| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Immunopathology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Correspondence: Larry M. Wahl, Ph.D., Immunopathology Section, Building 30, Room 325, National Institute of Dental and Craniofacial Reseacrh, National Institutes of Health, Bethesda, MD 20892-4352. E-mail: lwahl{at}dir.nidcr.nih.gov
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and GM-CSF, ox-LDL enhanced MMP-1 significantly by two- to threefold, increased MMP-9 slightly, and had no effect on TIMP-1 production. In contrast, HDL suppressed the induction of MMP-1 by TNF- and GM-CSF as well as the ox-LDL-mediated increase in MMP-1 production. The enhancement of MMP-1 production by ox-LDL occurred through, in part, a prostaglandin E2 (PGE2)-dependent pathway as indomethacin suppressed and PGE2 restored MMP-1 production. This conclusion was supported further by ox-LDL-mediated increases in PGE2 and cyclooxygenase-2 (COX-2) production. These data suggest that the interaction of primary monocytes with ox-LDL and proinflammatory cytokines may contribute to vascular remodeling and plaque rupture.
Key Words: atherosclerosis • prostaglandins • TNF- • GM-CSF
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
MMPs are comprised of a family of proteolytic enzymes that are capable of degrading all extracellular matrix components. MMP-1, an interstitial collagenase that degrades the fibrillar collagens I, II, III, VII, VIII, and X, and MMP-9/gelatinase B, which digests denatured or cleaved fibrillar collagens as well as basement membrane components, have been detected in atherosclerotic plaques but not in nonatherosclerotic vascular tissues [4 5 6 7 ]. Recent studies have demonstrated that within rupture-prone plaques, but not in stable plaques, monocytes/macrophages and MMP-1 colocalized in regions of enhanced collagenolysis [5 ]. Thus, MMPs may predispose vulnerable plaques to rupture by disrupting the connective tissue matrix within the vascular wall [5 , 6 ].
Factors within the vascular wall, such as lipids and cytokines, may contribute to the development, progression, and rupture of atherosclerotic plaques [5
, 8
9
10
]. Oxidized low-density lipoprotein (ox-LDL), but not native LDL, induced MMP-9 production and decreased its endogenous inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in 7-day human monocyte-derived macrophages [8
]. However, we do not know the effects of factors present in the vascular wall, including cytokines and ox-LDL, on the function of primary monocytes that enter the atherosclerotic lesion initially. We have shown previously that the combination of tumor necrosis factor (TNF-) and granulocyte macrophage-colony stimulating factor (GM-CSF), two cytokines found in the atheroma, induces monocyte MMP-1 production through a prostaglandin-dependent pathway and greatly enhances MMP-9 through a prostaglandin-independent pathway [11
]. In the present study, we evaluated the effects of ox-LDL, LDL, and high-density lipoprotein (HDL) on MMP-1, MMP-9, and TIMP-1 production in cytokine- and lipopolysaccharide (LPS)-stimulated monocytes. We demonstrate that although ox-LDL alone fails to influence monocyte MMP production, ox-LDL in the presence of TNF- and GM-CSF enhanced monocyte MMP-1 production significantly through a prostaglandin-dependent mechanism. MMP-9 was increased to a lesser degree by ox-LDL in the presence of cytokine stimulation, whereas TIMP-1 was unaffected.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Culture conditions
Purified monocytes were cultured in serum-free Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker, Walkersville, MD) supplemented with 2 mmol/L L-glutamine (Mediatech, Herndon, VA) and 10 µg/mL gentamicin sulfate (BioWhittaker). ox-LDL, LDL, and HDL were purchased from Intracel Corporation (Frederick, MD). LDL was oxidized by the manufacturer according to previously described methods [12
], and oxidation was verified by agarose gel electrophoresis [13
, 14
]. To examine the effects of the lipids on monocytes, the cells were plated at 5 x 106/mL DMEM per well in 12-well plates and adhered for 30 min at 37°C before reagents were added. The lipids were added to monocyte cultures alone or with TNF- (50 ng/mL; PeproTech, Rocky Hill, NJ) and GM-CSF (50 ng/mL; PeproTech) or LPS (Escherichia coli 055:B5; 100 ng/mL; Difco, Detroit, MI). Prostaglandin E2 (PGE2) and indomethacin (Sigma Chemical Co., St. Louis, MO) were added as indicated. Culture supernatants were then collected 24 or 36–48 h following stimulation for PGE2 and MMP-1/MMP-9 analyses, respectively. Each experiment was repeated a minimum of three times with different donors. Endotoxin levels in media and ox-LDL, LDL, and HDL were <10 pg/mL as determined by Limulus amoebocyte lysate assay.
