derived from distinct patients (Fig. 2A). By contrast, native LDL did not alter the expression of the acute-phase protein. Induction of PTX3 mRNA by atherogenically modified lipoproteins was accompanied by a concomitant increased release of the acute-phase protein by VSMC (Fig. 2B). In the presence of native LDL, release of PTX3 was not altered.
2.7. Statistical analysis Experiments were conducted in triplicate and results are depicted as mean values ± standard deviation (S.D.), unless otherwise stated. For the determination of the significance of differences the Kruskall–Wallis test for comparison of multiple groups, and the Mann–Whitney U-test for comparison of two different groups was used. P < 0.05 was considered significant.
3. Results 3.1. Lipoprotein-driven expression of the acute-phase protein PTX3 in VSMC foam cells Enzymatically degraded LDL (50 g/ml) induced foam cell transformation of human primary VSMC, as visualized by uptake of fluorescent dye labelled DiI-E-LDL after 24 h of exposure (Fig. 1). VSMC foam cell formation started af-
3.2. Comparison of E-LDL induced PTX3 expression in nine different VSMC The marked induction of PTX3 mRNA expression which accompanied E-LDL mediated foam cell formation was confirmed in nine further VSMC cultures, derived from eight different patients (Fig. 3A). In resting primary VSMC, absent or very minor constitutive expression of PTX3 mRNA was detected. Upregulation of PTX3 mRNA was paralleled by a concomitant increase of the acute-phase protein release of up to 211 ng/ml by VSMC foam cells, corresponding to a more than 70-fold increased PTX3 protein liberation after 24 h of incubation with E-LDL (Fig. 3B). 3.3. Dose-dependent induction of PTX3 mRNA expression by E-LDL-activated VSMC E-LDL induced a dose-dependent expression of PTX3 mRNA by VSMC, which started from only 25 g/ml E-LDL
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Fig. 2. Atherogenically modified lipoproteins induce expression of the acute-phase protein PTX3 in VSMC. (A) E-LDL and Ox-LDL mediated pronounced expression of PTX3, while native LDL had no effect in two different VSMC cultures (upper panel). Expression of the housekeeping gene cyclophilin B (CPB) (lower panel). (B) Marked induction of PTX3 protein release by modified lipoproteins, whereas native LDL did not alter pentraxin liberation. Lipoproteins were added at 50 g/ml based on cholesterol content for 24 h. Results depicted as 1% agarose gel; M: molecular weight marker; PTX3 (233 bp), nc: negative control RT-PCR.
(Fig 4A). Induction of PTX3 mRNA expression was determined by real time RT-PCR and quantified with an external PTX3 standard covering six-log phases of concentration (Fig. 4B). In the presence of 25 g/ml E-LDL, expression of PTX3 mRNA almost doubled compared to untreated controls. Maximum expression resulting in a more than 12-fold induction of PTX3 mRNA was induced by incubation with 50 g/ml E-LDL. 3.4. Kinetics of E-LDL induced PTX3 expression Expression of the acute-phase protein PTX3 after activation with enzymatically degraded LDL was delayed, but remained elevated over extended periods (Fig. 5). Detectable induction of PTX3 mRNA expression did not start until after 6 h of incubation with 50 g/ml E-LDL, and was sus-
Fig. 3. Comparison of E-LDL-induced PTX3 mRNA expression and protein release in VSMC derived from different patients. VSMC foam cell formation was paralleled by marked induction of PTX3 mRNA expression and protein release. (A) E-LDL (50 g/ml) induced PTX3 mRNA expression in nine VSMC cultures derived from eight different patients (upper panel). Expression of the housekeeping gene cyclophilin B (CPB) (lower panel). (B) Concomitant release of the acute-phase protein into the culture supernatant. Incubations were conducted for 24 h. Results depicted as 1% agarose gel; M: molecular weight marker, nc: negative control RT-PCR. Results are expressed as means of duplicate determinations ± S.D.
tained at five-fold elevated concentrations during the presence of E-LDL for 96 h (Fig. 5A). Maximum induction of PTX3 mRNA expression was noted after 24 h of continuous incubation with E-LDL, resulting in a more than 15-fold expression of PTX3 mRNA compared to untreated controls (Fig. 5B). 3.5. Release of the acute-phase protein PTX3 by cholesterol-loaded VSMC foam cells E-LDL induced a dose-dependent release of the acute-phase protein from primary VSMC (Fig. 6). Release of PTX3 started from only 10 g/ml E-LDL and peaked at 50 g/ml E-LDL for 24 h, as exemplified for VSMC25 (Fig. 6A). Under these conditions, VSMC liberated more than 85 ng/ml PTX3, representing a more than 16-fold increase compared with untreated controls. Release of the acute-phase protein PTX3 by VSMC was retarded follow-
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Fig. 4. PTX3 mRNA expression after E-LDL-induced foam cell formation. Enzymatically degraded LDL induced dose-dependent expression of PTX3 mRNA in primary VSMC. (A) Regulated PTX3 mRNA expression depicted as mean fluorescence intensities by real time RT-PCR. (B) Induction of PTX3 expression started at 25 g/ml E-LDL, and peaked at 50 g/ml after 24 h of incubation. For quantification, the internal PTX-3 standard was used and upregulation of PTX3 mRNA by E-LDL was expressed in relation to unstimulated control VSMC.
