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(Circulation. 2002;105:650.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Monocyte/Macrophage Regulation of Vascular Calcification In Vitro

Yin Tintut, PhD; Jignesh Patel, MD, PhD; Mary Territo, MD; Trishal Saini; Farhad Parhami, PhD; Linda L. Demer, MD, PhD

From the Departments of Medicine (Y.T., J.P., M.T., T.S., F.P., L.L.D.) and Physiology (L.L.D.), UCLA School of Medicine, Los Angeles, Calif.

Correspondence to Yin Tintut, PhD, Division of Cardiology, UCLA School of Medicine, 47-123 Center for the Health Sciences, 10833 Le Conte Ave, Los Angeles, CA 90095-1679. E-mail ytintut{at}ucla.edu

	Abstract

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Background— Calcification is a common complication of atherosclerosis and other chronic inflammatory processes that involves infiltration of monocytes and accumulation of macrophages.

Methods and Results— To determine whether these cells modulate vascular calcification in vitro, calcifying vascular cells (CVCs), a subpopulation of osteoblast-like cells derived from the artery wall, were cocultured with human peripheral blood monocytes for 5 days. Results showed that alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation, was significantly greater in cocultures than in cultures of CVCs or monocytes alone. Both ALP activity and matrix mineralization increased in proportion to the number of monocytes added. Activation of monocyte/macrophages (M/Ms) by oxidized LDL further increased ALP activity in cocultures. However, neither conditioned medium from oxidized-LDL–activated M/Ms or transwell coculture had this effect on CVCs, which suggests a need for cell-to-cell contact. In contrast, conditioned medium from lipopolysaccharide-activated M/Ms increased ALP activity of CVCs. ELISA showed that lipopolysaccharide-activated M/Ms secreted tumor necrosis factor-, and neutralizing antibody to tumor necrosis factor- attenuated the induction of ALP activity by the conditioned media.

Conclusions— These results suggest that M/Ms enhance in vitro vascular calcification via 2 independent mechanisms: cell-cell interaction and production of soluble factors such as tumor necrosis factor-.

Key Words: muscle, smooth • lipids • atherosclerosis • leukocytes

	Introduction

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Vascular calcification is a major independent predictor of cardiovascular morbidity and mortality.¹ Although pathologists have known for centuries that vascular calcification often includes fully formed bone tissue and even marrow within the artery wall,² little was known of the mechanisms until this decade. Like skeletal bone, human atherosclerotic calcification contains matrix vesicles, which are key elements in organized calcium phosphate crystal formation, and the stoichiometry of crystals in atherosclerosis matches that of the bone mineral hydroxyapatite.³ In addition, a series of reports beginning in 1992 provided evidence that many osteogenic regulatory factors are expressed in atherosclerotic lesions^4–6 and that the process resembles embryonic bone development.^7,8

In previous studies, we⁹ showed that calcifying vascular cells (CVCs), a subpopulation of cells isolated from the aortic medial layer, undergo osteoblastic differentiation and mineralization in vitro. We and others^10–12 have shown that vascular medial cells express bone differentiation markers, and the distinctive time sequence of expression mimics that in osteoblastic differentiation of skeletal bone cells.^13,14 One of the most widely used bone differentiation markers is the bone/liver/kidney isoenzyme of alkaline phosphatase (ALP).^14,15 The function of ALP in osteoblastic differentiation is believed to be hydrolysis of ester phosphates at sites of mineralization, providing ionic phosphate for incorporation into mineral. Although this enzyme is also expressed in liver and kidney cells among mesenchymal cells, induction of ALP is specific for osteoblastic differentiation.

