Skip Navigation

Cardiovascular Research 2003 59(3):723-733; doi:10.1016/S0008-6363(03)00514-5
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Vasudevan, S. S
Right arrow Articles by Goldschmidt-Clermont, P. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vasudevan, S. S
Right arrow Articles by Goldschmidt-Clermont, P. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Mac-1 and Fas activities are concurrently required for execution of smooth muscle cell death by M-CSF-stimulated macrophages

Sanjay S Vasudevana, Neuza H.M Lopesa, Puvi N Seshiahb, Tao Wanga, Clay B Marshc, Dean J Kereiakesd, Chunming Donga and Pascal J Goldschmidt-Clermonta,*

aDivision of Cardiology, Department of Medicine, Duke University Medical Center, Box 3845, Durham, NC 27700, USA
bDivision of Cardiology, Emory University, Atlanta, GA, USA
cDorothy M. Davis Heart and Lung Institute, Ohio State University, Columbus, OH, USA
dThe Lindner Research Center/Ohio Heart Health Center, Cincinnati, OH, USA

golds017{at}mc.duke.edu

* Corresponding author. Tel.: +1-919-668-1755; fax: +1-919-668-1861.

Received 9 August 2002; accepted 30 August 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We have previously shown that macrophage colony stimulating factor (M-CSF), a potent survival and mitogenic factor for monocytes/macrophages (MM), enables MM to induce vascular smooth muscle cell (VSMC) apoptosis. The killing requires the binding of MM to VSMC via Mac-1 (CD11b/CD18) on MM and intracellular adhesion molecule-1 (ICAM-1) on VSMC. We hypothesized that, in addition to Mac-1 binding, the killing process requires the activation of the Fas–death receptor pathway, which can be blocked at the level of Fas–Fas ligand interaction. Methods and Results: Human peripheral blood monocytes and VSMC were isolated and cultured as previously described. Soluble Fas (sFas) was overexpressed in VSMC by transduction using adenovirus specifying soluble Fas (Ad3hsFas). M-CSF markedly increased the expression of ICAM-1 in VSMC, resulting in enhanced clustering of MM on the surface of VSMC (≥3 MM per VSMC). MM, but not VSMC, expressed Fas-ligand (FasL), and VSMC apoptosis was inhibited by secretion of sFas by VSMC upon Ad3sFas transduction. Conclusions: MM and M-CSF-induced VSMC killing requires MM binding to VSMC mediated by Mac-1 and ICAM-1, and Fas–FasL interaction.

KEYWORDS Apoptosis; Smooth muscle; Infection/inflammation; Macrophages; Growth factors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Excessive VSMC apoptosis takes place in advanced atherosclerotic plaques [1], unstable lesions [2], abdominal aortic aneurysms [3] and restenosis following percutaneous coronary intervention (PCI) [4]. M-CSF is a hematopoietic growth factor that induces survival, proliferation and differentiation of monocytic cells. In the absence of M-CSF, monocytes are programmed to die within 24 to 48 h following their release from the bone marrow. M-CSF plays an important role in the progression of atherosclerosis. M-CSF (op–/op–)-deficient mice, when crossed with the LDL-R-deficient mice, demonstrated a dramatic reduction in fatty streak lesions at the aortic root and arch, in contrast to the LDL-R null control mice with normal M-CSF [5]. Furthermore, M-CSF mRNA and protein expression are present in atherosclerotic lesions, but not in normal vessels [6]. Moreover, M-CSF is found to be a predictor of unfavorable short and long-term outcome in patients with coronary heart disease [7,8]. Elevated M-CSF levels are also correlated with the elevation of inflammatory markers (CRP, IL6, IL1β) in both stable and unstable angina patients [8–10].

Recent work by Seshiah et al. [11] indicates that M-CSF is implicated in monocyte activation and VSMC apoptosis. Specifically, M-CSF induced VSMC apoptosis in a dose-dependent manner and within a wide physiological range of concentrations (0.1– 100 ng/ml). In addition, M-CSF may serve as a final common pathway for many cytokines involved in VSMC killing by MM in a paracrine fashion where these cytokines provoke M-CSF production by activated MM, leading to VSMC apoptosis.