ing E-LDL activation, as shown in time course experiments (Fig. 6B). While only minor liberation of the acute-phase protein was observed after up to 6 h of incubation with E-LDL, an increase by more than 70% of PTX3 release
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Fig. 6. PTX3 protein release by VSMC foam cells. (A) Dose-dependent release of PTX3 by E-LDL-loaded VSMC started from as low as 10 g/ml E-LDL, as exemplified for VSMC25. (B) Delayed kinetics of E-LDL (50 g/ml) induced release of the acute-phase protein. Quantification of PTX3 release by enzyme-immunoassay. Results are depicted as means of triplicate determinations ± S.D.
was observed after 24 h of incubation of VSMC with the degraded lipoprotein, as exemplified for VSMC19 (Fig. 6B). 3.6. PTX3 release in VSMC from different origin Comparison of PTX3 liberation revealed significant differences between spontaneous and E-LDL induced release of the acute-phase protein in the 11 primary VSMC cultures derived from distinct lesions of 10 different patients, as determined by the Kruskall–Wallis test (Fig. 7). E-LDL induced PTX3 release was significantly greater in VSMC foam cells derived from advanced lesions compared to cultures obtained from early lesions. It is of note that this discriminate behaviour was even evident when comparing VSMC derived from distinct lesions of the same patient (dashed lines). However, the spontaneous release of PTX3 by VSMC was not significantly different (n.s.) in VSMC derived from early or advanced lesions.
Fig. 5. Kinetics of PTX3 mRNA expression E-LDL induced a marked and sustained expression of PTX3 mRNA in VSMC. (A) Delayed induction of the acute-phase protein, as expressed by mean fluorescence intensities of PTX3 mRNA RT-PCR. (B) Expression of PTX3 mRNA became initially detectable after 6 h of E-LDL incubation, and remained elevated over the entire period of analysis of 96 h. Quantitative RT-PCR was used to calculate the amount of PTX3 mRNA generated in E-LDL-treated VSMC compared to untreated controls.
4. Discussion We identified enzymatically degraded LDL as an important regulator of the vascular acute-phase response by inducing marked expression of the acute-phase protein PTX3 in human VSMC. Our results show that human VSMC foam cells may acquire the potential to mount a local vascular
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Fig. 7. PTX3 release by cholesterol-loaded and na¨ıve VSMC. Significant induction of PTX3 release in E-LDL activated VSMC foam cells (∗ P < 0.05). Minor constitutive release of PTX3, which did not differ significantly (n.s.) in VSMC derived from early (white rhomb) or advanced (white square) lesions, by dot plot depiction. E-LDL mediated release of PTX3 was significantly more pronounced in VSMC obtained from advanced lesions (black squares) compared to early (black rhomb) lesions (∗∗ P < 0.008). Comparison of PTX3 release in VSMC derived from advanced and early lesions of the same patient (dashed lines).
acute-phase reaction, which delineates a previously unrecognised factor promoting atherogenesis. The continuous transudation of serum LDL followed by the subendothelial transformation into atherogenic lipoproteins by degradative processes, is believed to initiate and to maintain atherogenesis. Oxidisation of LDL is a well recognised atherogenic modification of LDL, allowing the binding and internalisation of Ox-LDL by scavenger receptors [1], the induction of macrophage foam cell formation [2,3] and cytokine expression [4]. Besides Ox-LDL, we have demonstrated the presence of enzymatically degraded LDL in human early atherosclerotic lesions [7]. E-LDL activated several central proatherogenic pathways, including foam cell formation [8–10], selective monocyte and lymphocyte transmigration [12], chemokine and proinflammatory cytokine generation [9–11]. In contrast to Ox-LDL, E-LDL is capable to activate complement [8]. Here, we report further evidence for a chronic vascular inflammatory process driven by the E-LDL-induced foam cell formation of VSMC and pronounced expression of the acute-phase protein PTX3. E-LDL induced a more than 12-fold increase of PTX3 mRNA expression and more than 70-fold release of the acute-phase protein by VSMC, while native LDL had no effect on the expression of the acute-phase protein. Similar to E-LDL, other atherogenic modifications, such as Ox-LDL, promoted PTX3 expression by VSMC. While the capacity of E-LDL to induce marked intracellular cholesterol accumulation and foam cell formation is undoubted [10], it is not clear whether cholesterol-loading itself or other biologically active compounds present in the enzymatically modified lipoprotein are responsible for PTX3 induction. The comparable PTX3 induction efficiency of E-LDL and E-LDL, which has been dialysed for substances of less than 10 kDa, a typical size of biologically active small molecules, does not identify small molecules as responsible for the induction of the acute-phase protein (data
