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European Journal of Heart Failure 2006 8(7):665-672; doi:10.1016/j.ejheart.2006.03.005
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© 2006 European Society of Cardiology

Toll-like receptor 4 modulates myocardial ischaemia–reperfusion injury: Role of matrix metalloproteinases

Heidi Stapela, Se-Chan Kimb, Steffen Osterkampb, Pascal Knuefermannb, Andreas Hoeftb, Rainer Meyera, Christian Grohéc and Georg Baumgartenb,*

a Institute of Physiology II, University of Bonn, Germany
b Department of Anaesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany
c Medizinische Universitätspoliklinik, University of Bonn, Germany

* Corresponding author. Tel.: +49 228 287 4124; fax: +49 228 287 4115. E-mail address: Georg.Baumgarten{at}ukb.uni-bonn.de (G. Baumgarten).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Toll-like receptor 4 (TLR4) mediates innate immune responses following endotoxemia and myocardial ischaemia–reperfusion (I/R) injury. Pre-treatment with the major TLR4 ligand lipopolysaccharide (LPS) reduces infarct size. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play a crucial role in endotoxemia possibly also determining I/R injury.

Aims: We investigated the influence of TLR4 on infarct size and assessed the influence of MMP and TIMP regulation on I/R injury.

Methods: Left anterior descending artery (LAD) occlusion was performed on wild-type (C3H/HeN) and TLR4-deficient (C3H/HeJ) mice. Animals were stimulated with LPS (1mg/kg) or PBS 16h ahead of 60min LAD ligation. After 24h of reperfusion, triphenyltetrazolium chloride staining was performed and infarct size was measured by planimetry. MMP- and TIMP-mRNA expression were determined by RPA after 3h of reperfusion. MMP zymographic activity was monitored 6h after occlusion.

Results: TLR4-deficient mice and LPS-treated wild-type mice showed significantly reduced infarct areas. LPS-stimulation significantly increased the overall MMP/TIMP mRNA expression ratio due to elevated MMP-3, -8, -9, and TIMP-1 in wild-type mice. I/R overall reduced the MMP/TIMP ratio due to increased MMP-1, TIMP-1, and -3 mRNA expression.

Conclusions: LPS pre-treatment and TLR4-deficiency led to a decreased infarct size. However, infarct area and MMP/TIMP ratio were not correlated. This means that in TLR4-deficient mice MMP/TIMP ratios are not determining the infarct size.

Key Words: TLR4 • MMP/TIMP • Myocardial ischemia–reperfusion • Ligand lipopolysaccharide

Received June 22, 2005; Revised December 16, 2005; Accepted March 16, 2006


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Coronary heart disease is a major cause of heart failure and myocardial infarction, thereby inducing relevant inflammatory cascades. It has been shown that inflammatory responses following ischaemia as well as endotoxaemia are modulated via Toll-like receptor (TLR) 4 [1,2]. TLR4 is an innate immune receptor, binding lipopolysaccharide (LPS) from gram-negative bacteria [3]. Via LPS-binding protein (LBP) LPS forms an LPS-LBP-CD14 complex which activates the TLR4-dependent signalling cascade. Furthermore, TLR4 recognizes endogenous ligands such as fibronectin or heat shock protein 60, which are released during oxidative stress, following inflammatory processes and ischaemia-reperfusion (I/R) injuries [4]. Two major TLR4-signalling pathways that regulate the inflammatory response have been described to date. The early phase pathway increases the expression of pro-inflammatory genes like tumor necrosis factor-alpha (TNF-P), interleukin-1 beta (IL-1β), and interleukin 6 (IL-6), the late phase pathway results in interferon beta (IFN-β) expression [5]. Early studies have shown that both CD14- as well as TLR4-deficient mice are protected against LPS-induced inflammation, left ventricular dysfunction and depressed cardiac myocyte contractility [1,6-8]. Previous results also indicate that lipopolysaccharide reduces myocardial infarct size in I/R experiments [9-11], but the precise mechanisms remain unknown. I/R and endotoxaemia induce multiple inflammatory processes including degradation of the extracellular matrix by regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) [12,13]. MMPs are largely involved in collagen degradation, remodelling of de novo connective tissue synthesis, but also cell migration and angiogenesis. Given the observations that TLR4 mediates the immune responses following endotoxaemia and myocardial I/R injury, we investigated the influence of TLR4 on mice pre-treated with LPS and then challenged in a closed-chest model of I/R. The purpose of this study was to determine the role of TLR4 after LPS-administration and/or I/R on infarct size, MMP and TIMP regulation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Study protocol
Surgery was performed on 229 animals, the mortality rate was approximately 20%. After 5 to 7days of recovery, animals were pre-treated with endotoxin (Escherichia coli LPS) or placebo (PBS) 16h prior to induction of ischaemia (Fig. 1). Following 1h of ischaemia, the myocardium was reperfused for 3, 6 or 24h. Hearts from 115 animals were harvested after 3h or 6h of reperfusion for mRNA or protein analysis. Triphenyltetrazolium chloride (TTC) staining of the myocardium was performed after 24h of reperfusion. Hearts from 48 mice were included in planimetric analysis.


