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(Circulation. 2000;102:2636.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Tumor Necrosis Factor- Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway

Yin Tintut, PhD; Jignesh Patel, MD; Farhad Parhami, PhD; Linda L. Demer, MD, PhD

From the Division of Cardiology, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, Calif.

Correspondence to Yin Tintut, 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—Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor- (TNF-), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF- in in vitro vascular calcification.

Methods and Results—A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF- for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF- for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A–specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase activity, and mineralization induced by TNF-. Consistent with this, the intracellular cAMP level was elevated after TNF- treatment. Electrophoretic mobility shift assay demonstrated that TNF- enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation.

Conclusions—These results suggest that TNF- enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.

Key Words: muscle, smooth • signal transduction • atherosclerosis

	Introduction

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Tumor necrosis factor- (TNF-), a pleiotropic cytokine, has been shown to play a role in both vascular and bone pathophysiology. TNF- is mainly secreted by macrophages in response to factors such as oxidized LDL,¹ acetylated LDL,² physically damaged extracellular matrix,³ or bacterial infection.⁴ TNF- influences many aspects of atherogenesis,⁵ including increasing permeability of endothelial cells,⁶ promoting monocyte adhesion,⁷ inducing macrophage differentiation,⁸ and promoting foam cell formation.⁹ In vivo, TNF- induces arteriosclerosis-like lesions in coronary arteries.¹⁰ TNF- also regulates bone turnover, inhibiting osteoblastic function^{11 12 13} and stimulating bone resorption.¹⁴

Vascular calcification is a pathological condition that occurs in many diseases, including atherosclerosis, diabetes, and uremia.¹⁵ We and others have provided evidence that atherosclerotic calcification resembles osteogenesis,^{16 17 18} and factors regulating bone mineralization have been demonstrated in calcified atherosclerotic plaques.^{16 17 19 20 21 22} In previous studies of vascular calcification, we cloned a subpopulation of vascular cells from the bovine aortic media.^{16 17} In long-term culture, these calcifying vascular cells (CVCs) express osteoblastic differentiation genes in the sequence reported for bone cells and form a mineralized matrix in vitro.²³ Similar in vitro models have been developed by other investigators.^{18 24 25 26} Agents that are present in atherosclerotic arteries have been shown to promote the differentiation and mineralization of these vascular cells.^{17 27}

Although TNF- has been detected in both human and mouse atherosclerotic lesions,^{28 29} its contribution to vascular calcification has not been assessed. We and others found TNF- immunoreactivity in arteries with calcification in C57BL/6J mice fed an atherogenic diet.²⁹ In contrast, TNF- is not found in normal vascular intima or fatty streaks.^{29 30} Additional evidence that TNF- may regulate vascular calcification arises from recent in vivo studies showing that mice lacking the osteoprotegerin gene, a novel soluble member of the TNF- receptor superfamily, develop vascular calcification in addition to premature osteoporosis.³¹ Therefore, we hypothesized that TNF- regulates calcification in atherosclerotic lesions, and we examined the direct role of TNF- in in vitro vascular calcification.

In the present report, CVCs were treated with TNF- and the effects of TNF- on differentiation and mineralization of CVCs were determined. The results indicate that TNF- promotes CVC mineralization by modulating the expression of genes important for extracellular matrix formation and mineralization. We also showed that the enhanced mineralization of CVCs by TNF- was mediated in part by activation of the cAMP pathway and enhancement of the activity of transcription factors that are important for osteoblastic differentiation.

	Methods

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Materials
Human recombinant TNF- was purchased from Sigma Chemical Co. [⁴⁵Ca]CaCl₂, [-³²P]dCTP, [-³²P]ATP, and [³H]thymidine were from Amersham. Oligonucleotide probes containing AP1 and cAMP-responsive element-binding protein (CREB) consensus sequences were purchased from Santa Cruz Biotechnology, Inc. Oligonucleotide probe containing Cbfa1 binding site (OSE2)³² from osteocalcin promoter was synthesized by GIBCO BRL. Pertussis toxin, chelerythrine chloride, phorbol 12-myristate 13-acetate (PMA), AACOCF3, indomethacin, and KT5720 were purchased from Calbiochem. Anti-TNF- antibody was obtained from BioSource International. Anti-Fos and anti-Jun antibodies were purchased from Santa Cruz Biotechnology, Inc. Antibody specific to Cbfa1/Osf2³³ was a generous gift from Dr Gerald Karsenty. Quantification of bacterial endotoxin in human recombinant TNF- was tested with quantitative chromogenic limulus amebocyte lysate (LAL; BioWhittaker, Inc).