Detection of MMP-1 and TIMP-1 by Western blot analysis
Proteins in the conditioned medium were precipitated 36–48 h after cytokine or LPS stimulation with cold ethanol (final concentration, 60%). Pelleted proteins (2500 g for 15 min) were lyophilized by rotary evaporation, resuspended in sodium dodecyl sulfate (SDS)-Laemmli-loading buffer (500 mmol/L Tris-HCl, pH 6.8, 10% SDS, 0.01% bromophenol blue, 20% glycerol), reduced with 1% 2-mercaptoethanol, and heated for 4 min at 100°C before being loaded on a 8–16% Tris-glycine gradient polyacrylamide gel (Invitrogen, Carlsbad, CA) in SDS buffer (25 mmol/L Tris-HCl, pH 8.3, 192 mmol/L glycine, 10% SDS). After electrophoresis, the proteins were transferred to 0.45 µm nitrocellulose (Schleicher & Schuell, Keene, NH) in a buffer containing 25 mmol/L Tris-HCl, pH 8.3, 192 mmol/L glycine, and 20% methanol, then blocked with 10 mmol/L Tris-HCL, pH 7.5, 20 mmol/L NaCl, and 0.2% Tween-20 (TBST) containing 5% nonfat dry milk, and incubated with a peptide-specific antibody for MMP-1 or TIMP-1 (generously provided by Dr. Henning Birkedal-Hansen, National Institute of Dental and Craniofacial Research, NIH). The blots were incubated with protein A-horseradish peroxidase (1:3000 dilution in TBST containing 5% nonfat dry milk; Amersham, Little Chalfont, UK) and developed with SuperSignal West Pico luminol/enhancer solution (Pierce, Rockford, IL).
MMP-1 was detected as the 55 and 53 kDa procollagenase (PCL) forms and the 45 and 43 kDa active collagenase (ACL) forms.
Detection of MMP-9 by zymography
Culture supernatants (20 µl) obtained 36–48 h following stimulation were added to loading buffer (5 µl) as described for Western blot analysis, except the samples were not heated or reduced. Samples were then loaded on 10% polyacrylamide gels (Invitrogen) containing 0.1% gelatin. Following electrophoresis, the gels were incubated in 2.5% Triton X-100 for 30 min at room temperature and digested in buffer containing 0.2 mol/L NaCl and 5 mmol/L CaCl2 for 2–3 h at 37°C. The gels were then stained with Coomassie blue (0.5% Coomassie blue, 10% glacial acetic acid, 25% isopropanol) and destained (10% glacial acetic acid, 25% isopropanol).
Cell protein isolation
Purified monocytes, 10 x 106/2 mL DMEM, were adhered to six-well plates (Falcon, Becton Dickinson, San Jose, CA) for 30 min at 37°C and then were incubated with 5 µg/mL ox-LDL in the presence or absence of TNF- (50 ng/mL) and GM-CSF (50 ng/mL). Cultures were incubated for 18–24 h, then were washed in phosphate-buffered saline with protease inhibitors (Complete Mini cocktail tablets, Roche Diagnostics, Nutley, NJ), and were scraped from the plates. The pellets were suspended in 250 mmol/L sucrose containing protease inhibitors and were sonicated (Ultrasonic Cell Disruptor, Kontes, Vineland, NJ). The nuclear debris was pelleted at 100 g for 15 min in a 4°C centrifuge, and the supernatant containing the membrane protein was pelleted at 2,500 g for 45 min in a 4°C centrifuge. Equal amounts of protein were loaded on a 10% Tris-glycine gel, and Western blot analysis was performed using an antibody against COX-2 (Cayman Chemical, Ann Arbor, MI).
