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Am J Physiol Heart Circ Physiol 292: H994-H1000, 2007. First published October 13, 2006; doi:10.1152/ajpheart.00798.2006
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Surface association of pregnancy-associated plasma protein-A accounts for its colocalization with activated macrophages

Cheryl A. Conover,1 Sean C. Harrington,1 Laurie K. Bale,1 and Claus Oxvig2

1Division of Endocrinology, Metabolism, and Nutrition, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota; and 2Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark

Submitted 25 July 2006 ; accepted in final form 10 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intense immunostaining for pregnancy-associated plasma protein-A (PAPP-A), a newly characterized metalloproteinase in the insulin-like growth factor system, colocalizes with activated macrophages in human atherosclerotic plaque. To determine macrophage regulation of PAPP-A expression, we developed two models of human macrophages with basal and activated phenotypes. THP-1 cells and peripheral blood monocytes could be differentiated into macrophages and activated upon specific treatment regimens with phorbol myristate acetate, macrophage colony-stimulating factor, and interleukin-1beta. Activation was assessed by cell secretion of tumor necrosis factor-{alpha}, which increased 30- to 100-fold with activation. Activated macrophages also secreted matrix metalloproteinase-9. However, no PAPP-A mRNA or PAPP-A antigen could be detected in these cells under any condition. Upon incubation with recombinant PAPP-A, we found that activated macrophages bound and internalized more PAPP-A than unactivated macrophages or monocytes. Internalization accounted for at least 50% of macrophage-associated PAPP-A, as assessed in studies with cytochalasin B. Membrane-bound PAPP-A retained protease activity, whereas internalized PAPP-A had little or no activity. Similar experiments carried out with a mutated variant of PAPP-A, which retains functionality as a protease but is unable to bind surface-associated glycosaminoglycan, showed no macrophage association or internalization. Absence of PAPP-A expression was confirmed in activated macrophages isolated from a hypercholesterolemic rabbit model of atherosclerosis. We therefore conclude that PAPP-A is not synthesized in, but rather is bound and internalized by, macrophages. Our findings likely account for the observed intense immunostaining for PAPP-A colocalizing with activated macrophages and may have physiological significance in the development of vulnerable plaque.

macrophages; atherosclerotic plaque

ATHEROSCLEROSIS AS A COMPLEX inflammatory and fibroproliferative process and vulnerable plaque as a major clinical complication are widely accepted concepts in cardiovascular disease (5, 13, 20, 24). However, the mechanisms involved are not fully understood. Pregnancy-associated plasma protein-A (PAPP-A), a newly discovered insulin-like growth factor-binding protein (IGFBP) metalloproteinase (4, 19), is considered a potential marker of acute coronary syndrome and a possible target for the treatment of vulnerable plaque (1, 2, 14, 23, 30). Interest is high, therefore, in understanding the regulation of PAPP-A expression and its role in plaque development. PAPP-A was found to be elevated in ruptured and eroded plaque, with immunostaining most intense in the inflammatory shoulder of the ruptured plaque colocalizing with vascular smooth muscle cells and, in particular, with activated macrophages (2, 30). Similarly, specific immunostaining for PAPP-A was found in healing human skin in association with activated macrophages and myofibroblasts (7). These studies suggest that upregulation of PAPP-A is an important response to injury, but the mechanisms of this regulation are unknown. The work so far in vascular injury has focused on the smooth muscle cells. PAPP-A gene and protein expression by human coronary artery smooth muscle cells has been documented under basal conditions, and expression is known to be further stimulated by proinflammatory cytokines, tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1beta (3, 9). To our knowledge, there are no data on PAPP-A expression and its regulation in macrophages.