not shown). The exact mechanism of PTX3 induction by E-LDL, however, remains to be elucidated. The recent immunohistochemical detection of PTX3 in intimal VSMC at the proliferative front immediately adjacent to the endothelium [25] supports the relevance of our findings. Similar to CRP [13] and to enzymatically degraded LDL [7], PTX3 was described to occur predominantly extracellularly [25]. Although macrophages were the predominant cell type reported to be associated with PTX3 expression in advanced lesions, our findings support the contribution of VSMC to PTX3 generation, which may be particularly important in early developing lesions before prominent inflammatory cell infiltration. While induction of PTX3 by proinflammatory cytokines has been described [19,20,28], expression induced by lipoprotein derivatives has not previously been demonstrated. We show that detectable levels of PTX3 mRNA arose only after prolonged exposure of VSMC to degraded lipoproteins, but once induced, remained elevated during the unhalted presence of the degraded lipoprotein. Comparable to resident vascular smooth muscle cells, delayed expression of the PTX3 was reported in macrophages in response to lipid constituents [29–31]. This points to a distinct regulation of the expression kinetics of the acute-phase protein PTX3 in the vascular acute-phase response as opposed to the rapid induction but limited duration of the expression of the classic pentraxin CRP in the liver [16,17]. Beyond the direct induction of PTX3 expression by VSMC, E-LDL-induced proinflammatory cytokines, including IL-1 and IL-6 [9,10], may contribute to the local acute-phase reaction by stimulating expression of the long pentraxin by infiltrating monocytes and by resident endothelial cells [19,20,30,31]. While the role of IL-1 in inducing PTX3 in monocytes, endothelial cells and fibroblasts is undisputed [19,20,30,31], the function of IL-6 on extrahepatic expression of the long pentraxin remains still unclear [21,22]. Previously, we have shown that E-LDL induced IL-6 mediated several proatherogenic effects, including induction of VSMC mitogenesis and chemokine production [32]. Recently, we have demonstrated that the viral homologue vIL-6 induced pronounced extrahepatic expression of PTX3 by neovascular Kaposi sarcoma cells [26]. Thus, E-LDL induced IL-1 and possibly IL-6 may add to the accumulation of the acute-phase protein in developing atherosclerotic lesions. PTX3 expression by VSMC may lead to an in situ vascular acute-phase-response, by binding to C1q and initiation of complement activation via the classic pathway [22,33]. In addition, PTX3 may contribute to the clearance of lipid-loaded macrophages and VSMC foam cells by opsonizing apoptotic cells, thus mediating their removal by dendritic cells [24,34]. We have shown previously that in contrast to native LDL, enzymatically degraded LDL is endowed with the capacity to bind CRP [13]. These E-LDL/CRP complexes displayed an altered functional capacity, including enhanced activation of complement
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[13], and promotion of monocyte transmigration (Klouche, unpublished observations). Recently, structural alterations of LDL leading to exposure of phosphorylcholine were identified to be responsible for complex formation with CRP [35]. Since, in contrast to CRP, PTX3 does not bind phosphorylcholine or phosphorylethanolamine [23], it is conceivable that the long pentraxin plays a distinct role in the inflammatory pathogenesis of atherosclerosis. We show for the first time the expression of the acute-phase protein PTX3 by VSMC. The capacity of human VSMC foam cells to mediate a vascular acutephase response provides a rationale for the recently described role of the long pentraxin PTX3 as an early indicator of atherosclerotic complications, such as acute myocardial infarction [36], and of the activity of generalized vascular inflammatory processes [27,37,38]. Our results reveal a lipoprotein-driven expression of the acute-phase protein PTX3 by VSMC, which may contribute to the inflammatory pathogenesis of atherosclerosis.
Acknowledgements We thank Franziska Walther for expert technical assistance and Tabea Peußer for technical support. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Bonn, Germany, KL1353) and by the Robert-Bosch-Medical Society to M.K.
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[34] Rovere P, Peri G, Fazzini F, et al. The long pentraxin PTX3 binds to apoptotic cells and regulates their clearance by antigen-presenting dendritic cells. Blood 2000;96:4300–6. [35] Chang MK, Binder CJ, Torzewski M, Witztum JL. C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: phosphorylcholine of oxidized phospholipids. Proc Natl Acad Sci USA 2002;99:13043–8. [36] Peri G, Introna M, Corradi D, et al. A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans. Circulation 2000;102:636–41. [37] Fazzini F, Peri G, Doni A, et al. Querini PR. PTX3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum 2001;44:2841– 50. [38] Napoleone E, Di Santo A, Bastone A, et al. Long pentraxin PTX3 upregulates tissue factor expression in human endothelial cells: a novel link between vascular inflammation and clotting activation. Arterioscler Thromb Vasc Biol 2002;22:782–7.
