Skip Navigation


Cardiovascular Research Advance Access originally published online on July 24, 2008
Cardiovascular Research 2008 80(2):246-254; doi:10.1093/cvr/cvn201
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
80/2/246    most recent
cvn201v2
cvn201v1
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 Velot, E.
Right arrow Articles by Wagner, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Velot, E.
Right arrow Articles by Wagner, D. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Activation of the adenosine-A3 receptor stimulates matrix metalloproteinase-9 secretion by macrophages

Emilie Velot1, Benjamin Haas1, Frédérique Léonard1, Isabelle Ernens1, Magali Rolland-Turner1, Chantal Schwartz1, Dan Longrois2,3, Yvan Devaux1 and Daniel R. Wagner4,*

1 Laboratory of Cardiovascular Research, CRP-Santé, Luxembourg, Luxembourg
2 Department of Anesthesia and Intensive Care, Centre Hospitalier Universitaire de Nancy, France
3 INSERM U684, Université Henri Poincaré Nancy I, France
4 Division of Cardiology, Centre Hospitalier Luxembourg, 4 rue Barblé, L1210 Luxembourg, Luxembourg

* Corresponding author. Tel: +352 44 11 2221; fax: +352 44 11 6629.E-mail address: wagner.daniel{at}chl.lu

Received 29 April 2008; revised 10 July 2008; accepted 15 July 2008

Time for primary review: 29 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Matrix metalloproteinase-9 (MMP-9) plays an important role in ventricular remodelling after acute myocardial infarction (MI). The cardioprotectant adenosine (Ado) may be involved in ventricular remodelling. We have shown that Ado inhibits the secretion of MMP-9 by human neutrophils. This study investigated the effect of Ado on MMP-9 production by human macrophages.

Methods and results: Cells used in this study were monocytes of healthy volunteers, a human monocyte cell line, and leukocytes from patients following MI. Monocytes were differentiated into macrophages and treated with Ado. Ado enhanced MMP-9 secretion by human macrophages in a time- and dose-dependent manner. Increasing the level of endogenous Ado by inhibition of Ado deaminase or Ado transferase also increased MMP-9 secretion. Ado enhanced MMP-9 production when macrophages were activated by hypoxia or Toll-like receptor-4 ligands such as lipopolysaccharide, hyaluronan, and heparan sulfate. The effect of Ado was replicated by the A3 agonist IB-MECA and inhibited by silencing the A3 receptor. Ado improved monocyte capacity to migrate through a matrix of gelatin B, and this effect was blocked by inhibition of MMP-9 activity. The chemotactic capacity of macrophages was reduced by Ado through a loss of expression of the monocyte chemotactic protein-1 receptor. Finally, MMP-9 expression was higher in blood cells from patients with acute MI compared with healthy volunteers.

Conclusion: Adenosine activates MMP-9 secretion by macrophages through its A3 receptor. The effect is in contrast to that observed in neutrophils, where Ado inhibits MMP-9 secretion by the A2a receptor. These observations may have important implications for therapeutic strategies targeting Ado receptors in the setting of MI.

KEYWORDS Adenosine; Monocytes/macrophages; Ventricular remodelling; Matrix metalloproteinase-9; Cell migration


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Heart failure (HF) is a disease of epidemic proportion worldwide. It represents the fastest growing subclass of cardiovascular diseases over the past decade.1,2 Despite significant advances in the care of HF, its morbidity and mortality remain unacceptably high: patients diagnosed with acute decompensated HF have a 1-year survival rate of 37.5%.3 Post-infarct left ventricular (LV) remodelling is a leading cause of HF. Adaptive remodelling occurs in the early phase post-myocardial infarction (MI) and results in significant LV dilatation.

The transition from adaptive to maladaptive remodelling is mediated, at least partly, by dysregulated activation of the pro-inflammatory response involving both cellular and non-cellular mediators, among which matrix metalloproteinases (MMPs) play an important role. MMP-9, a member of the MMP family of zinc-endopeptidases, contributes to a large extent to maladaptive remodelling. Imbalance between MMP-9 and its inhibitory counterpart tissue inhibitor of matrix metalloproteinase (TIMP)-1 results in extracellular matrix (ECM) degradation. Direct inhibition of MMP has not yet been shown to have a clinical benefit.4 Cellular mechanisms involved in MMP-9 activation after MI are not thoroughly understood. Given the complex regulation of ECM biology, modulation of one of the upstream triggers of MMP-9 could be of potential therapeutic interest.

Adenosine (Ado) might be such an upstream regulator of MMP-9. Ado has been found to be cardioprotective in a range of cardiovascular disorders, particularly during myocardial ischaemia when Ado is massively released.5,6 We have previously reported that Ado inhibits TNF-{alpha} and IL-6 production by cardiac myocytes7–9 and MMP-9 secretion by neutrophils.10 Neutrophils are among the first wave of cells recruited to infarcted tissues. The second wave of inflammatory cells recruited to the infarcted tissues consists mainly of monocytes/macrophages. These cells migrate along a gradient of the chemokine monocyte chemoattractant protein (MCP)-1, which binds to its receptor CC chemokine receptor-2 (CCR2) expressed at the cell surface. Macrophages are the major contributor of MMP-9 secretion in the context of acute MI. So far, the effect of Ado on MMP-9 production by monocytes/macrophages has not been investigated.

Therefore, the present study was designed to test whether Ado could modulate MMP-9 production by monocytes/macrophages. In sharp contrast to our previous observations with neutrophils, we found that Ado consistently enhanced MMP-9 secretion by macrophages. This observation could help to explain why global activation of Ado receptors by Ado has no significant effect after MI and may direct further strategies aiming at targeting specific Ado receptors in the setting of MI.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
An expanded Materials and methods section can be found in the online supplement.

