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Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac-1 integrin

Sotirios N. Sotiriou, Valeria V. Orlova^*,, Nadia Al-Fakhri, Eveliina Ihanus, Matina Economopoulou^*, Berend Isermann, Khalil Bdeir^||, Peter P. Nawroth, Klaus T. Preissner^¶, Carl G. Gahmberg, Marlys L. Koschinsky^** and Triantafyllos Chavakis^*,,1

^* Experimental Immunology Branch, NCI, NIH, Bethesda, Maryland, USA;
Department of Internal Medicine I, University Heidelberg, Heidelberg, Germany;
Department of Clinical Chemistry and Molecular Diagnostics, Philipps-University, Marburg, Germany;
Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Finland;
^|| Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA;
^¶ Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany; and
^** Department of Biochemistry, Queen's University, Kingston, Ontario, Canada

¹Correspondence: Experimental Immunology Branch, NCI, NIH, 10 Center Drive, Rm. 4B17, Bethesda, MD 20892, USA. E-mail: chavakist{at}mail.nih.gov

SPECIFIC AIMS

Lipoprotein(a) [Lp(a)], consisting of LDL and apolipoprotein(a) [apo(a)], which contains multiple repeats resembling plasminogen kringle 4, is considered a risk factor for the development of atherosclerotic disorders, however, the underlying mechanisms for the atherogenicity of Lp(a) are not completely understood. We investigated whether the apo(a) component of Lp(a) might regulate adhesive and migratory functions of leukocytes, thereby providing a potential novel mechanism for the recruitment of inflammatory cells to the atherosclerotic vessel wall.

PRINCIPAL FINDINGS

1. Lp(a) is an adhesive substrate for monocytes
Based on the homology between the kringle 4-like domains of apo(a) with the plasminogen/angiostatin kringle 4 we recently found to interact with Mac-1, we hypothesized that Lp(a) and particularly apo(a) may serve as an adhesive substrate for inflammatory cells. We therefore tested the binding of monocytes and THP-1 cells to immobilized Lp(a). Monocytes and THP-1-cells adhered to immobilized Lp(a), and adhesion to Lp(a) was comparable in its extent to the adhesion of these cells to the established adhesive ligand, fibrinogen (Fig. 1 A). To identify the adhesive portion of Lp(a), the LDL moiety as well as recombinant apo(a) with increasing numbers of kringle 4 domains (6K, 12K, 17K) were tested for their adhesive properties. Significant adhesion of monocytes and THP-1 cells to immobilized apo(a) but not to LDL was observed.

View larger version (28K): [in this window] [in a new window]   Figure 1. Lp(a) mediates monocyte adhesion. A) Adhesion of peripheral blood monocytes (open bars) or PMA-stimulated THP-1 cells (filled bars) to immobilized BSA, Lp(a), LDL, the apo(a) variants 6K, 12K, and 17K as well as to fibrinogen is shown. Cell adhesion is represented as absorbance at 590 nm. B) Adhesion of peripheral blood monocytes to immobilized Lp(a) (open bars) or immobilized apo(a)-K6 (filled bars) was performed in the absence (–) or presence of EDTA (10 mM), or inhibitory mAb to CD29, CD18, LFA-1, Mac-1, or p150, 95 (each at 20 µg/mL), angiostatin kringle 4 (AS-K4) (50 µg/mL), or EACA (10 mM). Cell adhesion is shown as percent of control, which is represented by the adhesion to either Lp(a) or apo(a) in the absence of any competitor. All data are mean ± SD of 3 independent experiments each performed in triplicate.