Using an established in vitro model described by Seshiah et al. [11], we investigated the molecular players for MM binding to VSMC and the role of the Fas–death receptor pathway in VSMC apoptosis. We show that direct contact between MM and VSMC, mediated by Mac-1 and ICAM-1, is required for the activation of the Fas–death pathway. Secretion of sFas by adenovirus-transduced VSMC significantly inhibited the apoptosis of VSMC exposed to MM and M-CSF. This phenomenon was further confirmed in experiments using VSMC pretreated with Fas:Fc fusion protein. Thus, apoptosis of VSMC induced by M-CSF-activated MM is a two-step process: (1) M-CSF-stimulates the upregulation of ICAM-1 on VSMC, which, in turn, binds to the Mac-1 receptor on MM to establish cell-to-cell contact; and (2) FasL expressed on monocytes interacts with Fas death receptor, triggering the caspase-based apoptosis machinery, ultimately leading to VSMC apoptosis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Reagents
Recombinant human M-CSF was obtained from Research Diagnostics Inc. Anti-ICAM-1 blocking antibody (clone: BBIG-l1) and human sFas ELISA kit were from R&D systems. Anti-CD14-FITC (clone: M5E2), anti-CD45-PE (clone: HI30), anti-CD11b-PE (clone: D12), anti-CD54-PE (clone: HA58) were purchased from BD Pharmingen. Anti-FasL (clone: 33) and anti-Fas antibodies (clone: 13) were purchased from Transduction Laboratories. All blocking serum and secondary antibodies for immunohistochemistry and Western blotting were from Jackson Immunotech. Human monocyte isolation kit was from Miltenyi Biotec. The Vybrant apoptosis kit no. 5 was from Molecular Probes. The pan-caspase inhibitor Z VAD-fmk was from Calbiochem. The Fas:Fc fusion protein kit was from Alexis Corporation. The Ad3hsFas, Ad3Null and Ad3nBg were provided by Gene Therapy Inc.

2.2 Monocyte isolation
Human monocytes were isolated from buffy coat preparations. All procedures related to the procurement and handling of human blood products were approved by The Institutional Review Board of Duke University. Leukocytes were isolated by the Ficoll–Paque gradient method. Enriched populations of inactivated monocytes were further isolated by negative selection using an indirect magnetic labeling system and a monocyte isolation kit. Isolated monocytes were >90% pure, as determined by flow cytometry using double staining with antibodies CD14-FITC and CD45-PE.

2.3 Smooth muscle cell culture and co-culture with MM
VSMC were obtained from Clonetics and were cultured in SmGm-2 growth media (5% serum). At 50 to 70% confluence, cells (passage 5–7) were switched to 6-well plates with coverslips and cultured in DMEM/F12 serum-free media. Freshly isolated monocytes were added to VSMC in a ratio of 3:1 and cells were co-cultured for 48 h with or without M-CSF. Monocytes and M-CSF were added simultaneously to VSMC for co-culture. Where indicated, VSMC were pre-incubated with anti ICAM-1 antibody for 60 min at 37°C. Appropriate controls were included in all experiments involving inhibitors.

2.4 Quantification of monocyte clustering
After 24 and 48 h of co-culture (with or without M-CSF), cells were washed with phosphate-buffered saline (PBS). Cells were then visualized by differential interference contrast (DIC) microscope. The ratio of MM (±M-CSF stimulation) bound to VSMC to the total number of MM per high-power field (bound and unbound) was calculated and expressed as a percentage (monocyte binding index, MBI). Also the percentage of VSMC with three or more bound MM (±M-CSF stimulation) was calculated as the number of VSMC with three or more surface MM per high-power field to the total number of VSMC with bound MM.

2.5 Apoptosis assay
Following co-culture of MM and VSMC for 48 h, cells were washed with PBS and stained with Hoechst 33342 (Ho342, 5 mg/ml) and propidium iodide (PI, 1 mg/ml) contained in the Vybrant no. 5 apoptosis assay kit, for 50 min at 37°C and protected from light. Cells were then washed with PBS and fixed with 4% paraformaldehyde. The following criteria were used to define apoptosis: (1) cell shrinkage and blebbing; (2) alteration in nuclear–cytoplasmic ratio; (3) nuclear condensation (intense blue staining of nucleus with Ho342); (4) nuclear fragmentation; and (5) increased membrane permeability (loss of integrity of plasma and nuclear membrane, and intense PI-positive staining). The apoptotic index was calculated as a percentage of apoptotic VSMC vs. total number of VSMC per high-power field (live cells+apoptotic cells) by counting a total of 300 to 400 cells for each sample. All experiments were conducted in triplicates and repeated three times for statistical analysis with relevant controls.