PGE2 determination
Aliquots from three independent experiments were analyzed for PGE2 production by enzyme-linked immunosorbent assay (ELISA; Neogen Corporation, Lansing, MI), according to the manufacturer’s suggestions.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, a mediator of MMP production by monocytes/macrophages, compared with normal arteries [5
]. Additionally, previous studies have shown that the addition of TNF- or GM-CSF to monocytes enhances MMP-9 production, but will only induce MMP-1 production when added in combination [11
]. Therefore, we evaluated the role of TNF- and GM-CSF alone and in combination with ox-LDL on MMP and TIMP-1 production. We also compared the effects of LPS, a classic monocyte stimulant, in combination with ox-LDL on MMP and TIMP-1 production. As shown in Figure 1
, ox-LDL did not induce or enhance MMP-1 or MMP-9, respectively, when monocytes were stimulated with TNF- or GM-CSF. However, ox-LDL (5 µg/mL) enhanced the production of MMP-1 significantly by two- to threefold when added in combination with TNF- and GM-CSF or LPS. The production of MMP-9, which was found only in the 92 kDa proform, was relatively unaffected in this experiment by the addition of ox-LDL (5 µg/mL) in combination with TNF- and GM-CSF; however, ox-LDL increased MMP-9 production in LPS-treated monocytes (Fig. 1)
. ox-LDL had no effect on TIMP-1 production when added with TNF- or GM-CSF alone or the combination of the cytokines, whereas a slight increase in TIMP-1 was observed when ox-LDL was added with LPS (Fig. 1)
.
|
and GM-CSF. As shown in Figure 2
, optimal enhancement of monocyte MMP-1 and MMP-9 occurred when ox-LDL was added at the same time as the cytokines. Similar results were obtained with LPS.
|
|
and GM-CSF, but had no effect on MMP-9 production (Fig. 4
). In the presence of TNF- and GM-CSF, LDL at 5 µg/mL suppressed MMP-1 production, but enhanced MMP-1 production at 10 µg/mL. Similar to HDL, LDL had no significant effect on MMP-9 production (Fig. 4) .
|
|
and GM-CSF produced significantly more PGE2 compared with controls (Fig. 7B
). As COX-2 can mediate the release of PGE2, we also evaluated the effect of ox-LDL on COX-2 production. ox-LDL alone had no effect on COX-2 production; however, in combination with TNF- and GM-CSF, ox-LDL enhanced COX-2 production significantly (Fig. 7A)
.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
MMPs are a family of proteolytic enzymes produced by monocytes/macrophages that contribute to connective tissue remodeling in normal and inflammatory sites. Factors that regulate monocyte/macrophage MMP production, which may subsequently influence vascular remodeling, include cytokines [5
, 11
, 16
, 18
], extracellular matrix components [15
], cell-cell interactions [19
, 20
], ox-LDL [8
], and protease inhibitors [6
]. Our studies demonstrate that the interaction of ox-LDL with primary monocytes is not sufficient to induce MMP-1 or MMP-9 production; however, ox-LDL, in the presence of a specific cytokine combination—TNF- and GM-CSF—enhanced monocyte MMP-1 production significantly by two- to threefold (Fig. 1)
. MMP-13, an MMP that also degrades interstitial collagen, was not detected in the media of these cultures (data not shown). Additionally, we show that ox-LDL at 5–25 µg/mL in combination with TNF- and GM-CSF increased MMP-9 production slightly and when combined with LPS, caused a significant increase in MMP-9 (Fig. 3)
. However, at higher concentrations (50–100 µg/mL), ox-LDL decreased the optimal production of MMP-9 and MMP-1 in cytokine- and LPS-stimulated monocytes. Evidence from propidium iodide uptake analyses (data not shown), along with previous findings [21
, 22