Macrophages play a critical role in atherogenesis and participate in the pathogenesis of acute coronary syndrome. Resident macrophages of atherosclerotic plaque accumulate lipid and maintain a continuous state of activation during which they secrete proinflammatory cytokines, distinguishing these cells from their circulating monocytic progenitors (13, 20, 24). Cellular differentiation and activation of macrophages also increases their expression of matrix metalloproteinases (MMPs) such as MMP-1, -3, and -9, which may contribute to plaque instability (12, 16, 32). Both PAPP-A and the MMPs belong to the metzincin superfamily of metalloproteinases (4). Therefore, upregulation of PAPP-A expression in activated macrophages would fit with this known upregulation of MMP expression and with the observation of concentrated staining of these cells for PAPP-A in vulnerable plaque. To determine macrophage regulation of PAPP-A expression, we developed models of human macrophages with basal and activated phenotypes. We herein demonstrate that macrophages do not express PAPP-A in vitro or in vivo. Rather, macrophages bind and internalize PAPP-A, which may have functional consequences for vulnerable plaque.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Phorbol myristate acetate (PMA), cytochalasin B, and FCS were from Sigma (St. Louis, MO), macrophage colony-stimulating factor (MCSF) and IL-1beta were from R & D Systems (Minneapolis, MN), and tissue culture media and supplements were from Invitrogen (Carlsbad, CA). IGF-I and insulin were kind gifts from Dr. Martin Spencer (San Francisco, CA) and Eli Lilly (Indianapolis, IN), respectively. Recombinant human wild-type PAPP-A and PAPP-A, in which in the third short consensus repeat (SCR3) was replaced with PAPP-A2 sequence [designated chim6 (18)], were expressed in HEK 293T cells as previously described (18).

Cells. THP-1 cells were purchased from the American Type Culture Collection (Manassas, VA), and maintained in suspension culture in RPMI 1640 supplemented with 10% FCS, glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 2-mercaptoethanol (55 µM). For experiments, cells were plated with PMA (100 nM) to induce adherence and differentiation. After 2 or 3 days, media was changed to fresh PMA ± MCSF (100 ng/ml) and IL-1beta (1 nM) for another 5 days.

Human monocyte-derived macrophages were isolated from buffy coats from normal blood donors, which were obtained from Transfusion Medicine under a Mayo Institutional Review Board-approved protocol. Briefly, buffy coats were diluted 1:3 with PBS and layered on Ficoll-Paque (Amersham, Piscataway, NJ). After centrifugation at room temperature for 45 min at 1,350 rpm, the layer of mononuclear cells was removed, washed three times with RPMI, and counted by Trypan blue exclusion. The cells were then suspended and plated in RPMI 1640 medium without serum and allowed to adhere overnight. The cells were washed three times with RPMI, and medium was replaced with RPMI containing 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin without or with MCSF (100 ng/ml) to induce differentiation. After 2 or 3 days, media was changed to fresh MCSF ± PMA (100 nM) and IL-1beta (1 nM) for another 5 days.

Conditioned medium, whole cell lysate, membrane preparation. After indicated treatments, conditioned medium was collected and centrifuged 15 min at 2,500 rpm at 4°C. Supernatant was stored at –70°C until analysis. For whole cell lysates, medium was removed and washed three times in cold PBS and lysed in cold RIPA buffer (0.15 N NaCl, 0.5% Nonidet P-40, 0.1% SDS, and 50 mM Tris, pH 7.6) containing 2 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were sheared through 21- and 27-gauge needles and microfuged 2 min at 14,000 rpm; this supernatant was used for assays. Membranes were isolated as previously described (31). Cell lysate was centrifuged at 800 rpm for 5 min and 8,000 rpm for 20 min. The resultant supernatant was further centrifuged at 50,000 rpm for 60 min. The supernatants were saved for analysis of intracellular PAPP-A protein and protease activity. The membrane pellet was resuspended in RIPA buffer with 2 mM PMSF. This fraction was used to assay for PAPP-A membrane association. Protein content was determined by BCA Protein Assay (Pierce, Rockford, IL).

RNA isolation and PAPP-A mRNA expression. Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) and treated with DNase (DNA-free; Ambion, Austin, TX). RNA (400 ng) was reversed transcribed using TaqMan RT reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

Real-time quantitative PCR analyses for human PAPP-A and 28S were performed using the ABI PRISM 7700 Sequence Detection System and software, and for rabbit PAPP-A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using the 7900HT Fast Real-time Detection System and software (Applied Biosystems). Primer and probe sequences, as well as assay validations, were described previously (8, 9). All samples were run in triplicate. CT is the number of amplification cycles required by each gene to reach a fixed threshold of signal intensity. The higher the abundance, the lower the CT. CT values <35 are considered physiologically meaningful (8, 9). The positive control for human PAPP-A assay was a human epithelial ovarian cell line that expresses abundant PAPP-A (17). Rabbit kidney was used as a positive control in the rabbit PAPP-A assay. Relative RNA expression was determined using the 2Formula method (21).