2.6 Flow cytometry
Human monocytes were isolated as described above and stimulated with M-CSF for 24 h at 37°C. The supernatant (conditioned media) from these M-CSF-stimulated monocytes was collected and added to VSMC in DMEM/F12 media and cultured for another 24 to 48 h. Control VSMC was cultured for the same time frame in DMEM/F12 culture media (control media). These cells were then stained with a PE-conjugated anti-ICAM-1 monoclonal antibody and the surface expression of ICAM-1 on VSMC was measured by flow cytometry.

2.7 Construction of adenoviral vectors
cDNA for human soluble Fas (sFas), which contains the extracellular domain of the human Fas gene was sub-cloned into pbluescript SK (+) using previously described techniques [12]. The amplified sFas fragment was inserted into the Xbal/EcoRV sites of the backbone shuttle vector Pavs6a to create PavhsFas and then incorporated into the Ad3, a replication-deficient adenovirus with E1, E2a and E3 deletions, to generate Ad3hsFas. Ad3hsFas encoding human sFas cDNA and Ad3Null, containing no expression cassette, were generated. Ad3nBg encoding β galactosidase (β gal) was also generated to determine efficiency of VSMC transfection with the adenovirus. The average ratio of total viral particles (particles/ml) to plaque forming units (pfu/ml) was 25±4.3 (particles/pfu, mean±S.E.M.).

2.8 Adenoviral transfection of VSMC
VSMC were transduced with Ad3hsFas (pfu 7.4x107), Ad3Null (pfu 1.8x108) and Ad3nBg (pfu 7.0x108) at a multiplicity of infection (MOI) of 100 for 2 h. Cells were then washed with PBS (x1) and cultured in SmGm-2 media for another 24–36 h. Efficiency of transfection was determined by β-Gal staining. sFas expression from supernatant of Ad3hsFas-transduced VSMC was measured using a sFas ELISA kit. Ad3hsFas or Ad3Null-transduced VSMC were co-cultured with monocytes, with and without M-CSF, for 48 h and the apoptotic index was calculated as described earlier.

2.9 Immunofluorescent staining and Western blotting
Monocytes±M-CSF in co-culture with VSMC were stained at the end of 48 h with an anti-FasL monoclonal antibody or anti-Fas monoclonal antibody. A FITC-conjugated anti-CD14 monoclonal antibody was used to identify MM. Protein lysates were prepared from MM and 20 µg protein/well was electrophoresed on a 4–20% Tris–HCl polyacrylamide gel. Western blots were performed as previously described [13].

2.10 Statistical analysis
All experiments were performed in triplicates and repeated three or more times. Data are expressed as mean±S.E.M. Statistical analysis was performed using the Student’s unpaired t-test or ANOVA. A P-value of ≤0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 M-CSF upregulates ICAM-1 (CD54) expression on VSMC
We have demonstrated that Mac-1 binding is required for the killing of VSMC by M-CSF and MM [11,14]. To investigate whether ICAM-I might play a role in the MM binding to VSMC and subsequent VSMC apoptosis, we determined the expression levels of ICAM-1 in the different co-culture conditions. As shown in Fig. 1, exposure to M-CSF triggered marked ICAM-1 expression on VSMC, whereas the density of Mac-1 receptors on MM was not affected. We then incubated VSMC with an anti-human ICAM-1 blocking monoclonal antibody, which markedly reduced the VSMC apoptotic index (20.95±2.65%) compared to VSMC pretreated with a control IgG1 (54.11±1.11%, P≤0.007, or non-treatment control (54.70±2.3%, P=0.008) (Fig. 2). Although ‘activation’ of Mac-1 in response to M-CSF could not be ruled out, these data suggest that expression of ICAM-1 in response to M-CSF-activated MM is a prerequisite for VSMC apoptosis


Figure 1
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Expression of CD11b (Mac-1) by M-CSF-activated monocytes at 24 h is indicated by the orange line, while the black line represents an IgG control (a), Expression of CD54 (ICAM-1) on the surface of VSMC cultured in control media (DMEM/F12) (b), versus conditioned media from M-CSF-stimulated monocytes (c), (green line: CD54, blue line: IgG control). Pretreatment of VSMC with anti-ICAM-1 prior to co-culture with M-CSF-activated monocytes results in significant reduction in VSMC apoptosis (20.95±2.65%) compared to VSMC pretreated with an IgG1 control (54.11±1.11%, *P≤0.007) and M-CSF-induced VSMC apoptosis in the absence of antibody (54.70±2.30%, **P≤0.008; d).

