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Keywords:

  • anti-coagulant therapy;
  • cell signaling;
  • I/R injury;
  • inflammation;
  • mouse model myocardial I/R

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Summary. Background: Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. Objectives: To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. Methods: We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. Results: ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IκBα upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-κB (NF-κB) mediated cell signaling. Levels of nuclear activated NF-κB and proteins influenced by the NF-κB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). Conclusions: We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-κB mechanism.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Coronary heart disease remains the most important cause of death worldwide. While acute myocardial infarction is still associated with a near 10% death rate, chronic morbidity, particularly heart failure, develops in 25% of the survivors [1]. The salvage of ischemic myocardium is dependent on rapid reperfusion; however, the recovery in blood flow is accompanied by ischemia/reperfusion (I/R) injury [2,3]. Clinically, reperfusion strategies have been optimized over the past decades, but the paradox of I/R damage due to accelerated restoration of perfusion, cannot yet be specifically prevented in spite of promising pre-clinical studies [4].

Recent studies indicate that coagulation proteases affect I/R injury. One of these proteases is activated factor VII (FVIIa), which in complex with the cell membrane bound tissue factor (TF) not only triggers the generation of thrombin, but also affects intra-cellular signaling properties [5,6].

A study in a rabbit model of myocardial I/R injury showed that myocardial damage could be diminished with a specific inhibiting antibody against TF [7]. This inhibition of infarction was associated with reduced infiltration of neutrophils in the heart and with a trend towards lower levels of proinflammatory cytokines. Similar protection was obtained by infusion of a human recombinant active site inhibited activated FVII (ASIS) in a rabbit myocardial I/R model via inhibition of the procoagulant activity of TF [8]. These studies suggested a potential therapeutic intervention, but the spectrum of the cell protective effects remained largely unexplored. In addition, the duration of the protective effects was not addressed, which is relevant to assess the long-term impact of intervention in the acute phase of the I/R process.

To characterize the protective mechanisms of FVIIa-TF inhibition, we used recombinant mouse ASIS in an experimental mouse model of myocardial I/R. This study was the first to use species specific ASIS in a myocardial I/R model. We investigated the effects of mouse ASIS on early myocardial I/R injury, particularly related to anticoagulant, anti-inflammatory and anti-apoptotic mechanisms, and this in relation to cellular signaling.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Mouse myocardial ischemia/reperfusion model

All animal experiments were approved by the Animal Ethics Committee of Maastricht University. Male C57Black/6 mice (Charles River Laboratories, Sulzfeld, Germany), 8 weeks old and 22–28 g, were housed under normal conditions: temperature was kept constant at 20–24 °C and food and water were provided ad libitum. Myocardial I/R was induced according to the method of Jong et al. [9]. A brief description of the method and the specific time points of treatment are given in the online data supplement.

Tissue collection

A description is given online in the data supplements.

Determination of myocardial I/R injury: Evans Blue/TTC staining

The method is supplied online in the data supplements.

TF activity

A method description is supplied online in the data supplement.

Oligo GEArray® Mouse Signal Transduction Pathway Finder microarray

The microarray was used to analyze the expression of 113 genes representative of 18 signal transduction pathways. The technique is based on a side-by-side hybridization and was performed according to the manufacturer’s instructions (Tebu-Bio, Superarray, MD, USA). Data analysis was performed online using GEArray Expression Analysis Suite at http://www.sabiosciences.com/. Absolute and comparison analyses were conducted using the following settings: density with average, background with minimum value and normalization with interquartile.

Multiplex ligation dependent probe amplification (MLPA)

Coagulation, inflammation, cell signaling and apoptosis related gene expression levels were determined in isolated RNA of the mouse hearts (= 6 per group). Fifty nanograms per microlitre of RNA were analyzed according to the method described previously by Spek et al. [10]. Several genes involved in cellular signaling, inflammation and coagulation were analyzed: cell signaling, CD14, toll-like receptor (TLR)-2, TLR-4, IL-1 receptor-associated kinase (IRAK)-1, IRAK-3, IκBα, and protease activated receptor-1 (PAR-1); coagulation, TF, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator receptor (uPAR), tissue-type plasminogen activator receptor (t-PA), and protein C receptor; inflammation, interleukin (IL)-6, IL-4, IL-10, IL-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, hypoxia-inducible factor (HIF)-1α, ICAM-1, macrophage inflammatory protein (MIP)-1α, CxCl1, CCl3, and eNOS; apoptosis, Bcl-W, Bcl-Xl, Bcl-2, Mcl1, Bak, Bax, Bcl-Rambo, Bad, Bid, Bik, Bim, Map-1, Niap, AIF, Apaf-1, Smac/Diablo, Flip, Miap, Bruce, and p21. Only the genes that showed detectable expression levels are depicted in the results.