PAPP-A ELISA. Human PAPP-A levels were measured using an Ultrasensitive PAPP-A ELISA kit kindly provided by Diagnostic Systems Laboratories (Webster, TX). Minimum sensitivity is 0.24 mIU/l, with intra- and interassay coefficients of variation of 4.7 and 4.2%, respectively.

TNF-{alpha} ELISA. Cell culture supernatant was assayed for TNF-{alpha} using a Quantikine Human TNF-{alpha}/TNFSF1A Immunoassay (R&D Systems) following the manufacturer's instructions. Minimum sensitivity is 1.6 pg/ml, with intra- and interassay coefficients of variation of 4.9 and 5.3%, respectively.

IGFBP-4 proteolysis assay. Cell-free IGFBP-4 proteolysis was assayed as previously described (3, 8, 9, 19). Conditioned media, cell lysate fractions, or intact cells were incubated at 37°C for 6 or 24 h with 125I-labeled IGFBP-4 in the absence and presence of 5 nM IGF-II. IGF-II binds IGFBP-4 and appears to render this substrate more susceptible to proteolysis by PAPP-A (27). Reaction products were separated by SDS-PAGE and visualized by autoradiography.

Western immunoblot. Conditioned media were subjected to 7.5% SDS-PAGE, separated proteins were transferred to polyvinylidene difluoride, and the membrane was blocked in 5% milk in Tris-buffered saline containing 0.1% Tween 20. Membranes were incubated overnight at 4°C with mouse monoclonal anti-MMP-9 (Ab-1; Calbiochem, La Jolla, CA) at 2 µg/ml in 0.5% milk-Tris-buffered saline-0.1% Tween 20. Membranes were washed and incubated 1 h at room temperature with horseradish peroxidase conjugate (Transduction Laboratories, Lexington, KY), and bands were visualized using enhanced chemiluminescence reagents (Amersham).

Rabbit macrophages. New Zealand White male rabbits were fed a high-fiber laboratory rabbit diet supplemented with 4.5% coconut oil and 0.5% cholesterol (TestDiet, Richmond, IN) for 6 wk before implantation of two sterile micronova sponges (VWR, West Chester, PA) under the dorsal skin (6, 10, 11). Sponges remained in place for 3 wk to allow macrophage accumulation while the animal remained on the high-cholesterol diet. The recovered sponges were gently squeezed in sterile dishes, and the exudates were layered over 14.1% HistoDenz solution (Sigma) and centrifuged at 800 g for 25 min at room temperature. Foam cells were recovered from the interface and washed one time with PBS. Aliquots were prepared for oil red O staining to confirm lipid content, immunostained with rabbit macrophage-specific marker RAM-11 (Dako Cytomation, Carpinteria, CA), or plated for culture. These studies were approved by Mayo Clinic's Institutional Animal Care and Use Committee.

Statistical analysis. Statistical comparisons were performed using ANOVA, followed by multiple comparisons. Results are considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of macrophage model systems. THP-1 cells, derived from acute monocytic leukemia, have been shown to adhere and differentiate into macrophages following treatment with phorbol ester tumor promoters such as PMA. We confirmed the macrophage phenotype of THP-1 cells after PMA treatment by latex bead phagocytosis and accumulation of intracellular lipid (data not shown). For a model of activated macrophages, THP-1 cells required PMA followed by treatment with MCSF. Activation was assessed by TNF-{alpha} secretion. TNF-{alpha} secretion could be markedly enhanced 30- to 100-fold with the further addition of IL-1beta. These data are shown in Fig. 1A. Thus we considered untreated, nonadherent THP-1 cells as monocytes, PMA-treated THP-1 cells as differentiated macrophages, and PMA-, MCSF-, IL-1beta-treated THP-1 cells as activated macrophages. PAPP-A gene expression in macrophages was not affected by activation (Table 1). Indeed, essentially no PAPP-A mRNA, as measured by real-time PCR (see legend to Table 1), or protein, as measured by an ultrasensitive ELISA (data not shown), was detected in THP-1 cells under any of these conditions. On the other hand, these assays measured abundant PAPP-A mRNA and protein expression in human coronary artery smooth muscle cells that could be increased ~10- to 20-fold by IL-1beta treatment (Table 1 and see Refs. 3 and 9). Although IGF-I has been suggested to activate macrophages in vitro (29), there was no effect of IGF-I or insulin treatment on macrophage activation (Table 2) or on PAPP-A expression (data not shown). PAPP-A without or with IGF-I had no effect on macrophage activation (Table 2).