Figure 1
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  		Fig. 1 Study protocol: time points of surgery, LPS stimulation and ischaemia-reperfusion.

 
LPS hyporesponsive C3H/HeJ (TLR4-deficient) and LPS responsive C3H/HeN (wild-type, TLR4-responsive) mice (8-12weeks, male) were purchased from Charles River (Sulzfeld, Germany). C3H/HeJ mice carry a point mutation in the intracytoplasmic region of TLR4 resulting in a replacement of proline with histidine, leading to the LPS hyporesponsive phenotype [14]. Mice were housed in pathogen-free cages with free access to water and standard rodent chow. The animals were handled according to the principles of laboratory animal care (NIH Publication No. 85-23, revised 1996), and animal procedures were approved by the local committee for animal care.

2.2. LPS stimulation, myocardial ischaemia and reperfusion
We applied a closed-chest model as described previously to minimize elevated inflammatory reactions due to the operative trauma [15]. In brief, mice were anaesthetized with isoflurane 2.0vol.% (Forene®, Abbott GmbH, Wiesbaden, Germany), body temperature was controlled, animals were placed in a supine position and ventilation was adapted to physiological parameters (Mini Vent 845, Hugo Sachs Elektronik, March-Hugstetten, Germany). After thoracotomy, a ligature (8-0 prolene) was placed underneath the LAD, 2mm distal from the tip of the left atrium, passing the ends through a 1mm PE-tubing. The correct position of the suture was proven by observing paleness of the distal myocardium after brief occlusion of the LAD. Mice were allowed to recover for 7days, in order to avoid the effects of altered inflammatory mediators on ischaemia induced processes. E. coli LPS (1mg/kg bodyweight; Sigma Aldrich Chemie GmbH, Munich, Germany) or PBS (phosphate buffer solution; Life-Technologies, Gibco BRL, Karlsruhe, Germany) was injected intraperitoneally (i.p.) 16h ahead of infarction (Fig. 1). Animals were anaesthetized with propofol i.p. (Disoprivan® 1%, 100mg/kg bodyweight; Astra Zeneca, Wedel, Germany) prior to LAD occlusion and were randomly assigned to either I/R or sham group. The I/R group was subjected to 1h LAD occlusion. In sham animals, the ligatures were not occluded. In ECG recordings, ST segment elevation was documented to evaluate positive infarction signs (PowerLab, AD Instruments GmbH, Sperrbach, Germany; Software: Chart for Windows v 4.2.3).

2.3. Infarct area and area at risk
For measurement of infarct area (IA) and area at risk (AAR) in our experimental groups, ligatures were closed under anaesthesia and a 5% Phthaloblue solution was infused in vivo. Hearts were excised immediately and washed with PBS. The atria and right ventricle were removed. The left ventricle (LV) was sliced into 1mm sections after flash-freezing in isopentane at –190°C. Heart sections were incubated in 1.5% TTC (Sigma Aldrich, Munich, Germany) at 37°C for 20min followed by 30min in formalin. Viable tissue was stained red, infarct tissue appeared white. Slices were photographed (Olympus Camedia 3030). The AAR (red and white), infarct area (white) and non-AAR (blue) from each section was measured by blinded observers using Image J software (Version 1.29, NIH, USA) and ratios were calculated.