Activated NF-κB

Activated NF-κB levels (p50 and p105) were determined in the nuclear extract isolated according to Davis et al. [11]. Activated NF-κB levels were determined in nuclear extracts of 1 mg mL−1 protein content according to the manufacturer`s instructions (Colorimetric Enzyme Immunoassay for NF-κB; Oxford Biomedical Research, MI, USA).

IL-6, TNF-α and IL-1β antigen

IL-6, TNF-α and IL-1β antigen levels were determined in tissue homogenates containing 1 mg mL−1 protein using an ELISA reagent set. All analyses were performed according to the manufacturer’s manual (eBioscience, San Diego, CA, USA).

PAI-1 antigen

Total PAI-1 antigen levels were determined in tissue homogenates (1 mg mL−1 protein content) according to the manufacturer’s instructions (Innovative Research, Novi, MI, USA).

CD 45 staining

A method description is supplied online in the data supplements.

DNA laddering staining

Mouse heart paraffin sections were stained for DNA laddering using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit. Sections were stained according to the manufacturer’s manual (Chemicon, Billerica, MA, USA).

Statistical analyses

Data analysis was performed with Prism for Windows, version 5.00 (GraphPad Software Inc., San Diego, CA, USA). Values are mean ± SEM. Differences between groups were tested using a Mann–Whitney U-test and P values <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Determination of the optimal time point of administration

Mouse ASIS was administered at three different time points during I/R to determine the optimal administration time. Ischemia was induced for 60 min followed by a 2-h reperfusion period and ASIS was administered only during the ischemic phase (I), during the ischemic and reperfusion phase (I + R), or only during the reperfusion phase (R). Analysis of myocardial I/R injury by means of Evans Blue/TTC staining (focusing on LDH release) (Fig. 1A) and coagulation by means of TF activity (Fig. 1B), revealed that ASIS administration during both the ischemic and the reperfusion phase was the optimal time point of administration.

image

Figure 1.  (A) Area of infarction (AOI) as a percentage of the area at risk (AAR) after myocardial ischemia/reperfusion injury in three treatment groups. Active site inhibited factor VII (ASIS) was administered only during the ischemic phase (I), during the ischemic and reperfusion phase (I + R) or only during the reperfusion phase (R). Values are mean ± SEM, = 6. *Difference between ASIS-treated animals and their corresponding placebo-treated group (*I, **I + R, ***R), P < 0.05. #Difference between different ASIS groups (#ASIS I vs. ASIS R; ##ASIS I vs. ASIS I + R), P < 0.05. (B) TF activities in the left ventricles of hearts after ischemia/reperfusion injury. TF activity levels are depicted as percentages of the sham group, which is set at 100%. Values are mean ± SEM, = 6. *Difference between sham and placebo groups (*I, **I + R, ***R), P < 0.05. #Difference between ASIS treatment and the corresponding placebo (#I, ## I + R), P < 0.05. (C) Mouse Signal Transduction Pathway Finder Microarray. Two spots of the NF-κB pathway are highlighted. Spot 1, NF-κB-p50; Spot 2, IκBα. (D) The effect of ASIS administration on the expression of a number of genes involved in several pathways.

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Effects of mouse ASIS on cellular signaling pathways

To explore cell signaling effects affected by ASIS, a pathway finder microarray was employed. Different pathways were affected by ASIS as demonstrated by alterations in different mRNA expression levels after 2 h of reperfusion (only the pathways with a minimum of three genes detectable are mentioned; gene expression was defined as different compared to placebo when the expression values were more than 50% deviating): the NF-kB pathway (involved genes: Cxcl1, NF-κB, IκBα and TANK), the WNT signaling pathway (Jun, Myc and Pparγ), the stress pathway (Atf2, Fos and Hsp25) and the phospholipase C pathway (Egr1, Fos and Jun). As an example, two spots of the NF-kB pathway are highlighted in Fig. 1(C). RNA expression of NF-kB-p50 was increased threefold upon ASIS administration, whereas ASIS administration reduced the RNA levels of IκBα with only 32%. The variation in RNA expression levels of differentially expressed genes involved in several pathways, upon ASIS vs. placebo administration, is depicted in Fig. 1(D).