Figure 1
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  		Fig. 1. Characterization of in vitro models of activated macrophages. THP-1 cells (A) and peripheral blood monocytes (PBM; B) were untreated (Control, C) or treated with phorbol myristate acetate (PMA, P), macrophage colony-stimulating factor (MCSF, M), interleukin-1beta (IL), or indicated combinations. Details of the treatment strategies are provided in MATERIALS AND METHODS. Conditioned media were assayed for tumor necrosis factor (TNF)-{alpha} as a measure of macrophage activation. Results are means ± SE of 3 experiments. *Significant difference vs. control, P < 0.05.

 

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  		Table 1. PAPP-A mRNA expression

 

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  		Table 2. Effect of IGF-I, insulin, and PAPP-A on activation of THP-1 cells

 
To determine whether the lack of PAPP-A gene expression might be THP-1-specific, human macrophages were derived from peripheral blood monocytes. We found that these cells required a different treatment regimen than the THP-1 cells. For this model, monocytes separated by Ficoll gradient centrifugation were allowed to adhere overnight and then treated with MCSF for differentiation into macrophages, as visualized by cell spreading, and further with PMA plus IL-1beta for maximal activation (Fig. 1B). However, no significant PAPP-A mRNA (Table 1) or protein (data not shown) was detected in adherent monocytes or in the differentiated or activated macrophages. Other proposed stimulants of macrophage activation, e.g., lipopolysaccharide, PGE2, and granulocyte-MCSF, had no effect on PAPP-A expression (data not shown). As with the THP-1 cells, there was also no effect of IGF-I, insulin, or PAPP-A treatment on TNF-{alpha} secretion or PAPP-A expression (data not shown). However, THP-1 and monocyte-derived macrophages expressed and secreted MMP-9 (Fig. 2). Thus these THP-1 and monocyte-derived macrophage cell models have characteristic functions of activated macrophages (phagocytosis, TNF-{alpha} secretion, MMP expression) but do not express PAPP-A.


Figure 2
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  		Fig. 2. Matrix metalloproteinase (MMP)-9 expression. Conditioned media from experiments in Fig. 1 were immunoblotted with MMP-9 antibody as described in MATERIALS AND METHODS. U, unconditioned medium.

 
Cell-associated PAPP-A. So why is there intense immunostaining for PAPP-A in activated macrophages in atherosclerotic plaque (2, 30)? One idea would be that PAPP-A is not expressed by macrophages but that it is preferentially cell-associated and/or internalized by macrophages in the plaque. To test this hypothesis, THP-1 cells (monocytes, differentiated macrophages, activated macrophages) were incubated for 6 h at 37°C with recombinant wild-type PAPP-A. After incubation and washing, whole cell lysates and solubilized membrane preparations were assayed for PAPP-A by ELISA. PAPP-A was readily detected in lysates and membranes from the three different phenotypes (Fig. 3A and Table 3). Similar results were seen with the monocyte-derived macrophages (Fig. 3B and Table 3). Previous studies have shown that wild-type PAPP-A binds surface glycosaminoglycans mediated by its SCR3 in the carboxy terminal portion of the molecule (17). A mutated variant of PAPP-A (MUT PAPP-A) in which SCR3 is replaced is functional as a protease but does not bind glycosaminoglycan (17). No PAPP-A was detected in whole cell lysates or membrane preparations when similar incubations were carried out with MUT PAPP-A (Fig. 3, A and B). In addition, there was no IGFBP-4 protease activity in membranes from cells treated with MUT PAPP-A compared with the robust IGF-dependent IGFBP-4 protease activity following wild-type PAPP-A treatment (Fig. 4). Similar results were obtained if the assay was performed on intact cells (data not shown). The lack of detectable MUT PAPP-A in these experiments was not because MUT PAPP-A is not recognized in the assay, since wild-type PAPP-A and MUT PAPP-A are equally well detected in ELISA (data not shown). It was also not because of loss protease activity with mutation because proteolytic activity is not compromised in MUT PAPP-A (Fig. 5). Activated macrophages bound/internalized more wild-type PAPP-A than unactivated macrophages or monocytes. This was particularly evident in Fig. 3B with the monocyte-derived macrophage model. However, the amount of wild-type PAPP-A (per mg protein) associated with the membrane did not appear to be different among the cell types (Table 3). This membrane association is likely to involve glycosaminoglycan, as previously suggested (18), since binding was completely inhibited by 25 µg/ml heparin and heparin sulfate with lesser or no inhibition by chondroitin sulfates (Fig. 6). To distinguish internalization from membrane association, THP-1 cells were treated with 50 µM cytochalasin B, which inhibited phagocytosis by >95%, 30 min before incubation with wild-type PAPP-A. As shown in Fig. 7, internalization accounted for at least 50% of the macrophage-associated PAPP-A. Cytochalasin B had no affect on PAPP-A association with monocytes, which do not exhibit the phagocytotic phenotype. Cell fractionation studies indicated little or no proteolytic activity in cytoplasmic or nuclear-enriched fractions of cell lysates following incubation with wild-type PAPP-A despite measurable levels of immunoreactive PAPP-A (data not shown).