2.4. Ribonuclease protection assay
For ribonuclease protection assay, hearts were harvested after 3h of reperfusion, flash-frozen in liquid nitrogen and kept at –80°C. Hearts were homogenized and total RNA was extracted as described elsewhere [16].

mRNA levels of MMP-1, -2, -3, -8, -9, and TIMPs 1-4 per 20µg RNA sample were analysed with mMMP-1 multiprobe template set (BD Biosciences Pharmingen, San Diego, CA, USA). Signals were quantified with AIDA software v3.5 (Raytest, Straubenhardt, Germany) and normalized to the ribosomal housekeeping gene L32.

2.5. Zymographic measurement of MMP activity
For zymographic measurement of MMP activity after 6h of reperfusion, heart samples (60µg protein content, isolated with NE-PER protein isolation kit, Pierce, Bonn, Germany) were mixed with 2x Tris-glycine SDS sample buffer, and loaded on 10% polyacrylamide gels (SDS-PAGE) copolymerized with gelatine (each 0.3mg/ml type A from porcine skin and type B from bovine skin, Sigma Aldrich, Munich, Germany). Following 120min of electrophoresis at 125V, gels were washed with 2.5% Triton X-100 (Sigma) for 3x20min to remove SDS and then incubated for 48h at 37°C in developing buffer (50mM Tris-HCl, 0.2M NaCl, 5mM CaCl2, 0.02% Brij). Gels were then stained using 0.1% (w/v) Brilliant Blue (Sigma) in a mixture of water/methanol/acetic acid (5:5:1 v/v/v), destained in 45% methanol, and 3% acetic acid in water (v/v). Areas of protease activity were detected as transparent bands against blue background. All gelatinolytic activities reported could be inhibited by addition of EDTA to the incubation buffer. Zymograms were photographed and signals were quantified using AIDA software.

2.6. Statistics
All values are expressed as means (M)±S.E.M. Significant differences among experimental groups of p<0.05 are indicated. Two-way ANOVA was calculated for infarct size measurements and mRNA expressions in order to differentiate between the effects of genotype, LPS-stimulation and I/R. In case of significant differences, post-hoc testing was performed (Bonferroni's test). Statistics were calculated using Prism 3.03 (GraphPad Software Inc., San Diego, CA, USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
In this study eight different groups were compared: (1) sham-operated PBS injected; (2) I/R PBS injected; (3) sham-operated LPS injected; (4) I/R LPS injected. These four groups were formed in both genotypes resulting in eight groups.

First, we evaluated the infarct area in the different I/R groups. Occlusion of the LAD induced an average AAR of about 45% of LV area (Fig. 2). The AAR varied only within narrow limits (42.6% to 47.7% of LV) among the four I/R groups, indicating good reproducibility of the malperfusion. IA was measured after 60min of coronary artery occlusion and after 24h of reperfusion. The ratio between IA and AAR was decreased in TLR4-deficient mice compared to wild-type animals (30.3%±4.81 (n=17) vs. 49.8%±6.66 (n=13)). LPS treatment led to a significant reduction of the infarct zone in wild-type mice (24.8%±5.28; n=10), while in TLR4-deficient mice LPS-stimulation did modulate neither IA nor AAR. These data suggest that LPS can alter the I/R response in the intact mouse myocardium of wild-type mice. Interestingly, these alterations were not found in TLR4-deficient LPS hyporesponsive animals. Both, TLR4 deficiency as well as LPS pre-treatment resulted in a similar reduction of infarct size. This hints to an important role of the TLR4-dependent signalling pathway for the pathogenesis of injured myocardium.


Figure 2
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  		Fig. 2 Infarct area (IA) vs. area at risk (AAR) and AAR vs. left ventricular area (LV) in wild-type (TLR4+) and TLR4-deficient (TLR4–) mice (mean±S.E.M.). Significant differences in IA/AAR were detected between TLR4+ PBS vs. TLR4+ LPS and TLR4+ PBS vs. TLR4– PBS. *p<0.05. PBS=phosphate buffer solution (placebo), LPS=lipopolysaccharide (endotoxin).