All further experiments are carried out with a 1-h ischemia period, followed by a 2-, 6- or 24-h reperfusion period. ASIS was administered during both the ischemic and the reperfusion period.

Effects of mouse ASIS on myocardial I/R injury

Myocardial I/R injury decreased after prolonged reperfusion times upon administration of placebo: the percentage of AOI/AAR decreased by 20% after 6 h of reperfusion and further by 43% after 24 h of reperfusion. Administration of ASIS decreased I/R injury at all reperfusion times compared with placebo treated animals. After 2 h reperfusion, ASIS decreased I/R injury by 40%, whereas the % AOI/AAR decreased by 64 and 44% after 6 and 24 h reperfusion upon ASIS administration (Fig. 2A). A representative picture of the Evans Blue/TTC stained hearts is given in Fig. 2(B).

image

Figure 2.  (A) Area of infarction (AOI) as a percentage of the area at risk (AAR) after myocardial ischemia/reperfusion injury at varying reperfusion times. All values are mean ± SEM, = 6. *Differences between different reperfusion times after placebo or ASIS administration (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), = 6, P < 0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 h R), = 6, P < 0.05. (B) A representative picture of the Evans Blue/TTC staining after 2 h of reperfusion. The normal tissue is stained dark blue, whereas the ischemic area is stained brick red. The non-viable infarct area did not stain and remained pale. NIA, non-infarcted area (dark blue); AAR, area at risk (brick red); AOI, area of infarction (pale).

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Effect of mouse ASIS on cellular signaling pathways with varying reperfusion times

Based on the results of the signal transduction pathway finder, the NF-κB pathway was analyzed by MLPA. In Fig. 3, the mRNA expression levels of four different genes involved in cell signaling are shown. The expression levels of NF-κB-related genes TLR-4 (Fig. 3A) and IκBα (Fig. 3C) were increased upon I/R with varying reperfusion times, whereas IRAK-1 (Fig. 3B) levels were not influenced. The expression levels of TLR-4 were decreased to sham baseline levels when ASIS was administered after 2, 6 and 24 h of reperfusion, while IRAK-1 expression levels decreased after 2 and 6 h of reperfusion. ASIS administration decreased IκBα levels after 2 and 6 h of reperfusion. PAR-1 expression in the placebo group temporally increased after 2 h of reperfusion compared with the sham group; ASIS administration lowered PAR-1 expression after 2 and 6 h of reperfusion (Fig. 3D). The expression levels of CD14 and IRAK-3 are shown in Fig. S1 in the online data supplement.

image

Figure 3.  mRNA expression levels of cellular signaling-related genes determined by Multiplex Ligation dependent Probe Amplification (MLPA) analysis. All values are mean ± SEM, = 6. *Differences between different reperfusion times after placebo or ASIS administration (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), = 6, P < 0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 h R), = 6, P < 0.05.

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Results of the expression levels of inflammation- and coagulation-related genes are summarized in Fig. 4. TF mRNA expression levels were not influenced by I/R but were decreased by ASIS administration after 2 and 6 h of reperfusion (Fig. 4A). Inflammation-related genes IL-6 (Fig. 4B) and IL-1β (Fig. 4C) showed increased expression levels after 2, 6 and 24 h of reperfusion compared with sham animals. ASIS administration lowered IL-6 levels but did not affect IL-1β expression. ICAM-1 levels increased after I/R, which was reversed upon ASIS administration (Fig. 4D). The expression levels of TFPI, PAI-1, uPAR, HIF-1α and eNOS are shown in Fig. S2 in the online data supplement.

image

Figure 4.  mRNA expression levels of inflammation- and coagulation-related genes determined by MLPA analysis. All values are mean ± SEM, = 6. *Differences between different reperfusion times after placebo or ASIS (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), = 6, P < 0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 R), = 6, P < 0.05.

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Effects of mouse ASIS on activated NF-κB and NF-κB regulated proteins

Regarding the strong indications from the MLPA data for involvement of the NF-κB pathway in the mode of action of ASIS, nuclear activated NF-κB and a number of NF-κB regulated proteins were measured.