Modified atherogenic lipoproteins induce expression of pentraxin-3 by human vascular smooth muscle cells Mariam Klouche a,∗ , Giuseppe Peri b,c , Cornelius Knabbe d , Hanns-Henning Eckstein e , Franz-Xaver Schmid f , Gerd Schmitz a , Alberto Mantovani b,c a
Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauß Allee 11 93053 Regensburg, Germany b Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy c Istituto di Patologia Generale, Università di Milano, Milan, Italy d Robert-Bosch-Hospital, Stuttgart, Germany e Department of Vascular Surgery, Ludwigsburg, Germany f Department of Vascular and Thoracic Surgery, University of Regensburg, Regensburg, Germany Received 19 November 2003; received in revised form 1 March 2004; accepted 26 March 2004 Available online 10 June 2004
Abstract Inflammation is a critical contributing factor to the development and the progression of atherosclerosis. Recently, the acute-phase protein pentraxin-3 (PTX3), which has C-terminal sequence homology with the classic pentraxin C-reactive protein (CRP), was described to be increased in patients with myocardial infarction. In this study, we have investigated the capacity of human primary vascular smooth muscle cells (VSMC), derived from arterial specimens of ten different patients, to express PTX3 after incubation with atherogenic lipoproteins. Enzymatically degraded LDL (E-LDL), which is present in human early lesions, mediated a rapid cholesterol loading and foam cell transformation of primary VSMC, which was paralleled by a marked dose- and time-dependent expression of PTX3 mRNA and release of the acute-phase protein. Expression of PTX3 mRNA was delayed and remained almost undetectable for up to 6 h of incubation with E-LDL. However, during extended exposure to E-LDL for more than 24 h, PTX3 mRNA expression increased by more than 15-fold in VSMC foam cells, which was reflected by a concomitant release of up to 211 ng/ml PTX3 protein. We provide evidence for marked expression of PTX3 by VSMC induced by degraded lipoproteins, which may lead to an in situ vascular acute-phase reaction, contributing to the inflammatory pathogenesis of atherosclerosis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Smooth muscle; E-LDL; Acute-phase protein; PTX3
1. Introduction Inflammation plays a key role in the development of atherosclerosis and the related clinical manifestations. The progression of atherosclerosis is characterized by a continuing local inflammatory process, which is initiated by atherogenic lipoproteins and sustained by subendothelial acute-phase proteins and in situ activation of complement. A large body of literature supports oxidized LDL as the classic atherogenic lipoprotein [1]. Oxidized LDL is recognized by macrophage scavenger receptors and induced ∗ Corresponding author. Tel.: +49 941 944 6205; fax: +49 941 280 3210. E-mail address: [email protected] (M. Klouche).
foam cell formation [2,3], cytokine production [4], VSMC proliferation [5], and has been detected in atherosclerotic lesions [6]. We have demonstrated the presence of extensive extracellular deposits of different, enzymatically degraded forms of LDL in human fatty streaks, which appear well before the selective infiltration of monocytes or T-lymphocytes [7]. E-LDL was endowed with crucial properties of an atherogenic lipoprotein, including the induction of foam cell formation [8–10], proinflammatory actions, such as stimulation of chemokine and cytokine expression [9–11] and enhancement of the selective transmigration of monocytes and T-lymphocytes [12], as well as induction of VSMC interleukin (IL)-6 expression and mitogenesis [10]. Of note, lesional E-LDL colocalised with both the terminal complement complex C5b-9 [7] and with C-reactive protein (CRP) [13], and in contrast to Ox-LDL is endowed with the
0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.03.020
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capacity to activate complement [8], indicating an intimate interaction of the modified atherogenic lipoproteins with mediators of the innate immune system. The association of increased serum levels of acute-phase proteins with the progression of atherosclerosis and with the occurrence of atherosclerosis-related adverse events, such as coronary heart disease and myocardial infarction, has been well documented in several epidemiological studies [14,15]. In particular, elevated concentrations of the classic acute-phase protein CRP, as well as of serum amyloid A (SAA), have been implicated as an independent prognostic factor in atherogenesis [14,15]. CRP is the prototype of the family of a pentameric, evolutionary conserved classic pentraxins [16,17]. Members of this family play an important role in innate host defence against microorganisms and altered autologous substances. Classic pentraxins are predominantly expressed in the liver upon activation by IL-6 and other members of the IL-6 family [18], by interleukin (IL)-1 and tumor-necrosis-factor (TNF) ␣, or by foreign substances, such as LPS [16,17]. Thus, subendothelial deposits of CRP in developing atherosclerotic lesions result chiefly from passive transudation of elevated serum levels of this pentraxin. A decade ago, pentraxin 3 (PTX3) or TNF-stimulated gene (TSG)-14 was discovered as a cytokine-inducible gene in vascular endothelial cells [19] and in fibroblasts [20]. PTX3 is the prototype of the novel family of long pentraxins, which primarily encompasses members with unique tissue-restricted expression, such as the neuronal pentraxins NPTX2, NARP and NP, as well as apexin and Xenopus laevis XL-PXN1. Unlike CRP, the liver is not a major site of synthesis of the acute-phase protein PTX3 [21]. PTX3 exhibits C-terminal sequence homology with the classic acute-phase protein C-reactive protein, but displays a unique N-terminal sequence [22,23]. Similar to the classic pentraxins, PTX3 activates complement, and binds to microorganisms and to cellular debris. Moreover, PTX3 has been shown to bind apoptotic cells and to regulate their clearance by dendritic cells, marking its role as a novel scavenger molecule [24]. Recently, the presence of PTX3 protein was demonstrated in human advanced atherosclerotic plaques by immunohistochemistry [25]. In this study, we addressed the capacity of human vascular smooth muscle cells (VSMC) to express pentraxins and to excite a local acute-phase response. Vascular smooth muscle cells actively participate in the atherogenic process, and promote local inflammatory reactions. VSMC secrete proinflammatory cytokines and up-regulate adhesion molecules, migrate, and can be induced to proliferate after incubation with enzymatically degraded LDL [10]. We obtained primary human VSMC cultures from aortic specimens of eight different patients with early or advanced lesions. We then analysed the capacity of degraded lipoproteins to mount an acute-phase response in human primary VSMC, which could generate an in situ vascular inflammation independent from transsudated hepatic CRP. We demonstrate that athero-
genic lipoproteins activated human VSMC foam cells to express PTX3 mRNA and to release marked amounts of the acute-phase protein.