The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.1 Cell culture
All cell culture reagents were from Lonza (Verviers, Belgium) unless specified otherwise. Peripheral venous blood of healthy volunteers and patients after acute MI was used in this study. Patients with acute MI were enrolled in the Luxembourg Acute Myocardial Infarction Registry and were treated with primary percutaneous coronary intervention. Acute MI was defined by the presence of chest pain <12 h with significant ST elevation and positive cardiac enzymes. Blood samples were obtained at the time of mechanical reperfusion. All patients signed an informed consent and the protocol has been approved by the National Ethics Committee (reference no. 200504). Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient. Monocytes were purified by negative selection using the Monocyte Isolation Kit II (Myltenyi Biotec GmbH, Bergisch Gladbach, Germany) as described before.10 Differentiation was achieved with 50 ng/mL macrophage colony-stimulating factor (M-CSF) for 7 days. Macrophages were incubated for 15 min with 10 µmol/L Ado and 10 µmol/L erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or vehicle, then LPS (100 ng/mL) or vehicle was added, and cells were incubated for another 24 h before harvesting.

Cells from the monocyte-like line THP-1 were treated for 15 min with EHNA vehicle (DMSO), Ado (0.01–100 µM), Ado receptors agonists (0.1–10 µM), EHNA (10 µM), or dipyridamole (DIP) (10 µM). Cells were then differentiated into macrophages with 150 nM phorbol myristate actetate (PMA) for 24 h. Macrophages were subsequently treated with LPS (100 ng/mL), formyl-Met-Leu-Phe (fMLP) (10–7 M), hyaluronan (HA, 10 µg/mL), or heparan sulfate (HS, 10 µg/mL) for another 24 h. In some conditions, cells were incubated in a Gaspak anaerobic pouch system (GasPak 100/GasPak-Pouch, Becton–Dickinson, Erembodegem, Belgium) for 24 h to simulate hypoxia. For experiments involving RNA interference, 1.4 µg siRNA specific for each Ado receptor was transfected 48 h before Ado treatment by Nucleofection according to the manufacturer’s instructions (Amaxa Inc., Cologne, Germany). The following are the selected target mRNA sequences for Ado receptors: siRNA A2a: 5'-CAGGAGTGTCCTGATGATTCA-3'; siRNA A2b: 5'-CACGTATCTAGCTAATATGTA-3'; siRNA A3: 5'-CCCTATCGTCTATGCCTATAA-3'.

Cells were lysed in TriReagent® prior to RNA extraction. Cell-free conditioned medium was collected and mixed with protease inhibitors (Roche, Mannheim, Germany) and bovine serum albumin (0.2% final concentration) to perform ELISA or zymography. All samples were stored at –80°C until analysis.

2.2 Real-time quantitative polymerase chain reaction
Total RNA was isolated using TriReagent® and the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA from blood cells collected in PAXgene® blood RNA tubes was isolated using PAXgene® Blood RNA Kit as described by the manufacturer (Qiagen). One microgram of total RNA was reverse-transcribed using the Superscript® II Reverse Transcriptase (Invitrogen, Merelbeke, Belgium). PCR was performed using the iCycler® and the IQTM SYBR® Green Supermix (Bio-Rad, Nazareth, Belgium). β-Actin was chosen as housekeeping gene for normalization.

2.3 Flow cytometry
Macrophages were stained with the following antibodies: fluorescein iso thio cyanate-conjugated mouse anti-human Toll-like receptor-4 (TLR4) (clone HTA125, Abcam, Cambridge, UK), APC-Cy7-conjugated mouse anti-human CD14 (clone M {phi}P9, BD Biosciences), AlexaFluor647-conjugated mouse anti-human CCR2 (clone 48607, BD Biosciences), and appropriate isotype controls. Ten thousand events were analysed on a BD FACSCantoTM flow cytometer (BD Biosciences), and analysis was performed with the FACSDivaTM software (BD Biosciences).

2.4 Analysis of gelatinase activity
Gelatin zymography was performed on culture supernatants to assess secreted MMP-9 activity as described before.10

2.5 Enzyme-linked immunosorbent assay
Total MMP-9 and TIMP-1 concentrations in conditioned medium were measured by ELISA (R&D Systems, Abingdon, UK). Detection limits were 0.156 ng/mL for MMP-9 and 0.08 ng/mL for TIMP-1.

2.6 Migration assay
Migratory capacity of THP-1 monocytes was studied using a Transwell® system with polycarbonate microporous membranes coated with 15 µL of a 2% gelatin B solution. MCP-1 (10 ng/mL) was added in the bottom compartment. Cells were pre-incubated for 30 min with GM 6001 (10 nM or 1 µM) or TIMP-1 (10 ng/mL) before seeding into the upper compartment. After 24 h, cells that migrated through the membrane were stained by the CyQuant GR® dye (Invitrogen) and fluorescence was read.

2.7 Statistical analysis
Results are expressed as mean ± SD. Data with a Gaussian distribution were analysed by paired t-test. Mann–Whitney (unpaired data) or Wilcoxon (paired data) tests were used for non-Gaussian data. A P-value <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Adenosine increases matrix metalloproteinase-9 production by primary macrophages from healthy volunteers and myocardial infarction patients
Monocytes were isolated from PBMCs obtained from both healthy volunteers and MI patients by negative selection and differentiated along the macrophage lineage by M-CSF. The effects of Ado on MMP-9 production were measured by quantitative PCR and ELISA. Using both techniques, a mild but significant increase in MMP-9 secretion was detected in the Ado-treated group. Ado induced a 70% increase in MMP-9 mRNA expression and a 20% increase in MMP-9 protein secretion in cell supernatants obtained from both cultures of macrophages from healthy volunteers and MI patients (Figure 1A and B).