Cell Culture
CVCs were isolated from bovine aortic media layer and were identified as described previously.^{16 17} CVCs were grown in Dulbecco’s modified Eagle’s medium (DMEM; Irvine Scientific) containing 15% heat-inactivated 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 (passages 12 to 17) were treated at 80% confluence with appropriate agents in DMEM containing 5% FBS.

Alkaline Phosphatase Activity Assay
Cells were cultured in 24-well plates and treated with or without test agents at 80% confluence for 2 to 3 days. Cells were lysed and alkaline phosphatase (ALP) activity was measured as previously described.²⁷ ALP activity was normalized to total protein determined with the Bio-Rad protein assay solution (Bio-Rad Laboratories).

Reverse Transcription–Polymerase Chain Reaction
Total RNA (3 µg) extracted as mentioned above was reverse-transcribed, and polymerase chain reaction (PCR) with ALP and GAPDH-specific primers was performed as described previously.²³

⁴⁵Ca Incorporation Assay
Cells were cultured in 24-well plates and treated at 80% confluence with or without 10 ng/mL TNF-. In the experiments in which there were pretreatments, cells were treated with agents 2 hours before addition of TNF- to the media. After 3 to 4 days of incubation, media were replaced with fresh media containing TNF- (pretreated agents and TNF-), 4 mmol/L CaCl₂, and 5 mmol/L ß-glycerophosphate (ßGP) and incubated for an additional 3 to 4 days. Cells were washed 3 times and incubated in fresh media containing TNF-, ßGP, and [⁴⁵Ca]CaCl₂ (1.0 µCi/mL) for an additional 48 hours. The matrix-bound radiolabeled calcium incorporation was performed as described previously.²³

cAMP Assay
Cells were plated in 6-well plates and treated at 80% confluence with serum-free medium for 30 minutes before being treated with 50 ng/mL TNF- in serum-free medium supplemented with 1 mmol/L 3-isobutyl-1-methylxanthine (IBMX; Calbiochem) for an additional 30 minutes. After incubation, cells were washed and scraped in 20 mmol/L phosphate buffer (pH 7.0) containing 20 mmol/L EDTA and 1 mmol/L IBMX. The extracts were sonicated briefly and boiled for 7 minutes to precipitate the cellular proteins. The extract was clarified by centrifugation, and supernatant was assayed for cAMP level with the cAMP EIA kit (Stratagene).

Gel Mobility Shift Assay
Nuclear extracts were prepared as described previously.³⁴ Three to 5 µg of nuclear extracts was incubated with binding buffer containing 20 mmol/L Tris-HCl (pH 8), 10 mmol/L NaCl, 3 mmol/L EDTA, 0.05% Nonidet P-40, 5 mmol/L dithiothreitol, 5% glycerol, and 1 µg of poly(dIdC) for 15 minutes, after which labeled probe was added and incubated for an additional 15 to 20 minutes. The samples were subjected to electrophoresis on a chilled 5% polyacrylamide gel at 400 V for 14 minutes. In the supershift experiments, respective antibodies were preincubated with nuclear extracts for 30 minutes before addition of the labeled probe. In competition experiments, 100-fold excess of the respective cold oligonucleotides was preincubated with nuclear extracts for 15 minutes before addition of the labeled probe.

Statistical Analysis
Data are expressed as mean±SD, and means were compared by 1-way ANOVA. The Tukey-Fisher least significant difference (LSD) criterion was used to judge statistical significance.

