Published as doi: 10.1096/fj.06-6018fje.
(The FASEB Journal. 2006;20:2547-2549.)
© 2006 FASEB
Atherogenic properties of LDL particles modified by human group X secreted phospholipase A2 on human endothelial cell function
Sonia-Athina Karabina*,1,
Isabelle Brochériou*,
,
Gilles Le Naour,
Monique Agrapart*,
Hervé Durand*,
Michael Gelb
,
Gérard Lambeau
,2 and
Ewa Ninio*,2
* INSERM, UMRS 525; Institut Fédératif CMV; Université Pierre et Marie Curie (UPMC-Paris 6) and Faculté de Médecine Pierre et Marie Curie;
Laboratoire Central d’Anatomie Pathologique du Groupe Hospitalier Pitié-Salpêtrière, Paris, France;
Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington, USA; and
IPMC, CNRS UMR 6097, Sophia Antipolis, Valbonne, France
1Correspondence: INSERM UMRS 525, Faculté de Médecine Pierre et Marie Curie, 91, Boulevard de l’Hôpital, 75634 Paris, France. E-mail: akarampi{at}chups.jussieu.fr
SPECIFIC AIMS
Increasing evidence suggests that secreted phospholipases A2 (sPLA2s) play an important role in the pathophysiology of atherosclerosis. Among sPLA2s, the human group X (hGX) enzyme has recently attracted considerable interest because of its high catalytic activity toward phosphatidylcholine (PC), one of the major phospholipids of cellular membranes and low-density lipoproteins (LDL). Here we determined whether or not hGX sPLA2 is present in human atherosclerotic lesions and have investigated the ability of hGX modified LDL to alter human endothelial cell (HUVEC) function.
PRINCIPAL FINDINGS
1. Expression of hGX sPLA2 in human atherosclerotic lesions
Double immunofluorescence confocal microscopy of human carotid artery and aorta specimens (n=7) revealed hGX sPLA2 to be present in the intima where it colocalized with the majority of foam cells (Fig. 1
A) and phenotypically dedifferenciated smooth muscle cells (SMC) resembling myofibroblasts (Fig. 1B
). hGX sPLA2 was detected neither in T-lymphocytes nor in the lesion-free areas (data not shown). Interestingly, although hGX sPLA2 was detected in the extracellular matrix (ECM) of media (Fig. 1C
), we did not observe colocalization with SMC, raising the question of the origin of the enzyme in this area. The signal was specific for hGX sPLA2 because preincubation of the primary antibody (Ab) with 1µM recombinant hGX abolished the fluorescence (Fig. 1D
).
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Figure 1. Cellular localization of hGX sPLA2 in human carotid arteries containing atherosclerotic plaques. Sections of arteries were stained with anti-CD68 (green, A and D), anti-hGX sPLA2 (red, A–D) and anti  -actin (green, B and C). Double immunofluorescence confocal analysis was performed and colocalized regions are shown in white. In intima (A and B), hGX sPLA2 expression is colocalized with foam cells (A) and smooth muscle cells resembling myofibroblasts (B). Inserts represent colocalization plots between hGX sPLA2 (red) and specific cell markers cluster of differentiation (CD) 68, or -actin, (green). Blue dots represent colocalization points. In media (C), hGX sPLA2 expression is localized to ECM. No colocalization with smooth muscle cells (stained with -actin, green) of media was observed. In intima (D), incubation of sections with 1 µM recombinant hGX sPLA2 (rec hGX) abolished the hGX sPLA2 labeling.
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2. Hydrolytic effect of hGX sPLA2 on LDL results in a modified LDL particle
Hydrolysis of LDL by hGX sPLA2 results in a particle (hGX-LDL) that has increased electrophoretic mobility on agarose gel as compared to nontreated LDL. Electron microscopy (EM) analysis further showed that hGX-LDL particles have a smaller diameter (19.35±4.35 nm, n=3) as compared to untreated ones (21.87±4.65 nm, n=3). Incubation of hGX-LDL with human monocyte derived macrophages induces foam cell formation, one of the fundamental steps in atherosclerosis.
3. Arachidonic acid release from HUVEC exposed to hGX-LDL
Treatment of HUVEC with hGX-LDL (100 µg/ml) induced a significant time- and dose-dependent release of arachidonic acid (AA) as compared to cells treated with either LDL (100 µg/ml) or hGX sPLA2 alone (100 nM), suggesting in addition to the hGX sPLA2 triggered AA release, the hGX modified LDL by itself triggers a significant AA release. No AA release was observed when HUVEC were exposed to the hGX active site mutant H48Q added alone or when cells were exposed to LDL particles pretreated with the H48Q mutant. To further discriminate between a direct cellular action of hGX sPLA2 and an action of hGX due to the modification of LDL particles, we used LY 329722, a novel potent hGX sPLA2 inhibitor. Addition of 10 µM LY329722 to LDL prior to treatment with hGX sPLA2 fully inhibited AA release. Conversely, addition of LY329722 after treatment of LDL by hGX sPLA2 and before addition of the resulting modified LDL to cells had no significant effect on the hGX-LDL induced AA release. This clearly indicates that most of the AA release is due to the modified LDL particle and not to hGX sPLA2 directly acting on cells. Furthermore, extensive dialysis of LDL after treatment with hGX sPLA2 and before addition to cells did not affect AA release induced by hGX-LDL. Similarly, reisolation of hGX-LDL by ultracentrifugation followed by extensive dialysis did not affect the capability of hGX-LDL to induce AA release either in the absence or presence of the hGX sPLA2 inhibitor. These results demonstrate that hGX sPLA2 can release AA by acting directly on HUVEC cells but can also hydrolyze and thereby convert LDL into a modified particle that itself triggers additional release of AA from HUVEC cells. Using a specific and potent inhibitor of the cytosolic PLA2 group IVA (Wyeth-1) and MAP-kinase inhibitors (PD98059 and SB 203580), we found that the AA release induced by hGX-LDL is not dependent on this intracellular PLA2 but requires the ERK1/2 signaling pathway.