Lipids and monocyte/macrophages (M/Ms), known atherogenic factors, colocalize with calcium deposits in plaques.^3,16,17 Whereas lipids have been shown to regulate vascular calcification,¹⁸ the role of M/Ms is unknown. Factors in the subendothelial space of diseased arteries such as oxidized LDL (ox-LDL) and lipopolysaccharide (LPS) activate M/Ms to produce reactive oxygen species (ROS), transforming growth factor-ß (TGF-ß), osteopontin, and inflammatory cytokines including interleukins and monocyte chemotactic protein-1.^19–22 Of these factors, TGF-ß, oxidized lipids, tumor necrosis factor- (TNF-), and ROS are reported to enhance in vitro calcification of vascular cells.^9,18,23,24

In the present study, we hypothesized that M/Ms regulate vascular calcification. We tested whether M/Ms promote osteoblastic differentiation and mineralization of CVCs and whether this effect is modified by ox-LDL or LPS activation of M/Ms.

	Methods

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Materials
LPS (Escherichia coli 0111:B4), polymyxin B, and anti-TNF- antibody were purchased from Sigma. The ELISA kit for TNF- was purchased from Biosource International. The class-matched mouse anti-human antibody against pro-B-cell antigen CD19 was purchased from Dako. Ox-LDL was prepared from freshly isolated human LDL as described previously.²⁵

Cell Culture
CVCs were isolated from bovine aortic medial explant and were cloned and identified as described previously.^9,13 CVCs were grown in Dulbecco’s modified Eagle’s medium (DMEM; Irvine Scientific) containing 15% heat-inactivated fetal bovine serum (FBS; Hyclone Labs) and supplemented with sodium pyruvate (1 mmol/L), penicillin (100 U/mL), and streptomycin (100 U/mL), all from Irvine Scientific. Cells from passages 12 to 17 were used.

Coculture
CVCs (4x10⁴/well) and monocytes (13 to 15x10⁴/well) were cocultured in 24-well dishes for the indicated period. Test agents were applied 2 days after culture. Media were replenished every 3 to 4 days.

Conditioned Media
Freshly isolated peripheral blood monocytes from healthy donors were plated in 30% autologous serum. After 5 days in culture, M/Ms were treated with vehicle alone or ox-LDL or bacterial LPS in 5% DMEM for 4 to 7 hours. After treatment, cells were washed 3 times in 1xPBS and then placed in fresh 5% DMEM overnight. The medium was collected and centrifuged to remove cellular debris, and the supernatant was stored at -70°C before use. To ensure that the effect of conditioned medium on CVCs was not due to the direct effect of LPS carried over from the treated cells, the conditioned medium was tested with quantitative chromogenic limulus amoebocyte lysate (Biowhitaker, Inc). In addition, conditioned medium was also incubated with polymyxin B for 1 hour at 37°C to neutralize the LPS before application on CVC culture.

Transwell Coculture
CVCs (4x10⁴/well) were plated in 24-well dishes. Freshly isolated monocytes were plated in transwell chambers, and the chambers were placed in the wells above the CVCs. The transwell allows exchange of the medium; however, cell-cell interactions between CVCs and monocytes were inhibited by polycarbonate membranes with 0.4-µm pores. Two days after plating, cultures were treated with buffer or 50 µg/mL ox-LDL, and ALP activity was measured as described below.

ALP Activity Assay
Cells were cultured in 24-well plates at 80% confluence and were treated after 2 days of culture with or without test agents. Cells were incubated for the indicated period, and ALP activity was measured as described previously.^13,18 ALP activity was normalized to total protein determined with Bio-Rad protein assay solution (Bio-Rad Laboratories).

Von Kossa Staining
CVCs and monocytes were cocultured as described above. Media were replenished 2 days after fresh plating with media containing 4 mmol/L CaCl₂ and 5 mmol/L ß-glycerophosphate to accelerate CVC calcification. Von Kossa staining was performed as described previously.^13,18

ELISA
Conditioned media from M/Ms that had been treated with LPS for 4 to 7 hours were centrifuged, and the supernatant was immediately frozen at -70°C. Samples were thawed at room temperature immediately before the TNF- ELISA was performed. Data were from 2 representative experiments and are shown as mean±SD of triplicate samples.