2. Materials and methods 2.1. Isolation and culture of human primary vascular smooth muscle cells Human primary VSMC were derived from arterial specimens from 10 different patients (mean age 68 years, seven male, three female), obtained during vascular operations after informed consent. Primary VSMC cultures were prepared as described [10]. Briefly, VSMC were obtained by outgrowth from pieces of media after 2–6 weeks in medium (PromoCell, Heidelberg, Germany) supplemented with 1% FCS, recombinant human FGF (1 ng/ml) and EGF (25 g/ml), and 25 g/ml Gentamicin. The typical morphology and positive staining for smooth muscle cell-specific ␣-actin (clone 1A4, IgG2␣, Cy3; Sigma, Deisenhofen, Germany) was confirmed. Prior to experiments, VSMC were kept in medium without supplements for 16 h, and cells up to the fifth passage were used. 2.2. Lipoprotein isolation and generation of E-LDL and Ox-LDL Native LDL (d = 1.020–1.062 g/ml) was obtained by preparative ultracentrifugation from healthy blood donor plasma [9]. Enzymatically degraded LDL was generated by subjecting native human LDL to treatment with trypsin, cholesterol esterase and neuraminidase, resulting in extensively degraded apolipoprotein B, as described in [8]. Dialysed E-LDL was generated after dialysis against Tris-buffer (5 mmol/L Tris; 150 mmol/L NaCl; pH 7,4) using a 10 kDa exclusion membrane (Perbio Science, Bonn, Germany). Oxidized LDL was obtained by incubating native LDL with 50 mol/L CuSO4 for 20 h at 37 ◦ C, and used after dialysis [9]. 2.3. Intracellular lipid labelling and quantification of cholesterol uptake VSMC foam cell formation was visualized using lipoproteins labelled with a fluorescent lipid dye 1,1-dilinoleyl-3,3, 3,3-tetramethylindocarbocyanine perchlorate (FAST DiI, Molecular Probes, Leiden, The Netherlands). After incubation with DiI (20 g/ml) for 12 h at 37 ◦ C, unbound chromophore was removed by ultracentrifugation in KBr gradients (d < 1.063), and labelled lipoproteins were dialyzed against Tris-buffer. 2.4. RNA extraction and RT-PCR Total RNA was extracted from two confluent wells (8 cm2 ) (Qiagen), and conventional RT-PCR for PTX3
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was performed using 1 g total RNA, as described [26]. PTX3 (Accession number NM 002852 and X63053) specific primers were selected: 5 -tggctgccggcaggt-3 and 5 -tccacccaccacaaacactat-3 , and absence of binding to the classic acute-phase protein CRP (Accession number NM 000567) was ascertained. Cyclophilin B (CPB) was consistently expressed by human VSMC and was used as a housekeeping gene (Seach-LC, Heidelberg, Germany). The PTX3 amplificate was cloned into the pCR3.1 TA vector (Invitrogen, Groningen, Netherlands), the standard was used in the same RT-PCR reaction, allowing quantification of PTX3 mRNA from 5 × 100 to 5 × 105 fM by real-time one tube RT-PCR with the Light Cycler technology (Roche, Mannheim, Germany), as described [26]. 2.5. Quantification of PTX3 protein PTX3 protein was measured using an enzyme-linked immunosorbent assay method based on PTX3-specific monoclonal antibody MNB4 and on biotinylated rabbit PTX3-specific polyclonal IgG, as described [27]. The VSMC culture supernatants were collected from two confluent wells after stimulation with E-LDL, Ox-LDL, LDL or in the presence of medium without additives (control). The amount of PTX3 released was normalized to the cellular protein content (mean 99 ± 3.7 g), corresponding to a mean VSMC number of 3 × 106 in two confluent wells, as determined in triplicate (n = 9). 2.6. Cell viability testing Viability of VSMC after incubation with E-LDL was assessed by Trypan Blue (0.1% in NaCl, Sigma) exclusion and by determination of unaltered intracellular ATP content, as described [9,10].