	Results

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Effects of TNF- on Markers of Osteoblastic Differentiation and Mineralization
To determine the effects of TNF- on onset of osteoblastic differentiation, CVCs were treated for 3 days, and 2 established markers of early osteoblastic differentiation, morphological change³⁵ and ALP activity,³⁶ were assessed. TNF- induced a morphological change from an elongated to a cuboidal shape (Figure 1A). Treatment of CVCs with increasing concentrations of TNF- showed a dose-dependent increase in ALP activity (Figure 1B). We compared the mean values with 1-way ANOVA. A significant increasing trend by dose was observed (F=2117; P<0.00005). The effect of TNF- on ALP activity was not due to an artifact of normalization, because even without normalization for total protein, the ALP activity was increased 15-fold by TNF- (10 ng/mL) (data not shown). Further tests were performed to determine that the effects were specific to TNF-. Results from a quantitative chromogenic LAL assay showed an endotoxin (lipopolysaccharide) level of 4x10^-5 pg/µL, which is 5 orders of magnitude below the level required for activity in this system. In addition, treatment with TNF- antibody completely blocked the induction of ALP activity (data not shown), indicating that TNF- effect on ALP activity is not attributable to contamination with bacterial endotoxin.

View larger version (87K): [in this window] [in a new window]   Figure 1. Effect of TNF- on CVC differentiation and mineralization. CVCs were treated with control (no TNF-) or medium containing 10 ng/mL TNF- for 3 days. A, Phase contrast (magnification x40) showing change in CVC morphology from elongated (control) to cuboidal shape (TNF-) (n=5). B, ALP activity from whole-cell lysates of CVCs treated with increasing concentrations of TNF- as indicated. Enzyme activity was normalized for total protein concentration. Each bar represents mean of 4 wells (n=3). C, Semiquantitative PCR of 2 separate samples from control and TNF-–treated cultures for expression of ALP mRNA. GAPDH expression was used as internal control (n=2). D, Calcium mineral deposition assayed by incorporation of 45Ca in matrix. Cells were treated with medium containing increasing concentrations of TNF-. Each bar represents mean of 4 wells (n=3). *P<0.00005.

To determine whether the induction of ALP by TNF- was at the level of mRNA expression, total RNA was isolated from samples treated with TNF- for 3 days, and semiquantitative reverse transcription-PCR (RT-PCR) was performed. Results showed that TNF- also enhanced mRNA expression of the ALP gene (Figure 1C). TNF- did not enhance expression of the internal control gene GAPDH. Treatment of CVCs with TNF- for 3 days did not appreciably affect expression of other osteoblastic genes, type I procollagen, and osteocalcin (data not shown).

To determine whether enhanced ALP activity ultimately results in increased mineralization, CVCs were treated with increasing concentrations of TNF- for 9 to 10 days, and matrix calcium mineral incorporation was assayed with ⁴⁵Ca as reported previously.²³ The results showed that TNF- dose dependently increased mineralization in these cells (Figure 1D). A significant increasing trend by dose was observed (F=123; P<0.00005).

Second Messenger of TNF- in CVC Differentiation and Mineralization
In various cell types, the pleiotropic effects of TNF- are mediated by different intracellular signaling pathways. Therefore, we examined the signaling pathway used by TNF- in induction of CVC differentiation and mineralization. First, we assessed the role of pertussis toxin–sensitive G protein, which mediates TNF- signaling in other systems.^{6 37} CVCs were pretreated with pertussis toxin for 2 hours before TNF- treatment. Results showed that pertussis toxin did not inhibit TNF-–induced ALP activity (measured 2 days after TNF- treatment), indicating that TNF- does not signal through a pertussis toxin–sensitive G protein in CVCs (Figure 2A). Instead, pertussis toxin induced ALP activity synergistically with TNF- (Figure 2A). By 1-way ANOVA, the difference between control and TNF- plus pertussis toxin (100 ng/mL) was significant (P<0.05 level; LSD 83.8). We previously showed that treatment with pertussis toxin alone (without subsequent TNF- treatment) had relatively little effect on ALP activity.²³