Figure 1
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 Adenosine (Ado) increases matrix metalloproteinase-9 (MMP-9) production by primary and THP-1-derived macrophages. Monocytes isolated from peripheral blood mononuclear cells of healthy volunteers and myocardial infarction patients by negative selection were differentiated with 50 ng/mL M-CSF for 7 days. Macrophages were incubated for 24 h with Ado and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (10 µmol/L each) or vehicle. THP-1 cells were treated for 15 min with Ado and 10 µM EHNA or vehicle before differentiation into macrophages with 150 nM PMA for 48 h. EHNA, which inhibits Ado deaminase, and dipyridamole (DIP), which inhibits Ado transport, were used to enhance endogenous Ado concentration. MMP-9 mRNA levels were assessed by quantitative PCR and protein levels in cell supernatants by ELISA. Gelatinase activity was measured in cell-free conditioned medium by zymography and densitometry. (A) Ado increased MMP-9 mRNA and protein expression by primary human macrophages from healthy volunteers. Results are mean ± SD (n = 8). *P < 0.05 vs. control. (B) As assessed by zymography in conditioned medium, MMP-9 production by macrophages from MI patients was increased by Ado. Results are mean ± SD (n = 4). *P < 0.05 vs. control. (C–F) These were generated from experiments with THP-1-derived macrophages. (C) A representative zymogram is shown. Densitometric analysis revealed that Ado dose-dependently increased MMP-9 secretion. Results are mean ± SD (n = 8). *P < 0.05 vs. control, **P < 0.01 vs. control. (D) Zymography showed that both endogenous and exogenous Ado enhanced MMP-9 secretion. Alone, Ado, EHNA, and DIP triggered MMP-9 secretion and their effects were additive. Results are mean ± SD (n = 4). *P < 0.05 vs. control, **P < 0.01 vs. control. (E) Time-course experiment (n = 1). Measuring MMP-9 release in culture medium by zymography revealed that a differentiation period of 18 h was necessary to trigger MMP-9 secretion and that Ado was only effective after 18 h. (F) Quantitative PCR and ELISA showed that Ado increased MMP-9 mRNA and protein expression after 48 h of differentiation. Results are mean ± SD (n = 6). *P < 0.05 vs. control.

 
3.2 Adenosine increases matrix metalloproteinase-9 production by THP-1-derived macrophages
Having demonstrated that Ado consistently increases MMP-9 secretion by primary macrophages, we explored the mechanisms responsible for this effect in macrophages differentiated from the monocytic cell line THP-1. Cells were incubated for 15 min with increasing concentrations of Ado and 10 µM EHNA prior to differentiation with 150 nM PMA for 48 h. Ado increased MMP-9 production by THP-1 macrophages in a dose-dependent manner, reaching a three-fold increase with 100 µM Ado (Figure 1C). Ado also increased MMP-9 production when it was added after differentiation (data not shown). Concentrations of 10 µM Ado and 10 µM EHNA were used in further experiments. Considering that Ado concentrations found in vivo in conditions like HF11 and sepsis12 are in the micromolar range, our results suggest that the effect reported here is of biological relevance.

3.3 Both endogenous and exogenous Ado enhance matrix metalloproteinase-9 secretion
To characterize the relative contribution of endogenous and exogenous Ado in MMP-9 production, several modulators of Ado metabolism such as EHNA, which enhances endogenous Ado through inhibition of Ado deaminase, and DIP, which inhibits Ado transport, were used. When added alone, Ado, EHNA, or DIP induced MMP-9 secretion. When Ado was combined with EHNA or DIP, an additive effect was observed, resulting in a higher MMP-9 secretion than each substance alone (Figure 1D). These data show that both endogenous and exogenous Ado stimulate MMP-9 secretion.

3.4 Adenosine increases matrix metalloproteinase-9 mRNA and protein expression
To investigate the mechanism involved in enhanced MMP-9 secretion, we performed time-course experiments. An incubation period of at least 18 h was necessary to detect the effect of Ado on MMP-9 (Figure 1E). Using quantitative PCR, we observed that Ado induced a more than two-fold increase in MMP-9 mRNA expression in THP-1 cells, which was higher than in primary macrophages (Figure 1F). In parallel, we determined a three-fold increase in MMP-9 protein expression by ELISA (Figure 1F). Together, these observations suggest that the Ado-dependent release of MMP-9 is regulated at the transcriptional level.

3.5 Adenosine increases tissue inhibitor of matrix metalloproteinase-1 but not RECK expression
Imbalance between MMPs and TIMPs can result in the deregulation of ECM turnover and lead to the development of adverse remodelling. Ado mildly increased TIMP-1 secretion, the main endogenous inhibitor of MMP-9 (see Supplementary material online, Figure S1). This suggests the existence of a feedback loop to compensate for the increased MMP-9 production. However, considering the equimolar ratio of the MMP-9/TIMP-1 heterodimer, the low increase in TIMP-1 (40%) is probably insufficient to counterbalance the three-fold increase in MMP-9 secretion (see Supplementary material online, Figure S1).

The membrane-anchored inhibitor of MMP, RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), a key regulator of ECM turnover,13 inhibits pro-MMP-9 processing to the active MMP-9 form. As shown in the Supplementary material online, Figure S1, Ado did not modify RECK expression.