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Fig. 1. VSMC foam cell formation induced by degraded lipoproteins. Foam cell formation of VSMC after incubation with degraded lipoproteins was visualized by intracellular accumulation of fluorescent dye labelled (DiL-I) lipids. While E-LDL (upper right) and Ox-LDL (lower right) induced marked foam cell formation, native LDL had no effect on cholesterol-loading of VSMC (lower left). (Upper left) VSMC kept in medium alone served as controls. Lipoproteins were added at 50 g/ml based on cholesterol content for 24 h.
ter 30 min of E-LDL-induced cholesterol-loading. Similarly, Ox-LDL induced foam cell formation, while native LDL had no effect on intracellular lipid accumulation. VSMC kept in medium alone served as controls. E-LDL and Ox-LDL (each at 50 g/ml) mediated a pronounced upregulation of PTX3 mRNA in VSMC foam cells after 24 h of incubation, as shown for two different VSMC cultures derived from distinct patients (Fig. 2A). By contrast, native LDL did not alter the expression of the acute-phase protein. Induction of PTX3 mRNA by atherogenically modified lipoproteins was accompanied by a concomitant increased release of the acute-phase protein by VSMC (Fig. 2B). In the presence of native LDL, release of PTX3 was not altered.
2.7. Statistical analysis Experiments were conducted in triplicate and results are depicted as mean values ± standard deviation (S.D.), unless otherwise stated. For the determination of the significance of differences the Kruskall–Wallis test for comparison of multiple groups, and the Mann–Whitney U-test for comparison of two different groups was used. P < 0.05 was considered significant.
3. Results 3.1. Lipoprotein-driven expression of the acute-phase protein PTX3 in VSMC foam cells Enzymatically degraded LDL (50 g/ml) induced foam cell transformation of human primary VSMC, as visualized by uptake of fluorescent dye labelled DiI-E-LDL after 24 h of exposure (Fig. 1). VSMC foam cell formation started af-
3.2. Comparison of E-LDL induced PTX3 expression in nine different VSMC The marked induction of PTX3 mRNA expression which accompanied E-LDL mediated foam cell formation was confirmed in nine further VSMC cultures, derived from eight different patients (Fig. 3A). In resting primary VSMC, absent or very minor constitutive expression of PTX3 mRNA was detected. Upregulation of PTX3 mRNA was paralleled by a concomitant increase of the acute-phase protein release of up to 211 ng/ml by VSMC foam cells, corresponding to a more than 70-fold increased PTX3 protein liberation after 24 h of incubation with E-LDL (Fig. 3B). 3.3. Dose-dependent induction of PTX3 mRNA expression by E-LDL-activated VSMC E-LDL induced a dose-dependent expression of PTX3 mRNA by VSMC, which started from only 25 g/ml E-LDL
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Fig. 2. Atherogenically modified lipoproteins induce expression of the acute-phase protein PTX3 in VSMC. (A) E-LDL and Ox-LDL mediated pronounced expression of PTX3, while native LDL had no effect in two different VSMC cultures (upper panel). Expression of the housekeeping gene cyclophilin B (CPB) (lower panel). (B) Marked induction of PTX3 protein release by modified lipoproteins, whereas native LDL did not alter pentraxin liberation. Lipoproteins were added at 50 g/ml based on cholesterol content for 24 h. Results depicted as 1% agarose gel; M: molecular weight marker; PTX3 (233 bp), nc: negative control RT-PCR.
(Fig 4A). Induction of PTX3 mRNA expression was determined by real time RT-PCR and quantified with an external PTX3 standard covering six-log phases of concentration (Fig. 4B). In the presence of 25 g/ml E-LDL, expression of PTX3 mRNA almost doubled compared to untreated controls. Maximum expression resulting in a more than 12-fold induction of PTX3 mRNA was induced by incubation with 50 g/ml E-LDL. 3.4. Kinetics of E-LDL induced PTX3 expression Expression of the acute-phase protein PTX3 after activation with enzymatically degraded LDL was delayed, but remained elevated over extended periods (Fig. 5). Detectable induction of PTX3 mRNA expression did not start until after 6 h of incubation with 50 g/ml E-LDL, and was sus-
Fig. 3. Comparison of E-LDL-induced PTX3 mRNA expression and protein release in VSMC derived from different patients. VSMC foam cell formation was paralleled by marked induction of PTX3 mRNA expression and protein release. (A) E-LDL (50 g/ml) induced PTX3 mRNA expression in nine VSMC cultures derived from eight different patients (upper panel). Expression of the housekeeping gene cyclophilin B (CPB) (lower panel). (B) Concomitant release of the acute-phase protein into the culture supernatant. Incubations were conducted for 24 h. Results depicted as 1% agarose gel; M: molecular weight marker, nc: negative control RT-PCR. Results are expressed as means of duplicate determinations ± S.D.