 

Figure 2
View larger version (94K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 M-CSF activated monocytes demonstrated significantly increased binding to VSMC (70.08±2.17%, 64.31±2.79%, at 24 and 48 h, respectively, measured by calculating the MBI in comparison with unstimulated monocytes (33.34±2.80%, *P≤0.0001; 31.88±2.29%, **P≤0.005; respectively, a). VSMC cultured with MM and M-CSF demonstrated increased clustering of ≥3 MM/VSMC (23.59±1.12%, 21.09±2.99%, at 24 and 48 h, respectively, calculated as a percentage of VSMC with ≥ 3 bound monocytes to total VSMC with bound monocytes) relative to co-cultures without M-CSF stimulation (7.67±0.50%, *P≤0.0005; 6.80±2.64%, **P≤0.001; respectively, b). High magnification (x400) DIC image of MM and VSMC in the absence (c) and the presence of M-CSF (d) are shown.

 
3.2 M-CSF promotes monocytes cluster on the surface of VSMC
We then examined binding of monocytes to VSMC (MBI) in the presence or absence M-CSF. M-CSF-activated MM showed a significant increase in binding to VSMC (70.08±2.17%, 64.31±2.79%, at 24 and 48 h, respectively), more than twice of the MBI observed in the absence of M-CSF (33.34±2.80%, P≤0.0001; 31.88±2.29%, P≤0.005), at 24 and 48 h, respectively (Fig. 2). Furthermore, the number of VSMC with three or more bound MM was calculated. At 24 and 48 h, in the presence of M-CSF, the percentage of VSMC with three or more clustered monocytes were 23.59±1.12% and 21.09±2.99%, respectively. In the absence of M-CSF, the percentages were 7.67±0.50% (P≤0.0005) and 6.80±2.64% (P<0.001) at these two time points, respectively (Fig. 2). These data indicate that the clustering of MM on VSMC is increased in the presence of M-CSF, probably via upregulated ICAM-1.

3.3 MM and VSMC show differential Fas and FasL expression
Previous studies indicate that MM, including those found in ruptured atherosclerotic plaques, express FasL [15]. As shown in Fig. 3, Western blot analysis confirmed the FasL expression in monocytes, but not in VSMC, irrespective of the presence or absence of M-CSF. In contrast, Fas was expressed by both MM and VSMC (data not shown). These data are consistent with previous observations [15,16].


Figure 3
View larger version (92K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 DIC and fluorescent overlay images (x400) of apoptotic VSMC with bound activated monocytes (a–c); Pictures demonstrate surface expression of FasL on M-CSF-activated monocytes (a and c, red staining indicates FasL). Cells were stained with Ho342 (blue nuclear staining). Monocytes were identified by co-localization with CD14-FITC (a and b, green staining). VSMC do not express membrane-bound FasL. Western blot for FasL expression in unactivated and activated monocytes (d).

 
3.4 Fas–fasL interactions are required to commit VSMC to apoptosis
Fas–FasL pathway has been implicated in VSMC apoptosis [17]and can promote apoptosis of cytokine-primed VSMC [18]. We have demonstrated that Fas-mediated apoptosis plays a critical role in the development of cardiac allograft vasculopathy [19]. In an attempt to determine whether VSMC apoptosis induced by M-CSF and MM required the participation of the Fas–FasL pathway, we first used a Fas:Fc fusion protein to block Fas–FasL interaction. As expected, the Fas:Fc fusion protein (1–4 µg/ml) reduced, in a dose-dependent fashion, VSMC apoptosis triggered by M-CSF-activated MM. Maximum reduction in VSMC apoptosis was detected at 4 µg/ml (13.67±1.08%) relative to cells pretreated with an IgG1 control (63.00±3.74%, P≤0.0007), or without pretreatment (58.18±2.60%, P≤0.001, Fig. 4).


Figure 4
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Fas:Fc fusion protein (1–4 µg/ml) pretreated VSMC in co-culture with M-CSF-activated monocytes showed a dose-dependent inhibition of VSMC apoptosis. Maximum inhibition was noted at a concentration of 4 µg/ml (13.67±1.08%) vs. IgG1 pretreated VSMC in co-culture (63.00±3.74%, *P≤0.0007) and VSMC co-cultured with M-CSF-stimulated monocytes in the absence of Fas:Fc fusion protein (58.18±2.60%, **P≤0.001).