3.6 Adenosine increased matrix metalloproteinase-9 production by activated macrophages
We next determined whether Ado could also increase MMP-9 production by macrophages activated by stimuli such as the bacterial superantigen LPS or the peptide fMLP. In addition, several potential activators of the TLR4 such as HS and HA were tested. Finally, macrophages were subjected to hypoxia. MMP-9 production was above the control condition when macrophages were treated with LPS, HA, and hypoxia. Pre-treating the cells with Ado resulted in an exacerbation of MMP-9 secretion in all conditions tested, although this effect did not reach significance with fMLP (Figure 2). Together with data from Figure 1D, these results suggest that the effect of Ado on MMP-9 production appears to be independent of the presence of LPS, HA, fMLP, or hypoxia.


Figure 2
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Adenosine (Ado) increases matrix metalloproteinase-9 (MMP-9) production by activated macrophages. THP-1 macrophages were treated by Ado as described in the figure. Then cells were treated with 100 ng/mL LPS, 10–7 M fMLP, 10 µg/mL hyaluronan (HA), 10 µg/mL heparan sulfate (HS), or were incubated in hypoxia. For all conditions, MMP-9 concentration in conditioned media was measured by ELISA after 24 h. LPS, HA, and hypoxia enhanced MMP-9 production. Ado exacerbated MMP-9 secretion in all conditions tested. Results are mean ± SD (n ≥ 3). *P < 0.05 vs. control, **P < 0.05 vs. no Ado/EHNA.

 
3.7 Adenosine modulates the Toll-like receptor-4 pathway
Since Ado enhanced MMP-9 production when macrophages were treated with LPS or endogenous activators of TLR4, we investigated the effects of Ado on the TLR4 pathway. LPS binds to TLR4 on the surface of macrophages, and a complex between LPS, TLR4, the receptor CD14, and the accessory protein MD-2 is formed. Quantitative PCR revealed that Ado induced a three-fold increase in MD-2 mRNA expression by macrophages (Figure 3A). CD14 and TLR4 are expressed on the cell surface or in the cytoplasm of macrophages, and cells can be primed by receptor exocytosis for further activation. Flow cytometry showed that Ado increases the expression of CD14, but not TLR4, on the cell surface in the presence of LPS (Figure 3B). Collectively, these data suggest that Ado primes macrophages for increased responsiveness to LPS through enhanced MD-2 and CD14 expression. This observation may explain why Ado exacerbates MMP-9 secretion by LPS-activated macrophages LPS (Figure 2).


Figure 3
View larger version (7K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Adenosine (Ado) modulates the LPS recognition system. THP-1 macrophages were treated by Ado as described in Figure 2. (A) Quantitative PCR revealed that Ado increased MD-2 mRNA expression by macrophages, whereas CD14 and TLR4 expression remained unchanged (n = 6). (B) Twenty-four hours after differentiation, cells were treated with 100 ng/mL LPS and were harvested 24 h later. Ado alone did not change the cell surface expression of TLR4 and CD14 but increased CD14 expression on LPS-treated macrophages (n = 3). Results are mean ± SD. *P < 0.01 vs. control.

 
3.8 Adenosine increases matrix metalloproteinase-9 production through its A3 receptor
To address which type of receptor mediates the effect of Ado on MMP-9 secretion, we used both pharmacological and molecular approaches. First, we determined the relative expression of each of the four Ado receptors by macrophages. Quantitative PCR showed that receptors of the A2 type were the predominant form (Figure 4A). No A1 receptor mRNA was detected in our experiments. Ado induced a more than two-fold increase in A2b and A3 receptors expression. Secondly, experiments using agonists of Ado receptors showed that only the A3-specific agonist IB-MECA was able to increase MMP-9 secretion to the same extent than Ado (Figure 4B). This result has been confirmed in primary macrophages (see Supplementary material online, Figure S3). Finally, a genomic approach using siRNA specific for A2a, A2b, and A3 receptors was used. The specific down-regulation of Ado receptors by their respective siRNA was verified by quantitative PCR (data not shown). Zymography revealed that only the A3-specific siRNA was able to significantly inhibit the Ado-mediated increase in MMP-9 secretion (Figure 4C). Taken together, these results show that the A3 receptor mediates the effect of Ado on MMP-9 secretion.


Figure 4
View larger version (7K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 Adenosine (Ado) increases matrix metalloproteinase-9 (MMP-9) production through its A3 receptor. (A) THP-1 macrophages were treated by Ado as described in Figure 2. Quantitative PCR revealed that A2a and A2b were the predominant forms of Ado receptors in macrophages. Ado induced a more than two-fold increase in A3 and A2b expression. Results are mean ± SD (n = 6). *P < 0.05 vs. control. (B) THP-1 cells were treated for 15 min with 0.1, 1, or 10 µM of Ado receptors agonists (CPA, A1-specific; CGS21680, A2a-specific; IB-MECA, A3-specific). EHNA was omitted to specifically study the effect of exogenous Ado. After 48 h of differentiation with 150 nM phorbol myristate actetate (PMA), gelatinase activity was assessed in conditioned medium. The A3 agonist mimicked the effect of Ado on MMP-9 secretion. The experiment was performed twice with similar results. (C) Cells were transfected with siRNA specific for A2a, A2b, or A3 Ado receptors, incubated for 48 h to achieve down-regulation of Ado receptors expression, treated for 15 min with 10 µM Ado and 10 µM EHNA or vehicle before differentiation into macrophages with 150 nM PMA for 48 h. Conditioned medium was analysed by gelatin zymography. The A3-specific siRNA inhibits the Ado-mediated increase in MMP-9 secretion. Results are mean ± SD (n = 3). *P < 0.05 vs. Ado.