We sought to define which receptor is responsible for the adhesion of monocytes to Lp(a) and apo(a). Blocking monoclonal antibodies (mAb) against ß1-integrins and ß2-integrins, the major adhesion receptors on monocytes, were tested. Only blocking mAb against ß2-integrins but not against ß1-integrin significantly reduced monocyte adhesion to Lp(a) and apo(a). Monocyte adhesion to immobilized Lp(a) was inhibited by a blocking mAb against Mac-1 but not by mAb against LFA-1 or p150,95 (Fig. 1B ). The adhesion of monocytes to apo(a) decreased as the number of kringles increased (Fig. 1A ). Moreover, adhesion of monocytes to immobilized Lp(a) or apo(a) was inhibited by plasminogen/angiostatin kringle 4 and was abolished by -aminocaproic acid (EACA), indicating a role of lysine binding sites in mediating the proadhesive effect of Lp(a) (Fig. 1B ). These data demonstrate that Lp(a) through its apo(a) moiety mediates monocyte adhesion through interaction with the ß2-integrin Mac-1.

2. Direct interaction between apo(a) and Mac-1
We investigated whether Lp(a) and apo(a) directly bound to Mac-1. Lp(a) and apo(a) bound to immobilized Mac-1 but not to LFA-1 in a dose-dependent and saturable manner. Mac-1 also interacted with its known ligands fibrinogen and ICAM-1 whereas LFA-1 bound only to ICAM-1 (Fig. 2 ). Binding of Lp(a) and apo(a) to Mac-1 was blocked by EACA or angiostatin kringle 4. A direct interaction between Lp(a)/apo(a) and the I-domain of Mac-1 was detected.

View larger version (20K): [in this window] [in a new window]   Figure 2. Direct interaction of Lp(a)/apo(a) with Mac-1. A) The total binding of fibrinogen (2 µg/mL, open bars), ICAM-1 (10 µg/mL, filled bars), Lp(a) (100 nM, gray bars), or apo(a)-K6 (100 nM, hatched bars) to immobilized BSA, Mac-1 or LFA-1 is shown. B) Dose-dependent specific binding of Lp(a) (filled squares) or apo(a)-K6 (open circles) to immobilized Mac-1 is shown. Total or specific binding (binding to BSA subtracted) is expressed as absorbance at 405 nm. Data are mean ± SD of 3 independent experiments each performed in triplicate.

3. Homocysteine increases the interaction between Mac-1 and apo(a)
Moderate increases in blood homocysteine predispose to atherosclerosis and homocysteine interacts with Lp(a), increasing the binding of the latter to fibrin. We therefore tested whether homocysteine might affect the Mac-1/Lp(a) interaction. Preincubation of Lp(a) or apo(a) with increasing concentrations of homocysteine rendered Lp(a) and apo(a) more adhesive for monocytes. Mac-1-dependent adhesion of monocytes or Mac-1-transfected CHO-cells to Lp(a) or apo(a) was enhanced 2- to 3-fold after preincubation of Lp(a) or apo(a) with homocysteine. Accordingly, the direct interaction between Lp(a)/apo(a) and Mac-1 was stimulated by homocysteine.

4. Chemotactic activity of Lp(a) and apo(a) on monocytes
Since Mac-1 plays an important role in the transendothelial migration of monocytes, we tested whether Lp(a)/apo(a) might affect this process. Monocyte chemoattractant protein-1 (MCP-1) -stimulated monocyte transmigration via cultured endothelial cells was blocked by mAb to Mac-1 as well as mAb to LFA-1. Lp(a) and apo(a) significantly stimulated the transendothelial migration of monocytes and the effects were blocked by mAb against Mac-1 but not by mAb against LFA-1. Combined stimulation with MCP-1 and Lp(a)/apo(a) resulted in an additive effect on monocyte transmigration.

5. Lp(a) and apo(a) stimulate the activity of the transcription factor nuclear factor-B (NF-B) and the expression of tissue factor in monocytes
Since ligation and activation of Mac-1 can result in the activation of NFB, we sought to investigate whether Lp(a)/apo(a) affect NFB activity. Lp(a) and apo(a) stimulated NFB activity, and this activity was prevented in the presence of blocking mAb against Mac-1 but not mAb against LFA-1. Moreover, incubation of THP-1 cells with Lp(a) or apo(a) resulted in a two-fold increase in tissue factor expression. Together, Lp(a)/apo(a) stimulate NFB activity and tissue factor expression in monocytes in a Mac-1-dependent manner.