 
Next, we overexpressed soluble Fas (sFas) in VSMC as another means to block Fas–FasL interaction to further confirm the involvement of Fas receptor-mediated pathway in VSMC apoptosis induced by M-CSF and MM. Transduction efficiency was determined by X-gal staining of Ad3nBg-transduced cells and by ELISA assay for human soluble Fas. At 100 MOI, VSMC transduced with Ad3hsFas demonstrated a significant increase in sFas expression (3137.25±51.38 µg/g) compared to control cells (36.57±3.30 µg/g) and Ad3Null transduced VSMC (36.67±0.52 µg/g, P≤0.00007, Fig. 5).


Figure 5
View larger version (95K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Photomicrograph demonstrates X-gal staining of VSMCs transduced with Ad3bngal (100 MOI, a); VSMC transduced with Ad3hsFas (100 MOI) showed a significant increase in sFas expression (measured by ELISA) at 36 h post-transduction (3137.25±51.38 µg/g) in contrast to VSMC transduced with Ad3Null (36.67±0.52 µg/g, *P≤0.00007, b).

 
When Ad3hsFas-transduced VSMC were co-cultured with M-CSF-activated MM, a significant reduction in VSMC apoptosis was observed (17.33±4.40%), relative to co-cultured Ad3Null-transduced VSMC (58.50±6.55%) and nontransduced VSMC (56.30±3.65%, Fig. 6). Such overexpression of soluble Fas did not antagonize the clustering of MM on the surface of VSMC, as multiple MM were found adherent to VSMC (Fig. 7i and j).


Figure 6
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Ad3hsFas (100 MOI)-transduced VSMC in co-culture with activated monocytes were significantly protected from VSMC apoptosis (17.33±4.40%) relative to co-cultured VSMC infected with Ad3Null (58.50±6.55%, *P<0.001) and uninfected VSMC in co-culture with activated monocytes (56.30±3.65%).

 

Figure 7
View larger version (110K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 (a–d) Images of co-cultured cells after 48 h. Control VSMC (x10) (a), VSMC+unstimulated monocytes (x20) (b) and VSMC +M-CSF-activated MM (x10 and b20, respectively, c and d) show cell shrinkage (d) and increased clustering of activated monocytes around apoptotic VSMC in contrast to image (b). (e–j) Overlay of fluorescent and DIC images (x400) of co-cultured cells stained with Ho342 (360 nm) and PI (555 nm) for apoptosis. Live Ad3hsFas-transduced VSMC (Ad3hsFas-VSMC) only (e); Ad3hsFas-VSMC co-cultured with unstimulated monocytes (f); AdNull-transduced VSMC with bound MM activated with M-CSF (100 ng/ml), demonstrating dense nuclear (blue-Ho342) staining (nuclear condensation), cytoplasmic condensation, cell shrinkage and alteration in the nuclear cytoplasmic ratio, as well as PI positivity (g and h). M-CSF-activated MM clustered around and attached to Ad3-VSMC, which are protected against apoptosis (i). Ad3hsFas-VSMC, although bound by many activated MM, demonstrate no features of apoptosis (j).

 
3.5 Caspase inhibitor blocks VSMC apoptosis
Caspases, including caspase-3, serve as executioners for Fas–FasL-induced apoptosis. To demonstrate that VSMC death was mediated by apoptosis, a pan-caspase inhibitor ZVAD-fmk (5 µmol/l) was added into our co-culture system. The ZVAD-fmk treatment completely abrogated VSMC apoptosis induced by M-CSF and MM. VSMC cultured with MM and M-CSF exhibited an apoptotic index of (63.33±0.88%), the pan-caspase inhibitor ZVAD-fmk decreased this index to 20.20±0.76%, P≤0.0005 (Fig. 8). Collectively, these data indicate that SMC apoptosis induced by M-CSF and MM is a two-step process involving MM binding to VSMC mediated by Mac1 and ICAM-1 and execution of VSMC death mediated by Fas–FasL interaction.