], suggests that ox-LDL-induced cytotoxicity at concentrations of 50 µg/mL and higher may contribute to the decrease in MMP-9 and MMP-1 production. These results differ from studies by Xu et al. [8
], who showed that increasing concentrations of ox-LDL in the absence of cytokine or LPS stimulation enhanced MMP-9 expression significantly in 7-day monocyte-derived macrophages. Functional and metabolic differences in primary monocytes compared with mature, differentiated macrophages may account for these differences. However, we cannot exclude the possibility that another mechanism may contribute to the down-regulation of MMP-9 production by primary monocytes exposed to increasing concentrations of ox-LDL. In Figure 4
, we show that the addition of LDL at a low concentration suppressed MMP-1 production, but at a higher concentration, LDL increased MMP-1 production slightly. This finding suggests that monocytes may mediate the enhancement of MMP-1 by oxidizing native LDL [21
]. Additionally, ox-LDL in the presence of TNF- or GM-CSF alone did not induce MMP-1 production (Fig. 1)
, suggesting that the cytokine milieu within the vascular wall is essential in the regulation of MMP-1 by monocytes. Previous studies have demonstrated that the interaction between monocytes and endothelial cells results in the production of GM-CSF [23
]. Moreover, atherosclerotic plaques have been shown to contain significantly more TNF- compared with nonatherosclerotic vascular tissue [5
]. Taken together, these data suggest that elements located within peripheral regions of the vascular wall, including monocytes, ox-LDL, TNF-, and GM-CSF, form a foundation that may influence connective tissue remodeling and plaque destabilization.
In calcified atherosclerotic plaques, collagen comprises approximately 60% of the total protein [24
]. The collagen composition in human atherosclerotic plaques consists primarily of interstitial collagen types I and III, and type I collagen accounts for two-thirds of the total collagen [25
, 26
]. TIMPs, biological regulators of MMPs, may limit connective tissue destruction within the vascular wall by binding to and limiting enzymatic activity of secreted MMPs. We show that there was no difference in TIMP-1 production when TNF- and GM-CSF were added in combination with ox-LDL (5 µg/mL), but TIMP-1 increased slightly when ox-LDL was added in combination with LPS (Fig. 1)
. When monocyte cultures were exposed to increasing concentrations of ox-LDL (10–100 µg/mL) in the presence of the TNF- and GM-CSF or LPS, TIMP-1 production was reduced significantly (unpublished results). These findings are consistent with previous studies [8
], which showed a reduction in TIMP-1 production in 7-day monocyte-derived macrophages exposed to increasing levels of ox-LDL (10–50 µg/mL). Our data demonstrate that the net effect on MMP-1 relative to TIMP-1 production in primary monocytes exposed to ox-LDL and a specific cytokine combination—TNF- and GM-CSF—favors the degradation of the extracellular matrix.
Similar to previous findings with MMP-9 [8 ], we show that HDL abrogated monocyte MMP-1 production induced by cytokine activation as well as the enhancement by ox-LDL. The mechanism by which HDL blocks cytokine-induced MMP-1 production is unclear and is currently being investigated. The ability of HDL to inhibit the enhancement of MMP-1 by ox-LDL may be due, in part, to some of its effects on the scavenger receptor, CD36. Previous studies have shown that CD36 is involved in the uptake of ox-LDL and ultimately in its internalization and degradation [27 ]. More recently, HDL and LDL, like ox-LDL, have been shown to have a high affinity for the CD36 receptor [28 ]. High concentrations of HDL may saturate CD36 receptor binding sites, thereby limiting the uptake of ox-LDL and thus the production of MMP-1.