6. Colocalization of Lp(a) and Mac-1 in atherosclerotic arteries
A histological analysis of Lp(a) and its possible association with Mac-1 on monocytes in the atherosclerotic plaque was then carried out. No immunostaining for apo(a) in normal arteries was seen. There were no monocyte or granulocyte infiltrations in the normal vessel wall, as demonstrated by negative Mac-1 immunostaining. In contrast, a strong expression of apo(a) was demonstrated in the neointima of 12 of 18 atherosclerotic vessels, and this was restricted to areas of atherosclerotic lesions that frequently showed foam cell accumulation, mostly in close proximity to the infiltrated inflammatory cells. These arteries showed positive Mac-1 immunostaining. The intima and media of the other 6 atherosclerotic arteries were negative for apo(a) and Mac-1. In the 12 apo(a) and Mac-1 positive atherosclerotic arteries, Mac-1 colocalized with apo(a) expression in almost all positive lesions (97% of positive lesions showed colocalization). Thus, expression and localization of Mac-1 and Lp(a) in atherosclerotic arteries is linked.

CONCLUSIONS AND SIGNIFICANCE

Our present work provides a novel link between inflammation and atherogenesis by demonstrating that proatherogenic Lp(a), via its apo(a) moiety, is a proinflammatory molecule that directly interacts with the leukocyte ß2-integrin Mac-1 and thereby facilitates the recruitment of inflammatory cells. In atherosclerotic plaques that were positive for Lp(a) expression, Lp(a) was found in close proximity to infiltrating mononuclear cells, and a high degree of colocalization was observed between Lp(a) and Mac-1, indicating these interactions may occur in vivo.

Another proatherogenic factor, homocysteine, can augment the interaction between Lp(a) and Mac-1, potentiating the proinflammatory action of Lp(a). Lp(a) and homocysteine synergize to increase the risk for coronary artery disease: when both risk factors are present, the associated risk is greater than what would be expected if these two risk factors were acting independently. Our data showing that homocysteine enhances the proinflammatory action of Lp(a) provide a novel clue as to how the interaction of these two risk factors increases cardiovascular risk.

The interaction of Lp(a)/apo(a) with the ternary complex is attributed at least in part to lysine binding sites within the kringle 4-like domains of apo(a). Thus, the interaction of apo(a) with Mac-1 shares similarities with the antifibrinolytic activity of apo(a). The fact that smaller isoforms of Lp(a) have been recognized as a risk factor for coronary heart disease independent of plasma Lp(a) concentrations has been partially attributed to the fact that smaller apo(a) isoforms bind more avidly to fibrin and are more effective in the inhibition of plasmin formation. Interestingly, we found an inverse relationship between the strength of Mac-1-dependent monocyte adhesiveness and the number of kringle 4 repeats in apo(a).

The present work describes a novel proinflammatory function of Lp(a)/apo(a) that directly interacts with Mac-1 and mediates inflammatory cell recruitment as well as concomitant up-regulation of the prothrombotic tissue factor. The proinflammatory mechanism of Lp(a) potentiated in the presence of homocysteine improves our understanding of the proatherogenic potential of these emerging cardiovascular risk factors.

View larger version (28K): [in this window] [in a new window]   Figure 3. Lp(a) recruits inflammatory cells to atherosclerotic plaques through its interaction with Mac-1. Lp(a) is an adhesive substrate for monocytes through a direct interaction with the integrin Mac-1 and may thereby promote recruitment of inflammatory cells to the atherosclerotic vessels. Moreover, Lp(a) may be prothrombotic as it induces increased expression of tissue factor in a Mac-1-dependent manner.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4857fje;

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