Figure 8
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 VSMC preincubated with ZVAD-fmk (5 µmol/l) significantly decreased M-CSF-stimulated monocytes-induced VSMC apoptosis (from 63.33±0.88% to 20.20±0.76%, *P≤0.0005).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Loss of VSMC by apoptosis has been shown in advanced atherosclerotic plaques [1] and in unstable lesions [2]. Multiple mechanisms and pathways have been implicated in inducing VSMC apoptosis. These include exogenous ROS [20], inflammatory cytokines [21], soluble FasL [22], p53 [23], and oxidized-LDL [24,25]. The mechanisms whereby inflammatory cells, including monocytes and T-lymphocytes induce VSMC apoptosis remain incompletely understood. We had shown that monocytes/macrophages accelerate the apoptosis rate of VSMC upon exposure to M-CSF [11]. In this study, we show that M-CSF and MM-induced VSMC killing is a two-step process. First, MM need to bind VSMC via Mac-1 interaction with ICAM-1. Then, FasL on the surface of MM interacts with the death receptor Fas on the surface of VSMC. Such interaction activates the caspase-based apoptosis machinery, leading to irreversible cell death.

It appears that the rate-limiting step in VSMC killing process is the aggregation of MM on the surface of VSMC. We show that the presence of ≥3 MM on the surface of an individual VSMC correlates positively with VSMC apoptosis. Importantly, the occurrence of MM binding to VSMC, especially the clustering of ≥3 MM on each individual VSMC, is significantly increased by M-CSF. These data indicate that M-CSF promotes VSMC apoptosis via increasing MM binding to VSMC.

Although the activation of Mac-1 by M-CSF cannot be ruled out, our data indicate that M-CSF does not alter the expression of Mac-1 on the surface of MM. Instead, it stimulates the expression of ICAM-1 on the surface of VSMC. An ICAM-1 blocking antibody inhibits MM binding to VSMC and prevents VSMC apoptosis, indicating that ICAM-1 upregulation is, at least in part, responsible for the potentiating effects of M-CSF in VSMC apoptosis. It is noteworthy that inhibition of VSMC apoptosis by the anti ICAM-1 antibody was not complete, indicating that mechanisms other than cell-to-cell contact, such as soluble cytokines, ROS and soluble FasL, may also be at play in M-CSF and MM-induced VSMC apoptosis.

The Fas–FasL pathway has been implicated in VSMC apoptosis in multiple settings, including atherosclerosis, restenosis and transplant vascular disease [19,22,26–28]. Using a replication incompetent adenovirus specifying the soluble form of Fas (sFas) to induce the truncated extra-cellular portion of Fas protein and Fas:Fc fusion protein to inhibit Fas–FasL interaction, we were able to abrogate the apoptosis of VSMC exposed to MM and M-CSF, without altering MM binding to VSMC. In addition, ZVAD-fmk, a pan-caspase inhibitor efficiently blocked VSMC apoptosis. These data support the notion that the Fas-death pathway serves as an executioner for VSMC apoptosis.

Boyle et al. [15] showed that monocytes induced VSMC apoptosis after co-culture for 6–8 days. The role of M-CSF was not addressed in their study. Also, the ratio of MM to VSMC used in their experiments was rather high (8:1). We used a considerably lower ratio of MM to VSMC (3:1) and demonstrated that MM resulted in VSMC apoptosis in a much shorter time interval (24 to 48 h) in the presence of M-CSF. Considering that only a 3–4-fold increase in the infiltrating MM was noted in complex, unstable plaques [29] and that the concentration of M-CSF used in our system represents the higher range of values determined by measuring serum and plasma from patients with unstable angina [9,10], our experimental system resembles the atherosclerotic milieu in vivo more closely. Hence, our findings are more pathophysiologically relevant.

It has been shown that M-CSF can also affect VSMC by stimulating the secretion of biologically active M-CSF [30] and, by inducing the expression of the M-CSF receptor (encoded by c-fms) at the protein and mRNA levels [30]. Such M-CSF effects on VSMC, especially in the presence of MM, may contribute to MM-induced VSMC killing, although these effects were not evaluated in our study.

Macrophage-derived foam cells in atherosclerotic plaques have shown a high killing potential by inducing oxidative damage and subsequent cell death [31]. In addition, recent work has shown that foam cells are protected from undergoing apoptosis by expressing high levels of macrophage scavenger receptor-A, thereby prolonging their presence in plaques to carry on important functions [32]. Macrophages can also induce VSMC apoptosis by activating the high-output NO system [33]. Whether direct cell-to-cell contact is also required for foam cell-induced VSMC death warrants further study.

In summary, we have demonstrated that M-CSF potentiates MM-induced VSMC killing by inducing ICAM-1 expression and subsequent MM binding to VSMC, which, in turn, facilitates the interaction of Fas–FasL interaction, resulting in VSMC apoptosis. These findings may lead to the development of novel inhibitors that disrupt cell-to-cell contact and Fas activation, preventing plaque rupture.