View larger version (30K): [in this window] [in a new window]   Figure 2. Involvement of signal transduction pathways on TNF-–induced ALP. A, ALP activity in CVCs treated with TNF- alone or TNF- with pertussis toxin (PT) for 2 days. B, ALP activity in CVCs treated with TNF- alone (10 ng/mL) or TNF- with increasing concentrations of cyclooxygenase inhibitor (indomethacin; Indo) or cPLA2 inhibitor (AACOCF3) for 2 days. C, ALP activity in CVCs treated with control or TNF- alone or TNF- with increasing concentrations of PKC inhibitor (chelerythrine chloride; ChCl) for 3 days. All inhibitors were applied 2 hours before addition of TNF-. Each bar represents mean of 4 wells (n=2). *P<0.05.

To determine whether TNF- activated phospholipase A₂ (PLA₂) and cyclooxygenase, as found in bone cells,³⁸ CVCs were pretreated with inhibitors of cytoplasmic PLA₂ (AACOCF3) or cyclooxygenase (indomethacin) for 2 hours before addition of TNF-. ALP activity (measured 2 days after treatment) showed that these agents also did not inhibit TNF-induced ALP activity (Figure 2B). Instead, the ALP activity induced by TNF- was further enhanced by the inhibitors. By 1-way ANOVA, the difference between control and each higher-dose treatment was significant (P<0.05 level; LSD 44.7). Treatments with inhibitor (AACOCF3 or indomethacin) alone had relatively little or no effect on ALP activity, CVC morphology, and total protein level (data not shown).

We also examined the role of the protein kinase C (PKC) pathway in TNF- effects in CVCs. Inhibition of the PKC pathway by pretreatment with a PKC-specific inhibitor, chelerythrine chloride (0.5 to 1.0 µmol/L), for 2 hours did not reverse the TNF-–induced ALP activity (Figure 2C). Instead, as seen with pertussis toxin and cyclooxygenase inhibitors, ALP activity induction by TNF- was further enhanced by the chelerythrine chloride. By 1-way ANOVA, the difference between chelerythrine chloride–treated CVCs and the controls was significant (P<0.05; LSD 65.6). The same response was observed when PKC was downregulated by prolonged treatment (24 hours) with 1 mmol/L PMA (data not shown). Treatment of CVCs with chelerythrine chloride or PMA alone without subsequent TNF- treatment had no effects on ALP activity or cell morphology (data not shown).

Because we have previously shown that the cAMP pathway promotes osteoblastic differentiation of CVCs,²³ we also examined the involvement of the cAMP/protein kinase A (PKA) pathway in response to TNF-. CVCs were treated with a PKA-specific inhibitor, KT5720.³⁹ Treatment of CVCs for 2 hours with KT5720 before addition of TNF- dose dependently inhibited the TNF-–induced morphological change (Figure 3A). KT5720 also inhibited TNF-–induced ALP activity (measured 2 days after treatment) (Figure 3B) and mineralization (measured 9 days after treatment) (Figure 3C) in a dose-dependent manner. Consistently, intracellular cAMP level was increased 2-fold in TNF-–treated cells (Figure 3D).

View larger version (82K): [in this window] [in a new window]   Figure 3. Effect of PKA specific inhibitor, KT5720, on TNF-–induced CVC differentiation and mineralization. CVCs were pretreated for 2 hours with increasing concentrations of KT5720 before addition of TNF- (10 ng/mL) to media. A, Phase contrast (magnification x40) of control, TNF- alone, TNF- + 5.0 µmol/L KT5720, or TNF- + 10.0 µmol/L KT5720 (n=2). B, ALP activity measured 2 days after treatments. All samples contain 10 ng/mL TNF-. Each bar represents mean of 4 wells (n=2). *P<0.001 vs TNF- alone. C, 45Ca incorporation in matrix measured 9 days after treatment. All samples contain TNF-. Each bar represents mean of 4 wells (n=2). **P<0.05 vs TNF- alone. D, Growth-arrested CVCs were treated with TNF- (50 ng/mL) in serum-free medium supplemented with IBMX for additional 30 minutes, and intracellular cAMP levels were analyzed. Average cAMP levels from duplicate wells are shown (n=2). **P<0.05 vs control.