4. Analysis of mRNA levels and functional expression of adhesion molecules
An important increase in intracellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM)-1 expression was observed by quantitative-polymerase chain reaction analysis, when HUVEC cells were treated for 3 and 6 h with hGX-LDL, as compared to cells treated with LDL or hGX sPLA2 alone. A significant increase in the mRNA level of E- selectin was also apparent at 6 h, while there was no significant change in the expression of P-selectin and PECAM. The ICAM-1 and VCAM-1 mRNA up-regulation was accompanied by a time-dependent increase in protein expression as shown by western-blot analysis. The increased expression of these adhesion molecules was functionally relevant since the adhesion of fluorescent THP-1 cells to HUVEC was significantly higher when HUVEC were treated with hGX-LDL, as compared to those treated with LDL or hGX sPLA2 alone. The addition of the hGX sPLA2 inhibitor LY 329722 before treatment of LDL with the hGX sPLA2 completely blocked the effect, while the inhibitor had no effect on THP-1 adhesion when added to either LDL or hGX sPLA2 alone or after treatment of LDL with hGX sPLA2. Together, these results demonstrate that the modification of LDL by hGX sPLA2 is a prerequisite step for adhesion of THP-1 cells to HUVEC monolayers.
CONCLUSIONS AND SIGNIFICANCE
Our study is the first to demonstrate that hGX sPLA2 is expressed in the intima of human atherosclerotic lesions where it colocalizes with foam cells and phenotypically dedifferentiated smooth muscle cells. We currently do not know if the presence of hGX sPLA2 in the lesions is due to local biosynthesis in the vascular wall or to deposition from plasma lipoproteins after diffusion in to the vascular space or cellular internalization. In contrast to GIIA and GV sPLA2, hGX sPLA2 does not bind to proteoglycans and does not promote LDL particle aggregation, yet it efficiently hydrolyzes LDL. Here, we show that the lipolytic effect of hGX on LDL produces a modified particle that induces human macrophage foam cell formation in vitro. We thus hypothesize that the cellular pathways implicated in the uptake of hGX-LDL are different from those for GIIA- and GV-modified LDL. The precise structural alterations of hGX-LDL that lead to such enhanced lipid accumulation in vitro remain to be elucidated.
Our results demonstrating that the lipolytic action of hGX renders the LDL particles smaller in diameter are important, as the smaller the size of the LDL particle the higher its atherogenic potential. As hGX is the most active sPLA2 toward PC the major phospholipid constituent of lipoproteins, it would be of interest to examine the possible involvement of this enzyme in lipoprotein metabolism and small dense LDL formation.
hGX-LDL was able to induce a strong time- and concentration-dependent release of AA from HUVEC. AA release was not observed when hGX-LDL was pretreated either with the hGX sPLA2 inhibitor LY 329722 or with the H48Q hGX active site mutant, indicating that the enzymatic modification of LDL by hGX is prerequisite for AA release. Additionally, when hGX-LDL was reisolated by ultracentrifugation and extensively dialysed to remove all loosely bound molecules, it retained the ability to release AA from HUVEC cells even in the presence of the hGX sPLA2 inhibitor. The AA release due to hGX-LDL appeared to be independent of cPLA2-IVA activation but dependent on ERK1/2 activation.
We also show that the lipolytic effect of hGX sPLA2 on LDL results in the formation of an atherogenic LDL particle that increases both the mRNA and protein expression of major adhesion molecules including ICAM-1 and VCAM-1, which was in turn accompanied by increased adherence of monocytes to endothelial cell monolayers. This observation is of importance, as an activated endothelium is prerequisite for monocyte adherence and recruitment into the subendothelial space and is one of the major events in atherosclerosis.
As the "oxidation hypothesis of atherosclerosis" still remains inconclusive and oxidation alone cannot explain the accumulation of large amounts of lipids and lysophosphatidylcholine (LPC) in foam cells and fatty streak lesion formation, we propose that in addition to LDL oxidation the hGX sPLA2 lipolytic modification of LDL may present an alternative pathway to the progression of atherosclerosis. In the arterial wall, hGX sPLA2 may exert proatherogenic actions by inducing the release of lipid mediators such as LPC that can affect functions and properties of vascular cells at sites of lipoprotein accumulation. In addition, hGX sPLA2 may also modify lipoprotein particles by hydrolyzing PC, in a way that promotes foam cell formation independently of LDL oxidation.
In conclusion, our data suggest a strong atherogenic property of hGX sPLA2 in modifying LDL into a proinflammatory and proatherogenic particle that may contribute to the initiation and propagation of atherosclerosis (Fig. 2
).
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Figure 2. Schematic representation of the initiation of atherosclerosis by hGX sPLA2. hGX sPLA2 is found in arterial wall where it colocalizes with foam cells and dedifferentiated smooth muscle cells. These cells are potentially involved in secretion of hGX sPLA2 under an as yet unknown stimulus. Additionally, hGX sPLA2 hydrolyzes LDL releasing LPC and AA through MAPK activation. Both lipid mediators are important signaling molecules. hGX-LDL can promote: foam cell formation when taken up by monocyte derived macrophages and can induce expression of adhesion molecules thereby increasing monocyte adherence on surface of endothelial cells
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FOOTNOTES
2 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6018fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