Figure 3
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  		Fig. 3. THP-1 cells (A) and PBM (B) were incubated with 500 ng/ml recombinant wild-type (WT) PAPP-A, mutated PAPP-A (MUT), or supernatant from mock transfections (mock) for 6 h at 37°C (see RESULTS for definition of monocytes, macrophages, and activated macrophages). Cells were washed, and whole cell lysates were assayed for PAPP-A. Results are means ± SE of 3 experiments. *Significant difference vs. mock transfections, P < 0.05.

 

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  		Table 3. Membrane-associated PAPP-A

 

Figure 4
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  		Fig. 4. Membrane-associated pregnancy-associated plasma protein-A (PAPP-A)-insulin-like growth factor-binding protein (IGFBP)-4 proteolytic activity. Solubilized membrane preparations from experiments in Fig. 3 were incubated with 125I-labeled IGFBP-4 without (–) or with (+) IGF-II for 6 h. Reaction products separated by SDS-PAGE were visualized by chemiluminescence. Arrows indicate intact and cleaved 125I-IGFBP-4. Data are representative of all the protease assays run on the membrane preparations in Fig. 3.

 

Figure 5
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  		Fig. 5. Wild-type vs. mutated PAPP-A-IGFBP-4 proteolytic activity. Indicated concentrations of wild-type and mutated PAPP-A were incubated with 125I-IGFBP-4 without (–) and with (+) IGF-II in cell-free assay as described in MATERIALS AND METHODS.

 

Figure 6
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  		Fig. 6. Macrophage-associated PAPP-A: effect of glycosaminoglycans. Activated macrophages (THP-1 cells treated with PMA, MCSF, and IL-1beta) were incubated with 500 ng/ml wild-type PAPP-A in the absence (C) or presence of 25 µg/ml of heparin (Hep; A), heparan sulfate (HS; B), and chondroitin sulfate (CS; C). Whole cell lysates were assayed for PAPP-A. *Significant difference vs. control, P < 0.05.

 

Figure 7
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  		Fig. 7. Macrophage-associated PAPP-A: effect of cytochalasin B. THP-1 cells (see text for definition of monocytes, macrophages, and activated macrophages) were preincubated for 30 min with 50 µM cytochalasin B (gray hatch) before addition of 100 ng/ml wild-type PAPP-A. *Significant effect of cytochalasin B, P < 0.05.

 
To determine whether macrophage cell association and uptake of PAPP-A was specific to recombinant protein, activated macrophages were incubated for 6 h with medium conditioned by human coronary artery smooth muscle cells, which are known to secrete PAPP-A (3, 9). This PAPP-A specifically associated with the activated macrophages (28 ± 0.2 mIU/l PAPP-A, mean ± SE of 3 separate incubations), whereas there was no detectable PAPP-A associated with macrophages after 6 h incubation with unconditioned medium.