Effect of TNF- on Transcription Factors Cbfa1/Osf2, AP1, and CREB
Because TNF- modulated the gene expression in CVCs as shown above, we also examined the effects of TNF- on transcription factors important in osteoblastic differentiation: Cbfa1/Osf2, AP1, and CREB.^{36 40 41} Nuclear extracts from CVCs treated with TNF- were prepared, and an electrophoretic mobility shift assay was performed with probes containing Cbfa1/Osf2, AP1, or CREB binding sites. Results showed that treatment of CVCs with forskolin or TNF- enhanced the DNA binding of Cbfa1/Osf2 (Figure 4A, left). Dibutyryl cAMP also enhanced Cbfa1/Osf2 DNA binding, whereas the same concentration of dibutyryl cGMP had little or no effect (data not shown). Preincubation of anti-Cbfa1 antibody³³ with TNF-–treated nuclear extracts before addition of labeled probe resulted in a supershifted band and a decrease in the faster-migrating complex, indicating the specificity of Cbfa1/Osf2 (Figure 4A, right). TNF- also enhanced DNA binding of AP1 and CREB transcription factors (Figure 4 B and 4C). AP1 DNA binding was competed with 100-fold excess of cold AP1 oligo probe (Figure 4B), and CREB DNA binding was competed with 100-fold excess of cold CREB oligo probe (Figure 4C, left). Incubation of nuclear extracts from TNF-–treated cells with anti-CREB antibody resulted in a supershifted complex (Figure 4C, right). Incubation with anti-Fos or anti-Jun antibodies, however, did not result in a supershifted complex, indicating that AP1 family members other than c-fos and c-jun were activated in response to TNF- in CVCs (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of TNF- on DNA binding of transcription factors Cbfa1/Osf2, AP1, and CREB. DNA binding was analyzed by gel retardation assay. A, left panel, Nuclear extracts prepared from 2 separate samples treated for 3 days with either vehicle alone (control), forskolin (25 µmol/L), or TNF- (10 ng/mL) for 3 days were incubated with 32P-labeled probe containing wild-type Cbfa1 binding probe.32 Bracket indicates mobility shift (n=4). A, right panel, Nuclear extracts from TNF-–treated cells were preincubated with 2 µL of anti-Cbfa1/Osf2 antibody for 30 minutes before addition of labeled Cbfa1 binding probe (n=2). Arrow indicates supershifted complex. B, Nuclear extracts from CVCs treated with vehicle alone or TNF- (10 ng/mL) for 3 days were incubated with AP1 binding oligo probe (n=3). A 100-fold excess of cold oligo probe was preincubated with nuclear extracts (third lane, n=2). C, left panel, Nuclear extracts from CVCs treated with TNF- (10 ng/mL) for 3 days were incubated with CREB binding oligo probe (n=3). A 100-fold excess of cold oligo probe was preincubated with nuclear extracts (third lane, n=2). C, right panel, Nuclear extracts from TNF-–treated cells were preincubated with 1 µL of anti-CREB antibody for 40 minutes before addition of labeled CREB binding probe. Arrow indicates supershifted complex.

	Discussion

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The present study showed that TNF- enhanced in vitro calcification of vascular cells, providing further evidence that vascular and bone calcification share regulatory factors. TNF- promotes CVC mineralization by increased expression and activity of ALP, an enzyme that has been shown to be important for matrix mineralization.^{36 42} The enhanced ALP may be a result of enhanced DNA binding of transcription factors shown to play a role in osteoblastic differentiation.^{36 40 41} These data, together with the previous report associating TNF- with calcified vascular lesions in mice,²⁹ raise the possibility that TNF- facilitates mineralization in atherosclerotic lesions.