Nuclear activated NF-κB levels decreased upon ASIS administration after 2 and 24 h of reperfusion (1.1 ± 1.1 pg mL−1 and 6.9 ± 1.8 pg mL−1, respectively) compared with placebo treatment (7.5 ± 2.2 pg mL−1 and 39.0 ± 9.4 pg mL−1, respectively, P < 0.05). ASIS administration upon 6 h of reperfusion showed a trend towards decreased activated NF-κB levels, but the results showed no statistically significant effect (Fig. 5A).

image

Figure 5.  Activated NF-κB, TF activity, PAI-1 and IL-6 antigen levels in the left ventricles of sham mice and animals after ischemia/reperfusion injury mice. (A) Nuclear activated NF-κB. (B) Il-6 antigen levels. (C) TF activity. (D) PAI-1 antigen levels. Values are mean ± SEM, = 6. *Differences between different reperfusion times after placebo or ASIS administration (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), = 6, P < 0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 h R), = 6, P < 0.05.

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IL-6 antigen levels were increased after 2 and 6 h of reperfusion (19.5 ± 6.7 and 9.0 ± 3.6 pg mL−1) compared with the sham animals (0.8 ± 0.3 pg mL−1) but remained equal after 24 h of reperfusion (3.0 ± 2.2 pg mL−1). IL-6 antigen levels were diminished by the administration of ASIS after a 2-h reperfusion period (4.5 ± 1.1 pg mL−1, P < 0.05) but remained equal after 6 and 24 h of reperfusion (9.9 ± 2.9 and 3.7 ± 2.0 pg mL−1, respectively) (Fig. 5B). TNF-α and IL-1β protein levels were not detectable.

TF activity levels increased after 2, 6 and 24 h of reperfusion (6.2 ± 0.2 pmol L−1, 9.1 ± 1.0 pmol L−1 and 11.1 ± 1.2 pmol L−1, respectively) compared with the sham animals (4.1 ± 0.6 pmol L−1), while ASIS administration decreased TF activity for 2, 6 and 24 h of reperfusion (1.0 ± 0.08 pmol L−1, 3.4 ± 0.6 pmol L−1 and 7.0 ± 1.0 pmol L−1, respectively, P < 0.05) compared with the placebo-treated animals (Fig. 5C).

PAI-antigen levels were increased in a time-dependent way after I/R (2 h R, 7.1 ± 2.4 ng mL−1; 6 h R, 33.7 ± 4.0 ng mL−1; 24 h R, 57.8 ± 2.0 ng mL−1) compared with sham animals (0.2 ± 0.05 ng mL−1, P < 0.05). Administration of ASIS diminished PAI-1 antigen levels after 24 h of reperfusion (38.4 ± 8.5 ng mL−1) but not after 2 and 6 h of reperfusion (4.1 ± 0.8 and 27.1 ± 3.4 ng mL−1, respectively) (Fig. 5D).

The influence of mouse ASIS on leukocyte infiltration

To determine the effect of ASIS on leukocyte infiltration, paraffin sections were stained for CD45 and the number of CD45-positive cells per cm2 was determined. A representative picture of placebo and ASIS treatment after 24 h of reperfusion is depicted in Fig. 6(A) (20× magnification). The number of CD45-positive cells increased after 6 (334.0 ± 26.9 cells cm−2) and 24 h (323.9 ± 36.9 cells cm−2) of reperfusion compared with a 2-h reperfusion period (32.0 ± 14.4 cells cm−2). Administration of ASIS attenuated the infiltration of CD45-positive cells after 6 and 24 h of reperfusion (164.1 ± 35.4 and 109 ± 23.2 cells cm−2, respectively), whereas the number of CD45+ cells was not altered by ASIS after 2 h of reperfusion (61.5 ± 5.9 cells cm−2) compared with the placebo (Fig. 6B).

image

Figure 6.  Representative example of CD 45 infiltration into the left ventricles of hearts after 24 h of reperfusion. (A) Left, placebo 24 h R; right, ASIS 24 h R. (B) Number of CD45-positive cells per cm2. Values are mean ± SEM, = 4. *Differences between different reperfusion times after placebo or ASIS administration (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), = 6, P < 0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 h R), = 6, P < 0.05.