Time for primary review 25 days.


   Acknowledgements
 
This study was supported by a grant from Centocor Inc., Pennsylvania. The replication deficient adenovirus (Ad3hsFas, Ad3Null and Ad3nBg) was a generous gift from Susan Stevenson (Novartis Inc.). Walter C. Stone was helpful in preparing this manuscript.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Geng Y.J., Libby P. Evidence for apoptosis in advanced human atheroma. Colocalization with interleukin-1 beta-converting enzyme. Am. J. Pathol. (1995) 147:251–266.[Abstract]
  2. Bauriedel G., Hutter R., Welsch U., et al. Role of smooth muscle cell death in advanced coronary primary lesions: implications for plaque instability. Cardiovasc. Res. (1999) 41:480–488.[Abstract/Free Full Text]
  3. Henderson E.L., Geng Y.J., Sukhova G.K., et al. Death of smooth muscle cells and expression of mediators of apoptosis by T lymphocytes in human abdominal aortic aneurysms. Circulation (1999) 99:96–104.[Abstract/Free Full Text]
  4. Isner J.M., Kearney M., Bortman S., Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation (1995) 91:2703–2711.[Abstract/Free Full Text]
  5. Rajavashisth T., Qiao J.H., Tripathi S., et al. Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor-deficient mice. J. Clin. Invest (1998) 101:2702–2710.[Web of Science][Medline]
  6. Clinton S.K., Underwood R., Hayes L., et al. Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis. Am. J. Pathol. (1992) 140:301–316.[Abstract]
  7. Saitoh T., Kishida H., Tsukada Y., et al. Clinical significance of increased plasma concentration of macrophage colony-stimulating factor in patients with angina pectoris. J. Am. Coll. Cardiol. (2000) 35:655–665.[Abstract/Free Full Text]
  8. Rallidis L.S., Zolindaki M.G., Manioudaki H.S., et al. Prognostic value of C-reactive protein, fibrinogen, interleukin-6, and macrophage colony stimulating factor in severe unstable angina. Clin. Cardiol. (2002) 25:505–510.[Web of Science][Medline]
  9. Rallidis L.S., Thomaidis K.P., Zolindaki M.G., Velissaridou A.H., Papasteriadis E.G. Elevated concentrations of macrophage colony stimulating factor predict worse in-hospital prognosis in unstable angina. Heart (2001) 86:92.[Free Full Text]
  10. Ikonomidis I., Andreotti F., Economou E., et al. Increased proinflammatory cytokines in patients with chronic stable angina and their reduction by aspirin. Circulation (1999) 100:793–798.[Abstract/Free Full Text]
  11. Seshiah P.N., Kereiakes D.J., Vasudevan S.S., et al. Activated monocytes induce smooth muscle cell death: role of macrophage colony-stimulating factor and cell contact. Circulation (2002) 105:174–180.[Abstract/Free Full Text]
  12. Stevenson S.C., Rollence M., Marshall-Neff J., McClelland A. Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein. J. Virol. (1997) 71:4782–4790.[Abstract]
  13. Wickenden A.D., Kaprielian R., Parker T.G., Jones O.T., Backx P.H. Effects of development and thyroid hormone on K+ currents and K+ channel gene expression in rat ventricle. J. Physiol. (1997) 504(Pt 2):271–286.[Abstract/Free Full Text]
  14. Simon D.I., Xu H., Ortlepp S., Rogers C., Rao N.K. 7E3 monoclonal antibody directed against the platelet glycoprotein IIb/IIIa cross-reacts with the leukocyte integrin Mac-1 and blocks adhesion to fibrinogen and ICAM-1. Arterioscler. Thromb. Vasc. Biol. (1997) 17:528–535.[Abstract/Free Full Text]
  15. Boyle J.J., Bowyer D.E., Weissberg P.L., Bennett M.R. Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions. Arterioscler. Thromb. Vasc. Biol. (2001) 21:1402–1407.[Abstract/Free Full Text]
  16. Sata M., Suhara T., Walsh K. Vascular endothelial cells and smooth muscle cells differ in expression of Fas and Fas ligand and in sensitivity to Fas ligand-induced cell death: implications for vascular disease and therapy. Arterioscler. Thromb. Vasc. Biol. (2000) 20:309–316.[Abstract/Free Full Text]
  17. Imanishi T., Hano T., Nishio I., Han D.K., Schwartz S.M. Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from endothelial cells. Jpn. Circ. J (2001) 65:556–560.[CrossRef][Medline]