TNF- has been shown to signal through different pathways in different cell types. In hepatic stellate cells and in osteoblasts, the TNF- receptor is coupled to a pertussis toxin–sensitive G protein, leading to activation of PLA₂ and de novo synthesis of cyclooxygenase.^{6 37 38} In cultured rat mesangial cells and human fibroblasts, TNF- signals through increased intracellular cAMP.⁴³ Our results suggest that TNF- triggers CVC mineralization via the cAMP pathway, based on the findings that PKA inhibition blocks and cAMP elevation mimics²³ the TNF-induced effects. In addition, the finding that pertussis toxin synergizes with TNF- in ALP induction further supports the possibility that TNF- signals via the cAMP pathway in CVCs, because inhibition of G_i by pertussis toxin enhances adenylate cyclase activity.

The results also suggested a form of cross talk between signaling pathways in CVCs. Inhibition of the PKC pathway potentiated the TNF- effect on ALP activity, which suggests that the PKC pathway may act as an antagonist of PKA-mediated CVC differentiation. This may be due to involvement of PKC in cellular proliferation,^{44 45} the decline of which has been shown to be necessary for the onset of osteoblastic differentiation.³⁶ The results also showed that similar effects were seen with inhibitors of the cyclooxygenase pathway, because activation of the PKC pathway by products of the cyclooxygenase pathway has been reported.⁴⁶

Although CVCs share features with bone-derived osteoblasts in many aspects, we previously showed that their responses to certain agents such as oxidized lipids²⁷ and active vitamin D are directly opposite.⁴⁷ We and other investigators^{11 12 13 14} have found that TNF- inhibited bone cell differentiation (unpublished data). The present results suggest that TNF- has similar reciprocal effects (stimulation of CVCs and inhibition of bone cell differentiation). The coexistence of vascular calcification and osteoporosis has been reported recently by Bucay et al³¹ in mice lacking the osteoprotegerin gene, a novel soluble member of the TNF- receptor superfamily. These inverse effects on bone and vasculature may be a result of different signaling pathways induced by TNF- in the 2 cell types: stimulation of the cAMP pathway in CVCs versus stimulation of the pertussis toxin–sensitive G-protein–mediated pathway but not the cAMP pathway in osteoblasts.³⁸ These differential responses to the same inflammatory agent may contribute in part to the paradoxical coexistence of vascular calcification and osteoporosis in older patients.

	Acknowledgments

 
This work was supported by NIH grant HL30568. We thank Dr Judith Berliner for suggestions; Dr Gerald Karsenty for providing us with anti-Cbfa1–specific antibody; H. Huynh and V. Le for technical assistance; and A. Han and A. Gasparyan for general assistance. We also thank the Atherosclerosis Research Unit Core A for LAL assay and Dr J. Gornbein for statistical analysis. The graphic illustrations were prepared in the Biomedical Technology Research and Instructional Productions (BTRIP) facility, and we also thank its staff for their assistance.

Received February 11, 2000; revision received June 21, 2000; accepted June 22, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Jovinge S, Ares MP, Kallin B, et al. Human monocytes/macrophages release TNF- in response to ox-LDL. Arterioscler Thromb Vasc Biol. 1996;16:1573–1579.[Abstract/Free Full Text]

2. Barath P, Cao J, Forrester JS. Low density lipoprotein activates monocytes to express tumor necrosis factor. FEBS Lett. 1990;277:180–184.[Medline] [Order article via Infotrieve]

3. Hershkoviz R, Cahalon L, Gilat D, et al. Physically damaged extracellular matrix induces TNF-a secretion by interacting resting CD4+ T cells and macrophages. Scand J Immunol. 1993;37:111–115.[Medline] [Order article via Infotrieve]

4. Mizutani H, Ohmoto Y, Mizutani T, et al. Role of increased production of monocytes TNF-alpha, IL-1beta and IL-6 in psoriasis: relation to focal infection, disease activity and responses to treatments. J Dermatol Sci. 1997;14:145–153.[Medline] [Order article via Infotrieve]

5. Libby P, Sukhova G, Lee RT, et al. Cytokines regulate vascular functions related to stability of the atherosclerotic plaque. J Cardiovasc Pharmacol. 1995;25:S9–S12.