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Influences of mouse ASIS on apoptosis

DNA laddering and MLPA analysis was performed to determine the possible effect of ASIS on apoptosis. ASIS neither influenced the number of cells that stained positive for DNA laddering, nor affected the size of area stained positive within the left ventricle (data not shown). MLPA analysis revealed no significant differences in Bcl-2-family-related gene expression levels upon ASIS administration. The expression levels of Bcl-W, Bcl-Xl, Bcl-2, Bak, Bax, Bcl-Rambo, Bad, Bik and Bid remained equal upon ASIS administration compared with placebo administration (data not shown). These data indicate no significant contribution of an anti-apoptotic effect of ASIS in limiting myocardial I/R injury.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Myocardial I/R is a complex disease process and the ultimate process of lethal reperfusion injury is of major importance for the absolute infarct size as it offsets the beneficial effects of reperfusion after myocardial ischemia through the induction of cardiomyocyte death. Several cellular processes contribute to lethal reperfusion injury and intervention in these processes has shown a reduction of up to 50% of myocardial infarct size within several animal models [1]. Previous experimental animal studies showed that intervention in the FVIIa/TF complex yielded smaller myocardial infarcts in rabbits. These data also suggested an anti-inflammatory effect of inhibition of the TF mechanism in myocardial I/R injury but the exact mechanisms were not investigated [7,8].

Our study provides several new insights. First, we used mouse ASIS in a mouse myocardial I/R model, excluding species-specific differences in TF/FVIIa binding, which were previously shown to be relevant [12]. Utilizing mouse-specific ASIS, we show that inhibition of the TF/FVIIa pathway provides a substantial reduction in myocardial I/R injury and that this effect was sustained for up to 24 h of reperfusion. Second, we show that intervention in the TF/FVIIa complex is associated with anti-inflammatory effects in the sense that there was a marked reduction in leukocyte influx, also shown in previous studies [7,8], as well as attenuation in the proinflammatory cytokine IL-6, and this at varying reperfusion times. In contrast, we do not show any overt effect on apoptosis as a possible protective mechanism. Third, we explored the pathways involved in these anti-inflammatory effects and show a number of changes in the NF-κB (mediated) genes and proteins upon administration of ASIS.

In this study, administration of the anticoagulant ASIS reduced myocardial I/R injury based on LDH release (Evans Blue/TTC staining), revealing the most profound effect when administered during ischemia and reperfusion, suggesting that ASIS diminishes lethal reperfusion injury already after a short reperfusion period. Remarkably, upon prolonged reperfusion time, I/R injury decreased both in placebo- and ASIS-treated mice, although this effect was significantly more pronounced in ASIS-treated animals, suggesting a sustained protective effect of FVIIa-TF inhibition on cardiomyocytes after I/R. As the percentage of AAR/whole heart remained equal over time (data not shown), technical issues regarding the model were excluded. The decrease of I/R injury within time observed in the placebo-treated animals is an unexpected finding. Previous studies suggest an equal infarct size or even an increase in infarct size after a prolonged reperfusion time rather than a decrease observed in this study. However, the exact time points used in this study were not previously investigated [13,14]. The observed apparent reduction in infarct size in all animals might be explained by the phenomenon of cell shrinkage, which is considered the onset of apoptotic cell death [15]. From this study, however, we did not obtain any convincing evidence for a reduction in apoptosis by ASIS, indicated by a lack of change in TUNEL staining and MLPA analysis.

Based on the previous notion of reduced inflammation [7,8] and the fact that the NF-κB pathway plays a significant role in I/R injury-related cell signaling in the cardiac myocyte, we further explored NF-kB-related gene function. Our results reveal a coherent association of cell signaling-related genes CD14, TLR-4 and IRAK-1 in the down-regulation of IκBα under the influence of ASIS administration. TLR-4, one of the regulators of NF-κB signaling, is best known to be activated by LPS [16] but can also be regulated by other proteins and requires CD14 and MD2. TLR-4 is expressed in cardiomyocytes and its expression is increased after myocardial I/R injury, most likely due to oxidative stress [17]. TLR-4 expression leads to the recruitment of IRAK-1, causing activation of inhibitor κB (IκB) kinases, which regulate NF-κB translocation to the nucleus [18,19]. NF-κB in turn activates the transcription of IκBα, regulating its own inactivation [20]. As is shown in human and animal studies, the process of myocardial I/R activates the phosphorylation of IκBα, resulting in NF-κB translocation and subsequent activation of inflammation [21,22]. Several factors involved in NF-κB signaling contribute to I/R injury. Deficiency in TLR-4 protects against myocardial I/R injury [23], as well as ischemic renal damage, showing a diminished inflammatory response [24]. Inhibition of IKKβ, responsible for the IκBα phosphorylation, yielded smaller myocardial infarcts and an improved cardiac function [25], while mice depleted in the p50 subunit of the NF-κB gene, also showed less myocardial I/R injury accompanied by reduced neutrophil infiltration [26]. Hence, the observed effects of ASIS on NF-kB-related pathways may indeed be compatible with known protective effects related to specific alterations in this mechanism.