 
To further elucidate the mechanisms involved in this process, we analysed the MMP/TIMP system which plays a crucial role in inflammation and cardiac remodelling after ischaemia [17,18].

3.1. MMP mRNA expression
To characterise the influence of I/R on MMPs the mRNA expression of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 was monitored (Table 1). In sham groups, mRNA of MMP-1 was expressed at the lowest level and that of MMP-2 was expressed at the highest level. MMP-1 mRNA was generally increased by I/R, but reached significance in only two groups. However, even in these groups, MMP-1 mRNA remained low compared to the other MMPs. The high MMP-2 mRNA expression was independent of LPS and I/R insults. LPS pre-treatment but not I/R affected the expression of MMP-3 mRNA, i.e. MMP-3 mRNA was increased three fold only in LPS pre-treated wild-type sham mice (Fig. 3A). MMP-8 mRNA expression was changed in the same way as MMP-3. MMP-9 mRNA expression was increased only by LPS pre-treatment in both wild-type groups (Fig. 3B). After I/R we did not observe differences in MMP-9 regulation.


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  		Table 1 mRNA expression of MMPs and TIMPs as well as MMP/TIMP ratios in wild-type (TLR4+) and TLR4-deficient mice (TLR4–)

 


Figure 3
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  		Fig. 3 mRNA expression (A-C) and calculated mRNA MMP/TIMP ratios (D-F) in wild-type (TLR4+) and TLR4-deficient mice (TLR4–). (A) MMP-3 mRNA expression. (B) MMP-9 mRNA expression. (C) TIMP-4 mRNA expression. (D) MMP-2/TIMP-2 ratio. (E) MMP-9/TIMP-3 ratio. (F) Total MMP/TIMP ratio. M±S.E.M., n=5 except for PBS sham groups (n=3), #p<0.05, p-values from Bonferroni's post-hoc test. Values of mRNA expressions are normalized to L32 and expressed as arbitrary units (AU). PBS=phosphate buffer solution (placebo), LPS=lipopolysaccharide (endotoxin).

 
3.2. TIMP mRNA expression
mRNA of all known TIMPs (TIMP-1 to TIMP-4) has been detected under control conditions (sham PBS) in hearts of both genotypes. TIMP-2 was expressed at the highest level, whereas the heart-specific TIMP-4 was lowest. LPS-treated sham wild-type mice showed an elevated TIMP-1 mRNA expression, which was not further increased by I/R. In all other groups I/R induced an increased TIMP-1 expression. The high basic TIMP-2 mRNA level was not influenced by any of the interventions. Overall TIMP-3 mRNA expression was elevated after I/R injury. TIMP-4 mRNA expression was approximately two-fold higher in LPS-stimulated wild-type hearts compared to PBS wild-types (Fig. 3C) and remained unchanged in TLR4-deficient hearts.

3.3. Stoichiometry MMP/TIMP
Since the stoichiometry between the gene expression of MMPs and TIMPs takes part in the regulation of the remodelling of the extracellular matrix, we calculated the ratios of MMPs to TIMPs [19-21]. Only the most interesting combinations were incorporated in Table 1 (Fig. 3D-F). In hearts from wild-type mice a general increase of MMP/TIMP ratios after LPS pre-treatment was observed depending mainly on MMP-2/TIMP-2, MMP-3/TIMP-1, and MMP-9/TIMP-3, whereas the LPS treatment did not induce changes in TLR4-deficient hearts. On the contrary, ischaemia alone led to a general reduction of MMP/TIMP ratios in both wild-type and TLR4-deficient hearts depending mainly on MMP-2/TIMP-1, MMP-3/TIMP-1, and MMP-9/TIMP-1.

3.4. MMP zymographic activity
Finally, we investigated whether the changes in MMP/TIMP expression correlated with the zymographic activity of the MMPs.

Gelatine zymography revealed myocardial MMP-2 and -9 activation. Although MMP-2 mRNA is expressed at a much higher level than MMP-9 mRNA, enzymatic activity of MMP-2 and -9 measured by zymography did not differ in sham animals (Fig. 4A). LPS stimulation did not induce enhanced activities in sham animals. I/R increased MMP-9 activity in the hearts of all groups (Fig. 4B). However, non-stimulated wild-type mice showed higher enzymatic activity than untreated TLR4-deficient mice. LPS pre-treatment prior to I/R elevated MMP-9 activity 2.5 fold only in TLR4-deficient mice (p<0.01; Fig. 4C).