6. Brett J, Gerlach H, Nawroth P, et al. Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins. J Exp Med. 1989;169:1977–1991.[Abstract/Free Full Text]

7. Thorne SA, Abbot SE, Stevens CR, et al. Modified low density lipoprotein and cytokines mediate monocyte adhesion to smooth muscle cells. Atherosclerosis. 1996;127:167–176.[Medline] [Order article via Infotrieve]

8. Riches DW, Chan ED, Winston BW. TNF-a-induced regulation and signalling in macrophages. Immunobiol. 1996;195:477–490.[Medline] [Order article via Infotrieve]

9. Li H, Freeman MW, Libby P. Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines. J Clin Invest. 1995;95:122–133.

10. Fukumoto Y, Shimokawa H, Ito A, et al. Inflammatory cytokines cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs. J Cardiovasc Pharmacol. 1997;29:222–231.[Medline] [Order article via Infotrieve]

11. Panagakos FS, Fernandez C, Kumar S. Ultrastructural analysis of mineralized matrix from human osteoblastic cells: effect of tumor necrosis factor-alpha. Mol Cell Biochem. 1996;158:81–89.[Medline] [Order article via Infotrieve]

12. Mayur N, Lewis S, Catherwood BD, et al. Tumor necrosis factor alpha decreases 1,25-dihydroxyvitamin D3 receptors in osteoblastic ROS 17/2.8 cells. J Bone Min Res. 1993;8:997–1003.

13. Rosenquist JB, Ohlin A, Lerner UH. Cytokine-induced inhibition of bone matrix proteins is not mediated by prostaglandins. Inflammation Res. 1996;45:457–463.[Medline] [Order article via Infotrieve]

14. Bertolini DR, Nedwin GE, Bringman TS, et al. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factors. Nature. 1986;319:516–518.[Medline] [Order article via Infotrieve]

15. Shanahan CM, Proudfoot D, Farzaneh-Far A, et al. The role of Gla proteins in vascular calcification. Crit Rev Eukaryotic Gene Expression. 1998;8:357–375.[Medline] [Order article via Infotrieve]

16. Boström K, Watson K, Horn S, et al. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993;91:1800–1809.

17. Watson KE, Boström K, Ravindranath R, et al. TGF-beta 1, and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994;93:2106–2113.

18. Shioi A, Nishizawa Y, Jono S, et al. Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995;15:2003–2009.[Abstract/Free Full Text]

19. Tanimura A, McGregor DH, Anderson HC. Matrix vesicles in atherosclerotic calcification. Proc Soc Exp Biol Med. 1983;172:173–177.[Medline] [Order article via Infotrieve]

20. Ginsberg B, Haarlem LJM, Soute BAM, et al. Characterization of a GLA-containing protein from calcified human atherosclerotic plaques. Arteriosclerosis. 1990;10:991–995.[Abstract/Free Full Text]

21. Katsuda S, Okada Y, Minamoto T, et al. Collagens in human atherosclerosis. Arterioscler Thromb. 1992;12:494–502.[Abstract/Free Full Text]

22. Giachelli C, Bae N, Almeida M, et al. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686–1696.

23. Tintut Y, Parhami F, Boström K, et al. cAMP stimulates osteoblast-like differentiation of calcifying vascular cells: potential signaling pathway for vascular calcification. J Biol Chem. 1998;273:7547–7553.[Abstract/Free Full Text]

24. Schor AM, Allen TD, Canfield AE, et al. Pericytes derived from the retinal microvasculature undergo calcification in vitro. J Cell Sci.. 1990;97:449–461.

25. Brighton CT, Lorich DD, Kupacha R, et al. The pericyte as a possible osteoblast progenitor cell. Clin Orthop Rel Res. 1992;275:289–299.