Expression of PAPP-A in macrophages in vivo. The hypercholesterolemic rabbit model of atherosclerosis, which was developed to facilitate study of the physiology of macrophages characteristic of atherogenic arteries (6, 10, 11), was used to investigate PAPP-A expression in macrophage-derived foam cells in vivo. In this model, implantation of subcutaneous sponges triggers the formation of granulomas whose macrophages accumulate lipid under hypercholesterolemic conditions with similar phenotype characteristics as aortic macrophage-derived foam cells in the fatty lesions of the aorta (10, 11). Isolated cells from the granulomas were positively identified as macrophages with RAM-11 antibody (Fig. 8A), and oil red O staining confirmed lipid accumulation in the macrophages (Fig. 8B). However, there was no PAPP-A mRNA expression in purified (n = 3) or cultured (n = 3) macrophages as assessed by real-time PCR (CT PAPP-A = 35.6 ± 0.11, CT GAPDH = 18.6 ± 0.02). Rabbit kidney served as a positive control (CT PAPP-A = 29.4 ± 0.02; CT GAPDH = 19.4 ± 0.04). In this model, there was no IGFBP-4 protease activity associated with the macrophages (data not shown).


Figure 8
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  		Fig. 8. Activated macrophages from a hypercholesterolemic rabbit model. A: RAM-11, magnification x10. B: oil red O, magnification x40.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study established that activated macrophages do not express PAPP-A. This is an important new understanding, since PAPP-A is currently being evaluated as a circulating marker for acute coronary syndromes (1, 2, 14, 23, 30) and as a potential target for identifying and therapeutically alleviating vulnerable plaque. Thus there is strong interest in determining the cell sources, mechanism(s) of regulation, and the interactions involving PAPP-A and plaque development. We initially studied human coronary artery smooth muscle cells and reported active PAPP-A expression and secretion by these cells that could be further stimulated by proinflammatory cytokines TNF-{alpha} and IL-1beta (3, 9). Because the immunohisochemistry indicated more intense staining for PAPP-A in the activated macrophages than in smooth muscle cells of vulnerable plaques (2, 30), it was important to assess macrophages as a major source of PAPP-A in plaque.

We were faced with several questions about how best to study macrophages in vitro. What was the appropriate cell model? Macrophage-like cell lines, such as THP-1, are commonly used, but do they represent the peripheral monocyte-derived macrophages found in plaque? How do you activate macrophages in vitro? And how does in vitro-activated relate to in vivo-activated macrophages? We invested quite a bit of effort into characterizing the THP-1 cells along with monocyte-derived macrophages as to the specific treatments, time, and sequence necessary to get the desired phenotypes. In general, the specific treatments (PMA, MCSF, IL-1beta) turned out to be similar for the two cell types, but the treatment regimens and quantitative responses were somewhat different. Thus THP-1 cells required initial treatment with PMA for adherence to the culture dish, followed by MCSF and IL-1beta for activation, whereas monocyte-derived macrophages required MCSF first and then PMA and IL-1beta. Also, minor differences in phagocytosis and activation were noted. Some of the observed differences between THP-1 cells and the normal human monocyte-derived macrophages might be because of the neoplastic characteristics and/or origin of the THP-1 cell line. To validate that the findings in vitro represented the in vivo situation, we employed the hypercholesterolemic rabbit model. In this model, sponges are implanted subcutaneously in rabbits fed a high-cholesterol diet (10). After 3 wk, the sponges are removed and infiltrating macrophages are released from the sponge by gentle squeezing and purification by gradient density centrifugation. These lipid-filled macrophages were originally shown to have the same phenotype as foam cell macrophages in the aortic plaque and proved that this was an appropriate in vivo model of activated macrophages (10, 11). We similarly isolated a highly enriched population of lipid-filled macrophages (positive staining for oil red O and RAM-11). These in vivo-activated macrophages, like the in vitro-activated macrophages, did not express PAPP-A.

Macrophages secrete a variety of growth factors, cytokines, and proteolytic enzymes that are related to the progression of atherosclerosis (5, 13, 20, 24). In particular, macrophages secrete MMPs that have been implicated in plaque vulnerability (11, 12, 22). In human atherosclerotic tissue, MMP-9 colocalizes with lesional macrophages, and MMP-9 is a major macrophage-derived MMP in vitro (6, 22, 26). Both THP-1 and monocyte-derived macrophages expressed MMP-9 under our culture conditions. Therefore, we were initially surprised that macrophages did not express any PAPP-A, a metalloproteinase in the same superfamily as the MMPs (4). However, this finding draws attention to the different regulation and function of PAPP-A in spite of the close biochemical relationship to MMPs.