 
3.9 Adenosine improves monocyte migratory capacity
Infiltration of monocytes/macrophages into the myocardium post-MI is under the control of cell migration, which is facilitated by degradation of the ECM by MMP-9, and chemotaxis, which involves the binding of the chemokine MCP-1 to its cognate receptor CCR2. We therefore tested whether the increase in MMP-9 secretion by Ado could improve monocyte migration along a gradient of MCP-1. For this purpose, the top of a modified Boyden chamber was coated with gelatin B and MCP-1 was added in the bottom compartment. Monocytes were treated with Ado before seeding on the microporous membrane of the chamber. As shown in Figure 5A, Ado enhanced cell migration through the gelatin layer. This effect was inhibited by the synthetic MMP inhibitor GM6001 and the endogenous MMP-9 inhibitor TIMP-1 (Figure 5A). To test a potential effect of Ado on chemotaxis, CCR2 expression was measured by quantitative PCR. As shown in Figure 5B, Ado had opposite effects on CCR2 mRNA expression depending on the differentiation status of monocytes. Ado increased CCR2 expression by monocytes (50% increase vs. control), whereas it decreased CCR2 expression by macrophages (75% inhibition vs. control). When assessed at the cell surface by flow cytometry, Ado also potently reduced the expression of CCR2 on macrophages (Figure 5C). The mean fluorescence intensity of CCR2 staining was decreased by Ado from 800 to 500, and the percentage or cells expressing CCR2 was reduced by Ado from 60 to 6% (Figure 5C). Therefore, we concluded that Ado enhances monocytes migration through increased MMP-9 activity. In addition, our data suggest that the chemotactic capacity of macrophages is significantly attenuated by Ado through a loss of CCR2 expression.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 Ado increases monocytes migratory capacity. (A) THP-1 monocytes were treated for 24 h with 10 µM Ado and 10 µM EHNA or vehicle before seeding on the microporous membrane of a modified Boyden chamber pre-coated with gelatin B. About 10 ng/mL MCP-1 was added in the bottom compartment. When specified, cells were pre-incubated with tissue inhibitor of matrix metalloproteinase (TIMP)-1 (10 ng/mL) or GM6001 (10 nM or 1 µM) before seeding on the membrane. Cell migration was quantified by fluorescence after 24 h. Ado enhanced cell migration and TIMP-1 or GM6001 prevented this effect. Results are mean ± SD (n = 5). *P < 0.05 vs. no Ado. (B) THP-1 monocytes and monocytes differentiated into macrophages by 150 nM PMA were treated for 24 h with 10 µM Ado and 10 µM EHNA or vehicle before assessment of CCR2 mRNA expression by quantitative PCR. Ado enhanced CCR2 expression in monocytes and decreased CCR2 expression in macrophages. Results are mean ± SD (n = 5). *P < 0.05 vs. no Ado. (C) Ado decreases CCR2 expression in macrophages. Macrophages were treated for 6 h with 10 µM Ado and 10 µM EHNA or vehicle before assessment of CCR2 expression by flow cytometry. The P1 gate was used to determine the percentage of CCR2-positive cells. A representative experiment from three independent experiments is shown.

 
3.10 Blood cells isolated from patients with acute myocardial infarction show enhanced matrix metalloproteinase-9 expression
Since Ado is massively released in the heart after MI, we hypothesized that blood cells isolated from patients the day of MI could express high levels of MMP-9. The PAXgene® RNA blood system that allows RNA extraction of all blood cells from only 2.5 mL of blood was used in these experiments. This does not have a major impact on the accuracy of our results assuming that the monocytes/macrophages population is largely responsible for MMP-9 mRNA expression. Indeed, MMP-9 is also released in large amounts from neutrophils by degranulation, thus independent from de novo transcription.14 Cells from MI patients expressed significantly more MMP-9 mRNA than controls (Figure 6). These observations suggest that the increased secretion of MMP-9 by monocytes/macrophages in the presence of Ado occurs during ischaemia post-acute MI.


Figure 6
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6 Cells isolated from the blood of patients with acute myocardial infarction (MI) express more matrix metalloproteinase-9 mRNA than healthy controls. Blood samples from healthy volunteers (controls, open square) and patients with MI (filled square) were recovered in PAXgene® tubes. RNA was extracted and quantified by qPCR. Cells isolated from patients with MI have higher levels of MMP-9 than controls. Results were normalized to β-actin. Each square represents one patient and the means ± SD (n = 12 in each group) are displayed. *P < 0.0005 vs. controls.

 
A potential regulation of the expression of Ado receptors after MI has been tested in the same two groups of MI patients and healthy volunteers. As shown in the Supplementary material online, Figure S2, MI had no significant effect on the expression of Ado receptors.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
The main finding of the present study is that, in sharp contrast to our previous observations in neutrophils, Ado enhances MMP-9 secretion by macrophages.

The fact that Ado prevents MMP-9 release by neutrophils whereas it enhances MMP-9 secretion by macrophages has several implications. Neutrophils are recruited to the infarct area early after MI (<2 days), whereas macrophages invade the lesion 2–4 days post-MI. The mechanisms responsible for MMP-9 production by these two cell types are fundamentally different. Neutrophils rapidly release large amounts of MMP-9 by degranulation. In our experiments, however, MMP-9 secretion by macrophages was not detected before 8 h, which is consistent with a de novo protein synthesis. We previously demonstrated in neutrophils that Ado decreases MMP-9 secretion through the A2a receptor and this mechanism involved a quick signalling cascade with rapid Ca2+ mobilization.10 A recent study showed that hypoxia inhibits MMP-9 expression in monocyte-derived dendritic cells through the A2b receptor.15 Data presented here show that enhancement of MMP-9 production by macrophages after exposure to Ado is mediated through A3 receptor activation and involves a transcriptional effect, although a contribution of the A2b receptor cannot be fully excluded. Therefore, the effects of Ado on MMP-9 production by inflammatory cells are highly dependent on the type of Ado receptors and their respective signalling pathways. This might have important implications for therapies using Ado analogues in the inflammatory context of MI or HF.