Statistical Analysis
Data are expressed as mean±SD. Means were compared by 1-way ANOVA, with comparison of different groups by Fisher’s protected least significant difference test. A value of P<0.05 was considered significant.

	Results

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Effects of M/Ms on CVC Differentiation
To determine whether M/Ms have a functional role in osteoblastic differentiation of vascular cells, CVCs were cocultured with freshly isolated human peripheral blood monocytes for 2 or 5 days, and ALP activity, an established early marker of osteoblastic differentiation,^14,15 was measured. Results showed that ALP activity was significantly enhanced after 5 days in cocultures compared with cultures of CVCs alone (536±9.6 versus 209±9.4 U/mg protein, P<0.0001; Figure 1a). Monocytes cultured alone produced no detectable ALP activity (Figure 1a). The increasing numbers of monocytes added to the CVC culture correlated with the increasing level of ALP activity (r=0.98; Figure 1b).

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of monocytes on CVC differentiation and mineralization. a, ALP activity, normalized for total protein, from whole-cell lysates of CVCs alone, CVCs cocultured with freshly isolated peripheral blood monocytes, or monocytes alone. Each bar represents mean±SD of quadruplicate wells (n=3; *P<0.0001). b, ALP activity from CVCs cocultured with increasing numbers of monocytes per well for 6 days (n=2, r=0.98). c, Von Kossa staining of CVC culture (A), CVCs cocultured with increasing numbers of monocytes (B [250 000] and C [500 000]), or monocyte culture (D, 500 000).

To determine whether M/Ms also promote mineralization, CVCs were cocultured for 9 days with increasing numbers of monocytes, and matrix calcium mineral incorporation was demonstrated by von Kossa staining. Results showed that mineralization also increased with the number of monocytes added to the CVC cultures (panels A through C in Figure 1c). Monocytes cultured alone showed no mineralization (panel D in Figure 1c).

Effects of Activated M/Ms on CVC Differentiation
Because M/Ms are exposed to activating inflammatory factors in the subendothelial space, we tested the effects of M/M activation on CVC differentiation using ox-LDL and LPS.

Effects of Ox-LDL–Activated Macrophages on CVC Differentiation
Treatment of CVC/monocyte cocultures with ox-LDL (100 µg/mL) for 3 days resulted in a 6-fold (5.9±0.2-fold) increase in ALP activity compared with untreated CVCs alone and a 2-fold (1.9±0.1-fold) increase compared with untreated coculture (Figure 2). Ox-LDL treatment of CVCs alone increased ALP activity 1.5±0.4-fold (Figure 2). Because monocyte induction of ALP in CVC plateaus at 6 days (data not shown), the 3-day time point, where induction is submaximal, was chosen to discern the added effects of ox-LDL.

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of ox-LDL on CVC/monocyte coculture. ALP activity of CVCs cultured alone or with monocytes or ox-LDL (100 µg/mL; n=2; *P<0.05).