The production of MMP-1 by monocytes involves a PGE2 cyclic adenosine monophosphate (cAMP)-dependent pathway [11
, 15
16
17
]. Through an initial increase in phospholipase activity, arachidonic acid is released and metabolized by prostaglandin synthase-1 and -2, also known as COX-1 and -2, leading to the production of prostaglandins. PGE2, one of several prostaglandins generated through this cascade, interacts with Gs, thereby activating adenylyl cyclase to convert adenosine triphosphate to cAMP. cAMP-mediated activation of protein kinase A then leads to a series of down-stream phosphorylation events and contributes to the production of MMP-1 by monocytes. MMP-1 production via PGE2-dependent mechanisms can be suppressed by the addition of COX inhibitors such as indomethacin and aspirin.
In the present study, we demonstrate that the induction of MMP-1 by ox-LDL in cytokine-activated monocytes is mediated, in part, through PGE2 and COX-2. Similar to previous findings with 7-day macrophages [29
], we show that ox-LDL alone is not sufficient to induce COX-2 production by monocytes. However, when monocytes were exposed to ox-LDL in the presence of TNF- and GM-CSF, COX-2 production (Fig. 7A)
and mRNA expression (data not shown) increased. These in vitro findings are in agreement with recent clinical studies. For example, COX-2 has been isolated from atherosclerotic plaques, but not normal arterial tissue [30
, 31
]. In the investigation of PGE2 in the induction of MMP-1 by ox-LDL, we showed that MMP-1 production was suppressed by indomethacin and restored by exogenous PGE2 (Fig. 6)
. Moreover, we show that monocytes exposed to ox-LDL in the presence of TNF- and GM-CSF resulted in elevated levels of PGE2 compared with controls (Fig. 7B)
. Previous studies have demonstrated that moderately and advanced atherosclerotic plaques contained significantly more PGE2 compared with normal aortas [32
]. Additionally, PGE2 levels in developing plaques corresponded with disease progression: Advanced atherosclerotic plaques contained significantly more PGE2 than initial plaques [32
].
In summary, we demonstrate that ox-LDL increases MMP-1 production significantly by two- to threefold in cytokine-activated, primary monocytes through, in part, a COX-2- and PGE2-dependent pathway. Moreover, the ability of HDL to suppress MMP-1 further supports the beneficial effects of a high ratio of HDL to that of LDL. The findings presented here provided further support to the concept that the interaction of primary monocytes with ox-LDL and proinflammatory cytokines may contribute to vascular connective tissue remodeling and plaque rupture.
Received December 13, 2001; revised February 4, 2002; accepted February 5, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. Zhang and L. M. Wahl Synergistic enhancement of cytokine-induced human monocyte matrix metalloproteinase-1 by C-reactive protein and oxidized LDL through differential regulation of monocyte chemotactic protein-1 and prostaglandin E2 J. Leukoc. Biol., January 1, 2006; 79(1): 105 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Nicholls, B. Cutri, S. G. Worthley, P. Kee, K.-A. Rye, S. Bao, and P. J. Barter Impact of Short-Term Administration of High-Density Lipoproteins and Atorvastatin on Atherosclerosis in Rabbits Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2416 - 2421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Whatling, H. Bjork, S. Gredmark, A. Hamsten, and P. Eriksson Effect of macrophage differentiation and exposure to mildly oxidized LDL on the proteolytic repertoire of THP-1 monocytes J. Lipid Res., September 1, 2004; 45(9): 1768 - 1776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cipollone, B. Rocca, and C. Patrono Cyclooxygenase-2 Expression and Inhibition in Atherothrombosis Arterioscler. Thromb. Vasc. Biol., February 1, 2004; 24(2): 246 - 255. [Abstract] [Full Text]
|
|
|
|
 |
|
 D. K. Das Cardioprotection With High-Density Lipoproteins: Fact or Fiction? Circ. Res., February 21, 2003; 92(3): 258 - 260. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