We also determined whether Ado was able to enhance MMP-9 production by activated macrophages. When macrophages were treated by the widely used superantigen LPS, a strong release of MMP-9 was observed and this effect was amplified by Ado. This observation suggests that macrophages can be primed by Ado for enhanced responsiveness to LPS. Our results showing that Ado increased MD-2 and CD14 expression are in accordance with this hypothesis. This priming mechanism appears to be different from the priming induced by oxidative stress that recruits TLR4 to the plasma membrane.16 Whether these two mechanisms occur simultaneously following MI and result in exaggerated macrophage activation remains to be determined.

A direct link between TLR4 activation and the development of LV remodelling has recently been proven by Timmers et al.17 However, the prototypical activator of TLR4, the bacterial product LPS, is probably absent in the heart after MI, suggesting that TLR4 must be activated by other endogenous ligands. Several ligands from diverse origins appear to be able to bind and activate TLR4, among which some are potentially present in the ischaemic heart. These include HS, which is involved in endothelial barrier integrity,18 and HA, which is up-regulated in the kidney after ischaemia–reperfusion.19 Our data show that LPS and HA significantly triggered MMP-9 production and that this effect was amplified by Ado. Interestingly, hypoxia was also able to induce MMP-9 production, reaching maximal levels after Ado treatment (four-fold increase). This observation suggests that Ado, which is massively released during ischaemia, is a potent stimulator of MMP-9 production in the ischaemic heart.

Chemotaxis and cell migration are two distinct processes that orchestrate monocytes tissue infiltration. The role of the main monocyte chemoattractant MCP-1 in LV remodelling is controversial since both beneficial20 and deleterious21 effects have been found in mice models. Interestingly, a study by Dewald et al.22 highlighted the possibility that MCP-1 could, at the same time, promote healing of infarcted areas and contribute to LV remodelling. Two distinct subsets of monocytes have been identified in the murine infarcted heart: inflammatory monocytes bearing the CCR2 receptor are recruited during the early phase post-MI and monocytes lacking the CCR2 receptor and exhibiting healing properties are recruited during the late phase.23 Our results reveal a dual effect of Ado on monocytes/macrophages. First, Ado improves monocyte migration through up-regulation of MMP-9 activity, and secondly, Ado favours healing macrophages to inflammatory macrophages through a loss of CCR2 expression.

We24 and others25 have shown that MMP-9 is a biomarker of HF after MI. Therefore, we compared MMP-9 expression in blood cells from patients with acute MI and healthy volunteers and we observed that cells from patients with acute MI had higher levels of MMP-9. This is probably due to a priming of the macrophages by the large amounts of Ado released in the ischaemic heart following MI. However, the exact contribution of Ado to this priming and the mechanisms involved in this potential priming remain to be determined.

A study with MMP-9 deficient mice revealed MMP-9 as a potential therapeutic target to prevent LV remodelling after MI.26 However, the PREMIER (prevention of myocardial infarction early remodelling) phase II trial showed that non-specific inhibition of MMPs activity with PG-116800 failed to prevent LV remodelling and did not improve outcome after MI.4 In this study, the MMP inhibitor was given 24–72 h post-MI. The negative results of this trial might be explained either by the non-specificity of the MMP inhibitor used or by the suboptimal timing of drug delivery. Further experimental work is necessary to fully understand the implication of MMPs in LV remodelling.

The therapeutic potential of Ado or Ado analogues has been reported in both animal studies and clinical trials. In animals, cardioprotective activities of Ado were found during three windows of opportunity: as a pre-treatment of ischaemia, during ischaemia, or during reperfusion. A dichotomy between the types of receptors involved was first hypothesized, A1 receptors mediating the cardioprotective effects of Ado during pre-treatment and during ischaemia, mainly through metabolic changes, and A2 receptors being beneficial during reperfusion, mainly through inhibition of neutrophil activity.27 Our previous data supported the involvement of A2a receptors in the protection provided by Ado in post-ischaemic injury, through inhibition of MMP-9 release by neutrophils.10 So far, most studies have shown that A3 receptor activation is cardioprotective before or during ischaemia.27,28 This protection appears to be dose-dependent: mice with mild over-expression of A3 were partly protected from ischaemic injury whereas mice which highly over-expressed the A3 receptor developed a dilated cardiomyopathy.29 It can be speculated that this phenomenon may partly be related to the stimulation of MMP-9 by the A3 receptor. Indeed, while low doses of MMP-9 may favour angiogenesis,30 persistent high doses of MMP-9 will likely lead to matrix degradation and remodelling. Recent work by Feldman’s group31 demonstrated the association between a polymorphism in the coding region of the A3 receptor and the infarct size in a population of patients with ischaemic cardiomyopathy. However, this observation has to be confirmed in larger studies.

Few clinical trials have tested the therapeutic potential of Ado in the context of MI. Ado improved cardiac function of patients with MI when combined with lidocaine32 or primary angioplasty.33,34 However, these trials, although reporting a beneficial clinical outcome, were performed on a small number of patients. The AMISTAD I and II trials, with a larger sample size, demonstrated a reduction in infarct size by Ado as an adjunct to reperfusion therapy, when added early after infarction.35,36 A post hoc analysis of the AMISTAD-II trial revealed that Ado reduced mortality when added within 3 h of infarction.37 Of note, in this trial, Ado was administered for more than 48 h after MI. Based on our current findings, it can be speculated that some of the early benefit of Ado was offset by stimulation of MMP-9 and migration of macrophages later on.