We examined whether ox-LDL and monocytes enhanced ALP activity via secreted factors by evaluating conditioned media. CVCs were treated with conditioned media obtained from monocytes that had been activated with vehicle alone (unstimulated) or ox-LDL (100 µg/mL) for 7 hours. Before collection of the conditioned media, monocytes were washed thoroughly to remove ox-LDL and incubated in fresh medium overnight. Nonconditioned (fresh) media or overnight-conditioned media from unstimulated or ox-LDL–stimulated M/Ms were added to CVC culture, and ALP activity was measured after 3 days. Results showed that there was no significant difference in induction of ALP activity when conditioned media from ox-LDL–activated M/Ms were used versus conditioned media from unstimulated M/Ms (62.2±9.0 versus 57.1±5.1 U/mg protein, P=0.26; Figure 3, top). However, ALP activity was increased in both conditioned media from unstimulated and ox-LDL–stimulated M/Ms compared with nonconditioned (fresh) media (57.1±5.1 and 62.2±9.0 versus 40.0±2.3 U/mg protein, P<0.005; Figure 3, top), which suggests that conditioned media from unstimulated M/Ms secrete soluble factors that are not further enhanced by stimulation with ox-LDL.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of conditioned medium from ox-LDL–activated M/Ms on CVC differentiation. Top, ALP activity of CVC cultures treated for 3 days with nonconditioned (fresh) media, conditioned media from unstimulated M/Ms, or conditioned media from M/Ms activated with 100 µg/mL ox-LDL (n=2; *P<0.005). Bottom, ALP activity of CVCs cultured alone (CVC), in noncontact coculture in transwell, or in direct coculture with monocytes (coculture) in presence of 50 µg/mL ox-LDL as described in Methods (n=2; **P<0.0005). Note that smaller increase in ALP activity of coculture over CVC may be explained by lower concentrations of ox-LDL used in this experiment.

To determine whether close contact between monocytes and CVCs is necessary for ox-LDL–induced ALP, monocytes and CVCs were cocultured in separate chambers of transwells, monocytes on the polycarbonate 0.4-µm-pore filters of the upper chamber and CVCs on the tissue culture plastic of the lower chamber. These transwells allow the exchange of soluble factors without cell-cell contact. Cultures were treated with 50 µg/mL of ox-LDL, and ALP activity was measured after 3 days of treatment. Results showed that ALP activity was not enhanced in the transwell cocultures, whereas it was enhanced in direct coculture (Figure 3, bottom), which suggests that close contact between monocytes and CVCs was required for the ox-LDL enhanced effect.

Effects of LPS-Activated Macrophages on CVC Differentiation
Because we have previously found that LPS strongly induces CVC differentiation (unpublished data), further induction by monocytes was not expected to be detectable. Therefore, we pretreated monocytes separately with LPS, then collected the conditioned media to treat CVCs. Freshly isolated monocytes were activated with LPS for 7 hours, and conditioned media were collected as described above. To prevent residual LPS contamination, the conditioned media were neutralized with polymyxin B before treatment of CVCs. Results showed that conditioned media from LPS-activated M/Ms significantly enhanced ALP activity compared with controls (156.8±16 versus 40.0±2.3 U/mg protein, P<0.0001), which suggests that LPS activation leads to secretion of a soluble factor that induces CVC differentiation. A quantitative chromogenic limulus amoebocyte lysate assay showed that the concentration of residual LPS in conditioned media was <50 pg/mL, which is below the threshold concentration required (>100 pg/mL) to induce CVC ALP activity (data not shown).

Kornbluth et al²¹ showed that LPS induces TNF- in M/Ms, and we²³ previously found that TNF- is a potent inducer of CVC differentiation and mineralization. Therefore, we examined whether TNF- is responsible for the osteoinductive effect of the conditioned medium from LPS-activated monocytes. CVCs were incubated with conditioned media from LPS-activated M/Ms that had been pretreated for 1 hour with either control or TNF- neutralizing antibody, and ALP activity was measured after 2 days. Results showed that TNF- neutralizing antibody attenuated the induction of ALP activity (Figure 4, top). Class-matched irrelevant antibody (mouse anti-human CD19) had a small but not significant effect on ALP activity (data not shown). ELISA of the conditioned media from LPS-activated M/Ms showed immunoreactivity for TNF- (Figure 4, bottom) at a level previously shown to induce CVC differentiation.²³ Conditioned media from irrelevant cell types (CVCs or smooth muscle cells) showed no significant level of TNF- (Figure 4, bottom).