tained at five-fold elevated concentrations during the presence of E-LDL for 96 h (Fig. 5A). Maximum induction of PTX3 mRNA expression was noted after 24 h of continuous incubation with E-LDL, resulting in a more than 15-fold expression of PTX3 mRNA compared to untreated controls (Fig. 5B). 3.5. Release of the acute-phase protein PTX3 by cholesterol-loaded VSMC foam cells E-LDL induced a dose-dependent release of the acute-phase protein from primary VSMC (Fig. 6). Release of PTX3 started from only 10 g/ml E-LDL and peaked at 50 g/ml E-LDL for 24 h, as exemplified for VSMC25 (Fig. 6A). Under these conditions, VSMC liberated more than 85 ng/ml PTX3, representing a more than 16-fold increase compared with untreated controls. Release of the acute-phase protein PTX3 by VSMC was retarded follow-
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Fig. 4. PTX3 mRNA expression after E-LDL-induced foam cell formation. Enzymatically degraded LDL induced dose-dependent expression of PTX3 mRNA in primary VSMC. (A) Regulated PTX3 mRNA expression depicted as mean fluorescence intensities by real time RT-PCR. (B) Induction of PTX3 expression started at 25 g/ml E-LDL, and peaked at 50 g/ml after 24 h of incubation. For quantification, the internal PTX-3 standard was used and upregulation of PTX3 mRNA by E-LDL was expressed in relation to unstimulated control VSMC.
ing E-LDL activation, as shown in time course experiments (Fig. 6B). While only minor liberation of the acute-phase protein was observed after up to 6 h of incubation with E-LDL, an increase by more than 70% of PTX3 release
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Fig. 6. PTX3 protein release by VSMC foam cells. (A) Dose-dependent release of PTX3 by E-LDL-loaded VSMC started from as low as 10 g/ml E-LDL, as exemplified for VSMC25. (B) Delayed kinetics of E-LDL (50 g/ml) induced release of the acute-phase protein. Quantification of PTX3 release by enzyme-immunoassay. Results are depicted as means of triplicate determinations ± S.D.
was observed after 24 h of incubation of VSMC with the degraded lipoprotein, as exemplified for VSMC19 (Fig. 6B). 3.6. PTX3 release in VSMC from different origin Comparison of PTX3 liberation revealed significant differences between spontaneous and E-LDL induced release of the acute-phase protein in the 11 primary VSMC cultures derived from distinct lesions of 10 different patients, as determined by the Kruskall–Wallis test (Fig. 7). E-LDL induced PTX3 release was significantly greater in VSMC foam cells derived from advanced lesions compared to cultures obtained from early lesions. It is of note that this discriminate behaviour was even evident when comparing VSMC derived from distinct lesions of the same patient (dashed lines). However, the spontaneous release of PTX3 by VSMC was not significantly different (n.s.) in VSMC derived from early or advanced lesions.
Fig. 5. Kinetics of PTX3 mRNA expression E-LDL induced a marked and sustained expression of PTX3 mRNA in VSMC. (A) Delayed induction of the acute-phase protein, as expressed by mean fluorescence intensities of PTX3 mRNA RT-PCR. (B) Expression of PTX3 mRNA became initially detectable after 6 h of E-LDL incubation, and remained elevated over the entire period of analysis of 96 h. Quantitative RT-PCR was used to calculate the amount of PTX3 mRNA generated in E-LDL-treated VSMC compared to untreated controls.
4. Discussion We identified enzymatically degraded LDL as an important regulator of the vascular acute-phase response by inducing marked expression of the acute-phase protein PTX3 in human VSMC. Our results show that human VSMC foam cells may acquire the potential to mount a local vascular
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Fig. 7. PTX3 release by cholesterol-loaded and na¨ıve VSMC. Significant induction of PTX3 release in E-LDL activated VSMC foam cells (∗ P < 0.05). Minor constitutive release of PTX3, which did not differ significantly (n.s.) in VSMC derived from early (white rhomb) or advanced (white square) lesions, by dot plot depiction. E-LDL mediated release of PTX3 was significantly more pronounced in VSMC obtained from advanced lesions (black squares) compared to early (black rhomb) lesions (∗∗ P < 0.008). Comparison of PTX3 release in VSMC derived from advanced and early lesions of the same patient (dashed lines).