In conclusion, the Ado A3 receptor increases MMP-9 secretion by human monocytes/macrophages, in contrast to the A2a receptor that inhibits MMP-9 secretion by neutrophils. Therefore, therapy with Ado in the setting of ischaemic injury appears complex because the effects dramatically change with the type of receptor stimulated and the dosage. Since high doses of MMP-9, such as those released by neutrophils, may lead to matrix degradation, whereas moderate doses, such as those produced by monocytes/macrophages, may improve revascularization, our results suggest the testing of a dual therapeutic strategy with A2a agonists immediately after MI and A3 agonists during the remodelling phase.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
Société pour la Recherche sur les Maladies Cardiovasculaires; Ministère de la Culture, de l’Enseignement Supérieur et de la Recherche of Luxembourg.


   Acknowledgements
 
We thank Céline Jeanty, Mélanie Vausort, Bernadette Leners, Christelle Nicolas, Malou Gloesener, and Loredana Jacobs for expert technical assistance. The help of Nicolaas Brons and Wim Ammerlaan with flow cytometry is acknowledged.

Conflict of interest: none declared.


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

  1. Kannel WB. Vital epidemiologic clues in heart failure. J Clin Epidemiol (2000) 53:229–235.[CrossRef][Web of Science][Medline]
  2. Levy D, Kenchaiah S, Larson MG, Benjamin EJ, Kupka MJ, Ho KK, et al. Long-term trends in the incidence of and survival with heart failure. N Engl J Med (2002) 347:1397–1402.[Abstract/Free Full Text]
  3. Zannad F, Mebazaa A, Juilliere Y, Cohen-Solal A, Guize L, Alla F, et al. Clinical profile, contemporary management and one-year mortality in patients with severe acute heart failure syndromes: the EFICA study. Eur J Heart Fail (2006) 8:697–705.[Abstract/Free Full Text]
  4. Hudson MP, Armstrong PW, Ruzyllo W, Brum J, Cusmano L, Krzeski P, et al. Effects of selective matrix metalloproteinase inhibitor (PG-116800) to prevent ventricular remodeling after myocardial infarction: results of the PREMIER (prevention of myocardial infarction early remodeling) trial. J Am Coll Cardiol (2006) 48:15–20.[Abstract/Free Full Text]
  5. Jacobson KA, Gao ZG. Adenosine receptors as therapeutic targets. Nat Rev Drug Discov (2006) 5:247–264.[CrossRef][Web of Science][Medline]
  6. Willems L, Ashton KJ, Headrick JP. Adenosine-mediated cardioprotection in the aging myocardium. Cardiovasc Res (2005) 66:245–255.[Abstract/Free Full Text]
  7. Wagner DR, McTiernan C, Sanders VJ, Feldman AM. Adenosine inhibits lipopolysaccharide-induced secretion of tumor necrosis factor-alpha in the failing human heart. Circulation (1998) 97:521–524.[Abstract/Free Full Text]
  8. Wagner DR, Combes A, McTiernan C, Sanders VJ, Lemster B, Feldman AM. Adenosine inhibits lipopolysaccharide-induced cardiac expression of tumor necrosis factor-alpha. Circ Res (1998) 82:47–56.[Abstract/Free Full Text]
  9. Wagner DR, Kubota T, Sanders VJ, McTiernan CF, Feldman AM. Differential regulation of cardiac expression of IL-6 and TNF-alpha by A2- and A3-adenosine receptors. Am J Physiol (1999) 276:H2141–H2147.[Web of Science][Medline]
  10. Ernens I, Rouy D, Velot E, Devaux Y, Wagner DR. Adenosine inhibits matrix metalloproteinase-9 secretion by neutrophils: implication of A2a receptor and cAMP/PKA/Ca2+ pathway. Circ Res (2006) 99:590–597.[Abstract/Free Full Text]
  11. Funaya H, Kitakaze M, Node K, Minamino T, Komamura K, Hori M. Plasma adenosine levels increase in patients with chronic heart failure. Circulation (1997) 95:1363–1365.[Abstract/Free Full Text]
  12. Barnes CR, Mandell GL, Carper HT, Luong S, Sullivan GW. Adenosine modulation of tumor necrosis factor-alpha-induced neutrophil activation. Biochem Pharmacol (1995) 50:1851–1857.[CrossRef][Web of Science][Medline]
  13. Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, et al. The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell (2001) 107:789–800.[CrossRef][Web of Science][Medline]
  14. Lindsey M, Wedin K, Brown MD, Keller C, Evans AJ, Smolen J, et al. Matrix-dependent mechanism of neutrophil-mediated release and activation of matrix metalloproteinase 9 in myocardial ischemia/reperfusion. Circulation (2001) 103:2181–2187.[Abstract/Free Full Text]
  15. Zhao P, Li XG, Yang M, Shao Q, Wang D, Liu S, et al. Hypoxia suppresses the production of MMP-9 by human monocyte-derived dendritic cells and requires activation of adenosine receptor A(2b) via cAMP/PKA signaling pathway. Mol Immunol (2008) 45:2187–2195.[CrossRef][Web of Science][Medline]
  16. Powers KA, Szaszi K, Khadaroo RG, Tawadros PS, Marshall JC, Kapus A, et al. Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages. J Exp Med (2006) 203:1951–1961.[Abstract/Free Full Text]
  17. Timmers L, Sluijter JP, van Keulen JK, Hoefer IE, Nederhoff MG, Goumans MJ, et al. Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function after myocardial infarction. Circ Res (2008) 102:257–264.[Abstract/Free Full Text]
  18. Tang AH, Brunn GJ, Cascalho M, Platt JL. Pivotal advance: endogenous pathway to SIRS, sepsis, and related conditions. J Leukoc Biol (2007) 82:282–285.[Abstract/Free Full Text]