Whether the down-regulation of TF is directly affecting these pathways, or the signaling pathways directly influence TF levels, still remains an important question. TF gene expression is known to be regulated by transcriptional factors, including NF-κB [27]. The TF/FVII complex is, however, also able to activate PAR-2, or can via thrombin formation activate PAR-1 [28]. A recent study has shown an important role for TF within the cardiac myocytes in heart hemostasis [29]. In this study, TF activity increased after I/R, showing increasing levels after a prolonged reperfusion time. The administration of ASIS decreased TF activity levels as well as TF mRNA levels within the myocardial tissue, possibly via cellular signaling mechanisms through the influence of ASIS on NF-κB signaling. A possible link between TF and the TLR-4 pathway could be explained by this PAR-2 signaling as the PAR-2 receptor, which is activated is by the TF/FVIIa complex, is possibly able to activate TLR-4 as is shown in an inflammation model [30]. However, research involving the interactions between those signaling pathways needs to be expanded. Recently, also a role for PAR-1, activated by thrombin, was determined in myocardial as well as renal I/R injury [31,32]. NF-κB regulation is also linked to PAR-1, as shown in a cancer cell line [33]. In our experiments PAR-1 showed an increased expression after 2 h of reperfusion, and this was attenuated by ASIS administration. Longer reperfusion times had no effect on PAR-1 expression, suggesting an early effect of PAR-1 on I/R injury. TLR-4 on the other hand was activated after 6 and 24 h of reperfusion and decreased by the administration of ASIS. Those data suggest a possible role for TLR-4 signaling in the later stages of the reperfusion process.

Having observed significant changes in the NF-kB gene complex, we studied activated NF-κB levels and several downstream proteins that may be involved in myocardial I/R injury. Activated NF-κB levels were decreased upon ASIS administration. IL-6, a typical downstream effector of the NF-κB pathway is an important regulator of inflammation and is known to be increased in patients with myocardial infarction, heralding a poor outcome [34,35]. Upon I/R injury the levels of the proinflammatory genes for IL-6, IL-1β and MIP-1α were increased compared with sham-operated animals. Importantly, IL-6 mRNA expression levels were decreased by ASIS administration after 2 h of reperfusion and the same pattern was seen in heart-specific IL-6 protein levels.

Both PAI-1 mRNA and protein levels were decreased by ASIS administration after a 24-h reperfusion period. However, the specific contribution of PAI-1 in myocardial I/R is controversial [36,37].

The beneficial effect of ASIS on myocardial infarction was associated with reduced inflammation, indicated by reduced neutrophil influx as well as reduced expression of proinflammatory genes and proteins in the left ventricle. Gene array analysis showed that the NF-κB pathway is involved in the ASIS-induced effects on the heart and it may be surmised that the observed reduction in inflammation is in part related to the effects on the NF-κB system, although this will require further experimental studies.

From a clinical perspective, the favorable effects of ASIS on I/R injury after myocardial infarction may ultimately pave the way for clinical studies with such specific anticoagulants in situations of critical ischemic organ damage.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Concept and design, analysis and interpretation of data, critical writing: Loubele STBG, Spek CA, van der Voort D, Spronk HMH, ten Cate H. Analysis and interpretation of data: Leenders P, van Oerle R, Aberson HL. Concept and design: Hamulyák K, Petersen LC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

We kindly acknowledge J. Cleutjens, Department of Pathology, for critical comments. This project was funded by the Netherlands Heart Foundation (grant no. 2003-B065).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Figure S1. RNA expression levels of cellular signaling-related genes determined by MLPA analysis. All values are mean ± SEM, n=6. *Differences between different reperfusion times after placebo or ASIS administration (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), n=6, P<0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 h R), n=6, P<0,05.

Figure S2. RNA expression levels of inflammation- and coagulation-related genes determined by MLPA analysis. All values are mean ± SEM, n=6. *Differences between different reperfusion times after placebo or ASIS (*2 h vs. 6 h, **6 h vs. 24 h, ***2 h vs. 24 h), n=6, P<0.05. #Difference between placebo and ASIS treatment (#2 h R, ##6 h R, ###24 h R), n=6, P<0,05.

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