  18. Geng Y.J., Henderson L.E., Levesque E.B., Muszynski M., Libby P. Fas is expressed in human atherosclerotic intima and promotes apoptosis of cytokine-primed human vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. (1997) 17:2200–2208.[Abstract/Free Full Text]
  19. Wang T., Dong C., Stevenson S.C., et al. Overexpression of soluble fas attenuates transplant arteriosclerosis in rat aortic allografts. Circulation (2002) 106:1536–1542.[Abstract/Free Full Text]
  20. Rossig L., Dimmeler S., Zeiher A.M. Apoptosis in the vascular wall and atherosclerosis. Basic Res. Cardiol. (2001) 96:11–22.[CrossRef][Web of Science][Medline]
  21. Geng Y.J., Wu Q., Muszynski M., Hansson G.K., Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta. Arterioscler. Thromb. Vasc. Biol. (1996) 16:19–27.[Abstract/Free Full Text]
  22. Imanishi T., Han D.K., Hofstra L., et al. Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from monocytes/macrophage. Atherosclerosis (2002) 161:143–151.[CrossRef][Web of Science][Medline]
  23. Bennett M., Macdonald K., Chan S.W., et al. Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science (1998) 282:290–293.[Abstract/Free Full Text]
  24. Hsieh C.C., Yen M.H., Yen C.H., Lau Y.T. Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells. Cardiovasc. Res. (2001) 49:135–145.[Abstract/Free Full Text]
  25. Martinet W., Kockx M.M. Apoptosis in atherosclerosis: focus on oxidized lipids and inflammation. Curr. Opin. Lipidol. (2001) 12:535–541.[CrossRef][Web of Science][Medline]
  26. Dong C., Redenbach D., Wood S., et al. The pathogenesis of cardiac allograft vasculopathy. Curr. Opin. Cardiol. (1996) 11:183–190.[CrossRef][Web of Science][Medline]
  27. Hebert M.J., Masse M., Vigneault N., et al. Soluble Fas is a marker of coronary artery disease in patients with end-stage renal disease. Am. J. Kidney Dis. (2001) 38:1271–1276.[Web of Science][Medline]
  28. Okura T., Watanabe S., Jiang Y., et al. Soluble Fas ligand and atherosclerosis in hypertensive patients. J. Hypertens. (2002) 20:895–898.[CrossRef][Web of Science][Medline]
  29. Davies M.J., Richardson P.D., Woolf N., Katz D.R., Mann J. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage, and smooth muscle cell content. Br. Heart J. (1993) 69:377–381.[Abstract/Free Full Text]
  30. Herembert T., Gogusev J., Zhu D.L., Drueke T.B., Marche P. Control of vascular smooth-muscle cell growth by macrophage-colony-stimulating factor. Biochem. J. (1997) 325(Pt. 1):123–128.[Web of Science][Medline]
  31. Martinet W., Kockx M.M. Apoptosis in atherosclerosis: focus on oxidized lipids and inflammation. Curr. Opin. Lipidol. (2001) 12:535–541.[CrossRef][Web of Science][Medline]
  32. Liao H.S., Kodama T., Geng Y.J. Expression of class A scavenger receptor inhibits apoptosis of macrophages triggered by oxidized low density lipoprotein and oxysterol. Arterioscler. Thromb. Vasc. Biol. (2000) 20:1968–1975.[Abstract/Free Full Text]
  33. Boyle J.J., Weissberg P.L., Bennett M.R. Human macrophage-induced vascular smooth muscle cell apoptosis requires NO enhancement of Fas/Fas-L interactions. Arterioscler. Thromb. Vasc. Biol. (2002) 22:1624–1630.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Eur Heart JHome page
A. Niessner, P. J. Hohensinner, K. Rychli, S. Neuhold, G. Zorn, B. Richter, M. Hulsmann, R. Berger, D. Mortl, K. Huber, et al.
Prognostic value of apoptosis markers in advanced heart failure patients
Eur. Heart J., April 1, 2009; 30(7): 789 - 796.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
T. E. Morrison, J. D. Simmons, and M. T. Heise
Complement Receptor 3 Promotes Severe Ross River Virus-Induced Disease
J. Virol., November 15, 2008; 82(22): 11263 - 11272.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Vasudevan, S. S
Right arrow Articles by Goldschmidt-Clermont, P. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vasudevan, S. S
Right arrow Articles by Goldschmidt-Clermont, P. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?