acute-phase reaction, which delineates a previously unrecognised factor promoting atherogenesis. The continuous transudation of serum LDL followed by the subendothelial transformation into atherogenic lipoproteins by degradative processes, is believed to initiate and to maintain atherogenesis. Oxidisation of LDL is a well recognised atherogenic modification of LDL, allowing the binding and internalisation of Ox-LDL by scavenger receptors [1], the induction of macrophage foam cell formation [2,3] and cytokine expression [4]. Besides Ox-LDL, we have demonstrated the presence of enzymatically degraded LDL in human early atherosclerotic lesions [7]. E-LDL activated several central proatherogenic pathways, including foam cell formation [8–10], selective monocyte and lymphocyte transmigration [12], chemokine and proinflammatory cytokine generation [9–11]. In contrast to Ox-LDL, E-LDL is capable to activate complement [8]. Here, we report further evidence for a chronic vascular inflammatory process driven by the E-LDL-induced foam cell formation of VSMC and pronounced expression of the acute-phase protein PTX3. E-LDL induced a more than 12-fold increase of PTX3 mRNA expression and more than 70-fold release of the acute-phase protein by VSMC, while native LDL had no effect on the expression of the acute-phase protein. Similar to E-LDL, other atherogenic modifications, such as Ox-LDL, promoted PTX3 expression by VSMC. While the capacity of E-LDL to induce marked intracellular cholesterol accumulation and foam cell formation is undoubted [10], it is not clear whether cholesterol-loading itself or other biologically active compounds present in the enzymatically modified lipoprotein are responsible for PTX3 induction. The comparable PTX3 induction efficiency of E-LDL and E-LDL, which has been dialysed for substances of less than 10 kDa, a typical size of biologically active small molecules, does not identify small molecules as responsible for the induction of the acute-phase protein (data
not shown). The exact mechanism of PTX3 induction by E-LDL, however, remains to be elucidated. The recent immunohistochemical detection of PTX3 in intimal VSMC at the proliferative front immediately adjacent to the endothelium [25] supports the relevance of our findings. Similar to CRP [13] and to enzymatically degraded LDL [7], PTX3 was described to occur predominantly extracellularly [25]. Although macrophages were the predominant cell type reported to be associated with PTX3 expression in advanced lesions, our findings support the contribution of VSMC to PTX3 generation, which may be particularly important in early developing lesions before prominent inflammatory cell infiltration. While induction of PTX3 by proinflammatory cytokines has been described [19,20,28], expression induced by lipoprotein derivatives has not previously been demonstrated. We show that detectable levels of PTX3 mRNA arose only after prolonged exposure of VSMC to degraded lipoproteins, but once induced, remained elevated during the unhalted presence of the degraded lipoprotein. Comparable to resident vascular smooth muscle cells, delayed expression of the PTX3 was reported in macrophages in response to lipid constituents [29–31]. This points to a distinct regulation of the expression kinetics of the acute-phase protein PTX3 in the vascular acute-phase response as opposed to the rapid induction but limited duration of the expression of the classic pentraxin CRP in the liver [16,17]. Beyond the direct induction of PTX3 expression by VSMC, E-LDL-induced proinflammatory cytokines, including IL-1 and IL-6 [9,10], may contribute to the local acute-phase reaction by stimulating expression of the long pentraxin by infiltrating monocytes and by resident endothelial cells [19,20,30,31]. While the role of IL-1 in inducing PTX3 in monocytes, endothelial cells and fibroblasts is undisputed [19,20,30,31], the function of IL-6 on extrahepatic expression of the long pentraxin remains still unclear [21,22]. Previously, we have shown that E-LDL induced IL-6 mediated several proatherogenic effects, including induction of VSMC mitogenesis and chemokine production [32]. Recently, we have demonstrated that the viral homologue vIL-6 induced pronounced extrahepatic expression of PTX3 by neovascular Kaposi sarcoma cells [26]. Thus, E-LDL induced IL-1 and possibly IL-6 may add to the accumulation of the acute-phase protein in developing atherosclerotic lesions. PTX3 expression by VSMC may lead to an in situ vascular acute-phase-response, by binding to C1q and initiation of complement activation via the classic pathway [22,33]. In addition, PTX3 may contribute to the clearance of lipid-loaded macrophages and VSMC foam cells by opsonizing apoptotic cells, thus mediating their removal by dendritic cells [24,34]. We have shown previously that in contrast to native LDL, enzymatically degraded LDL is endowed with the capacity to bind CRP [13]. These E-LDL/CRP complexes displayed an altered functional capacity, including enhanced activation of complement
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[13], and promotion of monocyte transmigration (Klouche, unpublished observations). Recently, structural alterations of LDL leading to exposure of phosphorylcholine were identified to be responsible for complex formation with CRP [35]. Since, in contrast to CRP, PTX3 does not bind phosphorylcholine or phosphorylethanolamine [23], it is conceivable that the long pentraxin plays a distinct role in the inflammatory pathogenesis of atherosclerosis. We show for the first time the expression of the acute-phase protein PTX3 by VSMC. The capacity of human VSMC foam cells to mediate a vascular acutephase response provides a rationale for the recently described role of the long pentraxin PTX3 as an early indicator of atherosclerotic complications, such as acute myocardial infarction [36], and of the activity of generalized vascular inflammatory processes [27,37,38]. Our results reveal a lipoprotein-driven expression of the acute-phase protein PTX3 by VSMC, which may contribute to the inflammatory pathogenesis of atherosclerosis.
Acknowledgements We thank Franziska Walther for expert technical assistance and Tabea Peußer for technical support. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Bonn, Germany, KL1353) and by the Robert-Bosch-Medical Society to M.K.
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