Figure 4
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  		Fig. 4 Gelatine zymographic activity (A) after sham operation and (B) after ischaemia-reperfusion (n=3). 72kDa and 92kDa correspond to MMP-2 and MMP-9, respectively. The bands of molecular mass >92kDa are known to be induced in the typical zymography patterns of MMP-9 from neutrophils [36]. (C) Quantified proteolytic activity of MMP-9; *p<0.05 vs. TLR4-PBS I/R. Values are expressed as arbitrary units (AU). PBS=phosphate buffer solution (placebo), LPS=lipopolysaccharide (endotoxin).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In a closed-chest model of I/R, we investigated the influence of TLR4 on infarct area. Toll-like receptor 4 is relevant for myocardial function, and influences infarct size in a murine model of ischaemia and reperfusion [2,22]. TLR4 is the major LPS binding receptor, but there are also endogenous TLR4 ligands such as fibronectin, heat shock proteins, and extracellular matrix components, which are associated with tissue injury [4]. They may act as activating ligands for TLR4, thereby affecting infarct size.

Our data demonstrate a dependence of infarct area and TLR4 signalling. On one hand, preconditioning with LPS reduced infarct area; on the other, deficiency of TLR4 signalling also led to a decreased infarct area. In TLR4-deficient animals, preconditioning with LPS did not further reduce infarct area, which has been shown here for the first time. These findings seem to be contradictory, as LPS activates TLR4 signalling, whereas the lack of TLR4 signalling had the same effect. However, pre-treatment with LPS induces an inflammatory answer in the organism. A second challenge like ischaemia within a short time may cause a reduced inflammatory response leading to a smaller infarct area [11]. In case of a mutated TLR4 with deficient signalling capacities, ischaemia per se may be followed by a smaller inflammation 2. According to this hypothesis, both situations investigated may be explained by the same underlying processes.

AAR, indicating the region which is not sufficiently perfused during occlusion of the LAD, did not differ significantly among our study groups. Thus the detected differences in infarct area have to be ascribed to processes modulating the survival of the cardiac myocytes and not to differences in the primary oxygen deficiency. One modulating process of infarct size is the remodelling of the extracellular matrix. As MMP activities and the quantitative balance between MMPs and TIMPs regulate the myocardial matrix and are involved in pathological processes such as infarction, LV remodelling, and also endotoxaemia, we visualized alterations in MMP expression and activity in the present study [18].

Basic MMP and TIMP expression in the hearts of all investigated animals did not differ between wild-type and TLR4-deficient animals. After LPS application to wild-type mice, we observed a general increase in myocardial MMP/TIMP ratio, based on MMP-3, -8, and -9, and TIMP-1 and -4 mRNA expression, the latter being predominantly distributed in myocardium [23,24]. Elevated MMP-3, -8, and -9 mRNA levels after LPS treatment have been observed before [17,25]. We also found increased MMP-2/TIMP-2 mRNA ratios in LPS-treated wild-type mice. Here LPS pre-treatment reduced TIMP-2 expression, thereby potentially promoting MMP-2 activity. Few surveys have dealt with the function of myocardial MMP-2 during endotoxaemia. Lalu et al. found decreased protein activity and expression in the heart and plasma of rats which, however, returned to baseline within 24h [17]. This is in line with our zymographic results 23h post-LPS which did not reveal changes in MMP-2 activity compared to untreated mice although MMP-2/TIMP-2 mRNA expression remained elevated.

As expected LPS treatment did not change expression of any MMP or TIMP mRNA in TLR4-deficient hearts. Thus LPS induces changes in mRNA expression of MMPs and TIMPs only in wild-type hearts. However, these changes in mRNA expression were not accompanied by comparable differences in the zymographic activity of MMP-2 and MMP-9. This may be due to effects at different levels, as MMPs are regulated at the level of transcription, translation, secretion, activation of the zymogen to active protease, and through protease inhibitors.