Moreover, this study demonstrated that macrophages intensely immunostain for PAPP-A not because they express PAPP-A, but because PAPP-A binds to macrophage membranes and is internalized. This is a new concept in plaque physiology, but it may have been predicted based on recent information about the structure of the PAPP-A molecule. The carboxy terminal portion of PAPP-A contains SCR modules known to be involved in protein-protein interactions (18). PAPP-A has been shown to reversibly bind to cell surfaces (18) and is associated with membranes from human placental trophoblasts that express endogenous PAPP-A (31). We have now shown that recombinant PAPP-A and PAPP-A secreted by vascular smooth muscle cells bind to macrophages and that binding depends on SCR3. Competition by heparin and heparan sulfate suggests involvement of heparan sulfate proteoglycan (18). Binding is necessary for subsequent internalization, i.e., mutated PAPP-A unable to bind glycosaminoglycans is not internalized. PAPP-A binding does not appear to be specific to macrophages as a cell type, but the macrophage models described in this study would be suitable for further investigation into PAPP-A-binding sites and binding kinetics because they do not express PAPP-A.

What is the function of macrophage-associated PAPP-A? Possibilities include: 1) cell surface targeting of PAPP-A proteolytic activity. The preserved protease activity of membrane-associated PAPP-A could serve to amplify local IGF-I action. Macrophages have been reported to express IGF-I and respond to IGF-I with increased uptake and degradation of low density lipoproteins (15). In the plaque, the source of the macrophage-associated PAPP-A is presumably from smooth muscle cells. We have shown that, in vitro, activated macrophages can bind the PAPP-A produced by human coronary artery smooth muscle cells and that this membrane-associated PAPP-A is enzymatically active. Interestingly, the activated macrophages generated in the rabbit granulomas did not have associated IGFBP-4 protease activity. This is likely because of the lack of nearby cells producing PAPP-A. This finding supports the notion that macrophage-associated PAPP-A is derived from smooth muscle cells and/or other cells in the plaque and not from circulating sources; and 2) uptake and inhibition of PAPP-A. Cell-bound PAPP-A is internalized by activated macrophages, and intracellular PAPP-A appears to be enzymatically inactive. During human pregnancy, PAPP-A circulates as a covalent 2:2 complex with the proform of major basic protein (proMBP). In the PAPP-A-proMBP complex, proMBP functions as an inhibitor of PAPP-A proteolytic activity (25). On the other hand, the PAPP-A elevated in acute coronary syndromes circulates as a PAPP-A dimer, without proMBP, and is proteolytically active (28). If there is no physiological inhibitor such as proMBP in the lesion, then the macrophage may attempt to serve this function through uptake and degradation of the PAPP-A. Of course other possibilities exist, but these two are the most likely candidates based on what is known so far about PAPP-A and atherosclerotic lesion development.

From the data generated in this study, as well as from previous work in vascular smooth muscle cells, we suggest the following concept of dynamic interactions involving PAPP-A in vulnerable atherosclerotic plaque: activated macrophages in developing plaque synthesize proinflammatory cytokines that stimulate vascular smooth muscle cells to synthesize and secrete PAPP-A. PAPP-A can function as both an autocrine and paracrine factor by binding to cells in the plaque. This positions PAPP-A favorably for directed proteolytic activity and enhanced local IGF actions on both smooth muscle cells and macrophages, representing an important amplification point in atherosclerotic disease progression. Macrophages also phagocytose its membrane-bound PAPP-A, which could restrain further IGF action or have physiological consequences independent of IGFs. Further study is necessary to understand the role of PAPP-A in atherosclerostic plaque development. Nonetheless, the fact that PAPP-A colocalizes to activated macrophages through cell binding and internalization sets the stage for potential use of PAPP-A as an imaging target for vulnerable plaque.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Heart, Lung, and Blood Institute Grant HL-74871 (to C. A. Conover).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Conover, Mayo Clinic, 200 First St. SW, 5-194 Joseph, Rochester, MN 55905 (e-mail: Conover.Cheryl{at}mayo.edu.)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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S. C. Harrington, R. D. Simari, and C. A. Conover
Genetic Deletion of Pregnancy-Associated Plasma Protein-A Is Associated With Resistance to Atherosclerotic Lesion Development in Apolipoprotein E-Deficient Mice Challenged With a High-Fat Diet
Circ. Res., June 22, 2007; 100(12): 1696 - 1702.
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