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of conditioned medium from LPS-activated M/Ms on CVC differentiation and evidence for secreted TNF-. Top, ALP activity of CVC cultures treated with conditioned media (cdm) from unstimulated M/Ms, LPS-activated M/Ms, or LPS-activated M/Ms that were pretreated with neutralizing antibody to TNF- (TNF Ab) for 1 hour at 37°C at 1 or 2 µg/mL. Bottom, ELISA assay of TNF- in conditioned media (cdm) from unstimulated M/Ms, LPS-activated M/Ms, or conditioned media from irrelevant cells (CVCs or smooth muscle cells [SMC]) that had been activated with LPS. *P<0.001.

	Discussion

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The present results indicate that monocytes enhance osteoblastic differentiation of CVCs, as evidenced by increased ALP activity and increased matrix mineralization. Activation of M/Ms further enhanced CVC differentiation. Interestingly, mechanisms by which activated monocytes induced CVC differentiation varied depending on the activating agent; the effect of ox-LDL activation required close contact with the CVCs, whereas the effect of LPS activation was mediated at least in part through secretion of TNF-.

Macrophages play a critical role in pathogenesis of chronic inflammatory conditions, including atherosclerosis.²⁶ The present results suggest that both unstimulated monocytes and activated macrophages contribute to in vitro vascular calcification through effects on osteoblastic differentiation. This finding is supported by the report of Jeziorska and colleagues¹⁶ that macrophages are associated with various phases of calcification in atherosclerotic lesions. Interestingly, histological analyses identify macrophages and giant cells near the calcified regions in other chronic pathological conditions, including aortic valvular sclerosis^8,27 and calcifying tendinitis.²⁸

Chronic infection has been implicated in atherogenesis. For example, Chlamydia pneumoniae infection is associated with coronary artery disease,²⁰ and its antigen has been detected in atherosclerotic lesions.²⁹ Ericson et al³⁰ reported that the degree of immunoreactivity of C. pneumoniae correlates with the severity of atherosclerosis. This may be due to inflammatory changes in macrophages that contribute to lesion progress in concert with hyperlipidemia. Our data suggest that bacterial products such as endotoxin (LPS) may contribute to vascular cell calcification indirectly through production of cytokines by activated M/Ms. These results are in agreement with reports of the possible role of C. pneumoniae or other infectious agents in valvular calcification.^31,32

Our data also show that activation of M/Ms by ox-LDL enhances monocyte-induced calcification. Our findings suggest that cell-cell contact between CVCs and M/Ms is necessary for ox-LDL–enhanced osteogenic effects. A similar cell-cell contact requirement has been reported for M/M stimulation of prostanoid synthesis in vascular smooth muscle cells.³³ Cell-cell interaction between monocytes and CVCs could be operating through engagement of cell-surface integrins with extracellular matrix proteins. Integrins such as -2 regulate osteoblastic differentiation and mineralization in vitro through binding to the extracellular matrix protein osteopontin.³⁴ Osteopontin is abundant in several pathological conditions, including atherosclerosis and aortic stenosis, especially at sites of mineralization, and is synthesized by macrophages, smooth muscle cells, and CVCs.^6,12,28 Other possible mechanisms include production of TGF-ß or ROS by macrophages. These factors contribute to vascular calcification,^9,24 and cell-cell contact may be required because TGF-ß is likely to bind to extracellular matrix and because other cellular processes mediated by macrophage-derived oxygen radicals also require cell-cell contact, as reported by Mix and collegues.³⁵

Vascular calcification is a complex phenomenon whose regulation is not well understood. Our findings indicate a role for M/Ms in this process. Moreover, oxidized lipids deposited in the artery wall and LPS released by bacteria such as C. pneumonia may further enhance vascular calcification by activating M/Ms to induce osteoblastic differentiation of CVCs. Additional in vivo and human studies are required to extend the relevance of these data to the human condition.

	Acknowledgments

 
This work was supported by NIH grant HL30568 and the Laubisch and Sam Nassi funds. We thank the UCLA Atherosclerosis Research Unit Core Laboratory.

Received October 2, 2001; revision received November 9, 2001; accepted November 14, 2001.

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