An important question is whether I/R alone caused differences in MMP and/or TIMP expression as well as MMP activity in our model. In all groups, the overall MMP/TIMP ratio was down-regulated by I/R. Among the TIMPs TIMP-1 and TIMP-3 proved to be sensitive to I/R as both were up-regulated by I/R in both genotypes, thereby contributing to the lowering of the overall MMP/TIMP ratio. TIMP-3 is known to be an important regulator of inflammation and has been shown to be responsible for TNF signalling associated with tissue homeostasis and tissue response to injury [26]. I/R caused up-regulation of MMP-1 mRNA expression in our model; however, others have also demonstrated increased MMP-2, -3, -8 and -9 mRNA expression after I/R [27-30].

In contrast to the up-regulation of TIMP expression I/R induced an increase in MMP-9 activity, which was more robust in wild-type mice compared to TLR4-deficient animals. This difference may be caused by technical limitations of zymography, which are discussed in the next paragraph.

Of special interest was the combination of LPS pre-treatment and I/R. In wild-type hearts with and without I/R, total MMP/TIMP ratio was increased by LPS, whereas it remained unchanged in TLR4-deficient hearts. LPS prior to I/R elicited a more robust increase of MMP-9 zymographic activity in TLR4-deficient (C3H/HeJ) mice. In agreement with our results, an LPS-dependent up-regulation of MMP-9 activity has already been reported in isolated macrophages from C3H/HeJ mice [31]. In vivo LPS treatment induced expression of MMP-3, -9 and TIMP-1 in spleen but not in brain, liver or kidney of C3H/HeJ mice [21]. This surprising result cannot be explained easily; however, Ryan et al. postulated that not all cells from C3H/HeJ mice seem to be LPS-resistant; that is, alveolar macrophages but not peritoneal macrophages displayed TNF secretion after endotoxin stimulation [32].

However, the observed disparity between gene expression and gelatinolytic activity of MMP-2 and -9 still remains. This may be explained by secretion of stored MMP-9 from infiltrating immune cells, thus de novo synthesis requiring mRNA transcription may not be necessary [33]. In addition, MMP activity monitored by zymography may be higher than the activity in the intact tissue, as MMP-TIMP complexes dissociate during the procedure [21].

Infarct area and MMP/TIMP ratio were not correlated because the wild-type mice without LPS pre-treatment developed the most extensive infarcts, although their MMP/TIMP ratio was comparable to that of TLR4-deficient hearts with smaller infarct areas. In contrast, wild-type mice with LPS pre-treatment developed small infarcts accompanied by high MMP/TIMP ratios. This is in line with the idea that early MMP induction during reperfusion might have beneficial effects, such as removing matrix and necrotic myocytes, releasing growth factors, processing inflammatory mediators such as IL-1β, and increasing capillary permeability [34]. Lindsey et al. reported that neutrophil-derived MMP-9 is released in the myocardium within the first hour of reperfusion and Lalu et al. observed rapid MMP-9 elevations after LPS injection within 1h [17,34]. However, the function of MMP-9 in LPS response is unknown.

As mentioned above, overall MMP/TIMP ratio in TLR4 deficient animals with a small infarct size was comparable to that of wild-type animals with an extended infarct area. This result means that an elevated MMP/TIMP ratio is not necessary for a reduced infarct area. However, we cannot exclude that the high MMP/TIMP ratios in LPS pre-treated wild-type animals are helpful in reducing infarct area. Common among the groups with small infarct areas seems to be a reduced inflammatory response after the ischaemia, as a reduced inflammation has been shown after LPS pre-treatment and is also present in TLR4-deficient animals, independent of ischaemia [6,11].

The data presented provide important evidence for the relevance of TLR4 in myocardial infarction. We clearly demonstrate that TLR4 modulates infarct size. It is also evident, that the LPS pre-conditioning effect is mediated via TLR4. The TLR4 receptor pathway has already been identified as a novel therapeutic target in the clinical setting. The TLR-4 antagonist E5564 may be a therapeutic option suitable for further evaluation in ischaemia [35].


   Acknowledgements
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (BA 1725/2-1, KN 521/2-1), Deutsche Herzstiftung (F/10/02, F/16/03) and institutional grants of the University of Bonn (Bonfor). The authors thank P. Efferz and D. Böker for excellent technical assistance.


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

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