  19. Wu H, Chen G, Wyburn KR, Yin J, Bertolino P, Eris JM, et al. TLR4 activation mediates kidney ischemia/reperfusion injury. J Clin Invest (2007) 117:2847–2859.[CrossRef][Web of Science][Medline]
  20. Morimoto H, Takahashi M, Izawa A, Ise H, Hongo M, Kolattukudy PE, et al. Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice prevents cardiac dysfunction and remodeling after myocardial infarction. Circ Res (2006) 99:891–899.[Abstract/Free Full Text]
  21. Hayashidani S, Tsutsui H, Shiomi T, Ikeuchi M, Matsusaka H, Suematsu N, et al. Anti-monocyte chemoattractant protein-1 gene therapy attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation (2003) 108:2134–2140.[Abstract/Free Full Text]
  22. Dewald O, Zymek P, Winkelmann K, Koerting A, Ren G, Abou-Khamis T, et al. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res (2005) 96:881–889.[Abstract/Free Full Text]
  23. Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med (2007) 204:3037–3047.[Abstract/Free Full Text]
  24. Wagner DR, Delagardelle C, Ernens I, Rouy D, Vaillant M, Beissel J. Matrix metalloproteinase-9 is a marker of heart failure after acute myocardial infarction. J Card Fail (2006) 12:66–72.[CrossRef][Web of Science][Medline]
  25. Yan AT, Yan RT, Spinale FG, Afzal R, Gunasinghe HR, Arnold M, et al. Plasma matrix metalloproteinase-9 level is correlated with left ventricular volumes and ejection fraction in patients with heart failure. J Card Fail (2006) 12:514–519.[CrossRef][Web of Science][Medline]
  26. Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest (2000) 106:55–62.[Web of Science][Medline]
  27. Vinten-Johansen J, Thourani VH, Ronson RS, Jordan JE, Zhao ZQ, Nakamura M, et al. Broad-spectrum cardioprotection with adenosine. Ann Thorac Surg (1999) 68:1942–1948.[Abstract/Free Full Text]
  28. Safran N, Shneyvays V, Balas N, Jacobson KA, Nawrath H, Shainberg A. Cardioprotective effects of adenosine A1 and A3 receptor activation during hypoxia in isolated rat cardiac myocytes. Mol Cell Biochem (2001) 217:143–152.[CrossRef][Web of Science][Medline]
  29. Black RG Jr, Guo Y, Ge ZD, Murphree SS, Prabhu SD, Jones WK, et al. Gene dosage-dependent effects of cardiac-specific overexpression of the A3 adenosine receptor. Circ Res (2002) 91:165–172.[Abstract/Free Full Text]
  30. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, et al. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med (1999) 5:1135–1142.[CrossRef][Web of Science][Medline]
  31. Tang Z, Diamond MA, Chen JM, Holly TA, Bonow RO, Dasgupta A, et al. Polymorphisms in adenosine receptor genes are associated with infarct size in patients with ischemic cardiomyopathy. Clin Pharmacol Ther (2007) 82:435–440.[CrossRef][Web of Science][Medline]
  32. Garratt KN, Holmes DR Jr, Molina-Viamonte V, Reeder GS, Hodge DO, Bailey KR, et al. Intravenous adenosine and lidocaine in patients with acute mycocardial infarction. Am Heart J (1998) 136:196–204.[CrossRef][Web of Science][Medline]
  33. Leesar MA, Stoddard M, Ahmed M, Broadbent J, Bolli R. Preconditioning of human myocardium with adenosine during coronary angioplasty. Circulation (1997) 95:2500–2507.[Abstract/Free Full Text]
  34. Marzilli M, Orsini E, Marraccini P, Testa R. Beneficial effects of intracoronary adenosine as an adjunct to primary angioplasty in acute myocardial infarction. Circulation (2000) 101:2154–2159.[Abstract/Free Full Text]
  35. Mahaffey KW, Puma JA, Barbagelata NA, DiCarli MF, Leesar MA, Browne KF, et al. Adenosine as an adjunct to thrombolytic therapy for acute myocardial infarction: results of a multicenter, randomized, placebo-controlled trial: the Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial. J Am Coll Cardiol (1999) 34:1711–1720.[Abstract/Free Full Text]
  36. Ross AM, Gibbons RJ, Stone GW, Kloner RA, Alexander RW. A randomized, double-blinded, placebo-controlled multicenter trial of adenosine as an adjunct to reperfusion in the treatment of acute myocardial infarction (AMISTAD-II). J Am Coll Cardiol (2005) 45:1775–1780.[Abstract/Free Full Text]
  37. Kloner RA, Forman MB, Gibbons RJ, Ross AM, Alexander RW, Stone GW. Impact of time to therapy and reperfusion modality on the efficacy of adenosine in acute myocardial infarction: the AMISTAD-2 trial. Eur Heart J (2006) 27:2400–2405.[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
 J. Immunol.Home page
K. E. Barnholt, R. S. Kota, H. H. Aung, and J. C. Rutledge
Adenosine Blocks IFN-{gamma}-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation
J. Immunol., November 15, 2009; 183(10): 6767 - 6777.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Shaykhiev, A. Krause, J. Salit, Y. Strulovici-Barel, B.-G. Harvey, T. P. O'Connor, and R. G. Crystal
Smoking-Dependent Reprogramming of Alveolar Macrophage Polarization: Implication for Pathogenesis of Chronic Obstructive Pulmonary Disease
J. Immunol., August 15, 2009; 183(4): 2867 - 2883.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
80/2/246    most recent
cvn201v2
cvn201v1
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 Velot, E.
Right arrow Articles by Wagner, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Velot, E.
Right arrow Articles by Wagner, D. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?