26. Proudfoot D, Skepper JN, Shanahan MCM, et al. Calcification of human vascular cells in vitro is correlated with high levels of matrix Gla protein and low levels of osteopontin expression. Arterioscler Thromb Vasc Biol. 1998;18:379–388.[Abstract/Free Full Text]

27. Parhami F, Morrow A, Balucan J, et al. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation: a possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol. 1997;17:680–687.[Abstract/Free Full Text]

28. Barath P, Fishbein MC, Cao J, et al. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol. 1990;64:297–302.

29. Qiao J, Xie P, Fishbein M, et al. Pathology of atheromatous lesions in inbred and genetically engineered mice. Arterioscler Thromb. 1994;14:1480–1497.[Abstract/Free Full Text]

30. Kaartinen M, Penttila A, Kovanen PT. Mast cells in rupture-prone areas of human coronary atheromas produce and store TNF-. Circulation. 1996;94:2787–2792.[Abstract/Free Full Text]

31. Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998;12:1260–1268.[Abstract/Free Full Text]

32. Ducy P, Zhang R, Geoffroy V, et al. A transcriptional activator of osteoblast differentiation. Cell. 1997;89:747–754.[Medline] [Order article via Infotrieve]

33. Thirunavukkarasu K, Mahajan M, McLarren KW, et al. Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfß. Mol Cell Biol. 1998;18:4197–4208.[Abstract/Free Full Text]

34. Dignam J, Lebowitz R, Roeder R. Accurate transcription by RNA polymerase II in soluble extracts from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475–1489.[Abstract/Free Full Text]

35. Nefussi JR, Pouchelet M, Collin P, et al. Microcinematographic and autoradiographic kinetic studies of bone cell differentiation in vitro: matrix formation and mineralization. Bone.. 1989;10:345–352.[Medline] [Order article via Infotrieve]

36. Stein GS, Lian JB, Stein JL, et al. Transcriptional control of osteoblast growth and differentiation. Physiol Rev.. 1996;76:593–629.[Abstract/Free Full Text]

37. Hernandez-Munoz I, Torre PDL, Sanchez-Alcazar JA, et al. Tumor necrosis factor a inhibits collagen a1(I) gene expression in rat hepatic stellate cells through a G protein. Gastroenterology. 1997;113:625–640.[Medline] [Order article via Infotrieve]

38. Yanaga F, Abe M, Koga T, et al. Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3–E1. J Biol Chem. 1992;267:5114–5121.[Abstract/Free Full Text]

39. Bernabeu R, Bevilaqua L, Ardenghi P, et al. Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proc Natl Acad Sci U S A. 1997;94:7041–7046.[Abstract/Free Full Text]

40. Sakamoto MK, Suzuki K, Takiya S, et al. Developmental profiles of phosphorylated and unphosphorylated CREBs in murine calvarial MC3T3–E1 cells. J Biochem. 1998;123:399–407.[Abstract/Free Full Text]

41. Rodan GA, Harada S. The missing bone. Cell. 1997;89:677–680.[Medline] [Order article via Infotrieve]

42. Klein BY, Gal I, Segal S. Studies of the levamisole inhibitory effect on rat stromal-cell commitment to mineralization. J Cell Biochem. 1993;53:114–121.[Medline] [Order article via Infotrieve]

43. Baud L, Perez J, Friedlander G, et al. Tumor necrosis factor stimulates prostaglandin production and cyclic AMP levels in rat cultured mesangial cells. 1988;239:50–54.

44. Lew DB, Brown ER, Dempsey BK, et al. Contribution of PKC to beta hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol. 1997;272:L639–L643.[Abstract/Free Full Text]

45. Quarles LD, Haupt DM, Davidai G, et al. Prostaglandin F2 alpha-induced mitogenesis in MC3T3–E1 osteoblasts: role of protein kinase-C-mediated tyrosine phosphorylation. Endocrinology. 1993;132:1505–1513.[Abstract/Free Full Text]

46. Fyrnys B, Claus R, Wolf G, et al. Oxidized low density lipoprotein stimulates protein kinase C (PKC) activity and expression of PKC-isotypes via prostaglandin-H-synthase in P388D1 cells. Adv Exp Med Biol. 1997;407:93–98.[Medline] [Order article via Infotrieve]

47. Watson K, Abrolate M, Malone L, et al. Active serum vitamin D levels are inversely correlated with coronary calcification. Circulation. 1997;96:1755–1760.[Abstract/Free Full Text]

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