Active site–inactivated factor VIIa inhibits nuclear factor kappa B activation in intestinal ischemia and reperfusion

Abstract

Background

Intestinal ischemia and reperfusion (I/R) injury is a pivotal mechanism in critical illness and in the development of multiple organ dysfunction syndrome, in which the nuclear factor kappa B (NF-κB) activation plays a central role. Intestinal I/R injury initiates the extrinsic tissue factor or factor VIIa–dependent pathway of coagulation, also of importance in multiple organ dysfunction syndrome. Our aim was to analyze NF-κB activation in I/R injury in the rat intestine and in two main “shock” organs, that is, the liver and lungs. Pretreatment with active site–inactivated factor VII (FVIIai), an inhibitor of the extrinsic pathway, was evaluated.

Materials and methods

NF-κB activation was analyzed using enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assay (EMSA) studies of nuclear extracts from the intestine, liver, and lungs in rats subjected to intestinal I/R injury. FVIIai was given 90 min before the induction of intestinal ischemia.

Results

I/R induced NF-κB p65 activation in all three organs, especially in the liver. Pretreatment with FVIIai counteracted NF-κB activation in all three tissues studied. A commercially available ELISA for (human) NF-κB p65 and EMSA gave parallel results.

Conclusions

I/R injury in the rat intestine induces a pronounced activation of NF-κB p50 or p65 in the small intestine and in the liver and lungs. The NF-κB activation is especially pronounced in the liver and plays a central role in the regulation of transcription of cytokines, adhesion molecules, and chemokines. ELISA for (human) NF-κB p65 and “gold standard” EMSA gave parallel results. Pretreatment with FVIIai completely counteracted NF-κB activation in the intestine and liver, although not in the lungs.

Introduction

Intestinal ischemia and reperfusion (I/R) injury is a pivotal mechanism in several pathophysiological conditions such as major trauma, acute pancreatitis, sepsis, bleeding, and burn injuries. Under these circumstances, a circulatory collapse and a redistribution of microcirculation frequently precede the I/R injury, leading to a local and a systemic inflammatory response. The ultimate result may be single or even multiple organ dysfunction syndrome (MODS), often associated with considerable morbidity and mortality [1], [2], [3], [4]. Also, after surgery of abdominal aortic aneurysm or small bowel transplantation and cardiopulmonary bypass, intestinal I/R injury may represent a significant clinical problem. Intestinal I/R causes gut dysfunction characterized by histologic evidence of mucosal injury, increased intestinal epithelial permeability, and impaired motility [1]. The pathophysiological mechanisms of I/R are, however, still not fully characterized although several mediators of potential importance have been suggested [1], [2], [3]. A variety of experimental treatment interventions have shown effects in experimental models of I/R, regarding both a decrease in inflammatory response and a decrease in mortality [1], [4], [5]. Corresponding clinical findings are lacking.

The expression of inflammatory mediator genes of importance in I/R injury and in many other critical illness diseases is controlled by nuclear factor kappa B (NF-κB). In particular, NF-κB plays a central role in regulating the transcription of cytokines, adhesion molecules, chemokines and other mediators involved in the acute respiratory distress syndrome, sepsis, and MODS [6], [7]. The NF-κB activation has been shown to occur locally in the intestine in experimental I/R injury [6], [8], [9], [10], although the response in systemic organs is scarce. This lack of information may be at least partly because of the fact that previous studies have used the time-consuming and difficult method of electrophoretic mobility shift assay (EMSA) [11]. Today, a commercially available enzyme-linked immunosorbent assay (ELISA) for NF-κB exists for human p65 and p50, based on the method developed by Renard et al. [12]. The NF-κB p65 ELISA detects human, rat, and mouse p65, whereas the NF-κB p50 ELISA detects only human p50. Studies have been made using the NF-κB p65 ELISA in different cell cultures, but there is, to our knowledge, only two published studies using ELISA on whole tissues in vivo [13], [14]. There is, however, no comparison made between the two methods in these studies.

A prominent early feature of intestinal I/R injury is the tissue destruction and destruction of vascular vessel walls. After vessel wall injury, the tissue factor (TF) is exposed and forms complexes with activated factor VIIa (FVIIa) in circulating blood, initiating the hemostatic coagulation cascade via the extrinsic pathway, in which TF and FVIIa are central initial mediators [15]. TF is also rapidly induced on blood mononuclear cells by endotoxin and tumor necrosis factor-alpha [15], released during I/R. Activation of the (extrinsic) TF or FVIIa-dependent pathway of coagulation is a pivotal mechanism in disseminated intravascular coagulation [16], central in the development of MODS. Furthermore, more recent data have renewed the interest for various cascade system proteinases as being central in critical illness, suggesting signaling between the various systems such as inflammation, coagulation, fibrinolysis, and the immune system through proteinase-activated receptors (PARs) [17], [18]. We have previously shown several pronounced serine protease–antiprotease interactions during I/R injury in the rat [1]. An effective inhibitor of the early events of the extrinsic pathway is active site–inactivated factor VII (FVIIai), and experimental studies have shown that pretreatment with FVIIai attenuates the inflammatory response in I/R [19], acute pancreatitis in rats [13], [20], and endotoxemia in baboons [21], [22].

The aim of the present study was to analyze the effect of pretreatment with FVIIai on possible NF-κB activation during initial I/R injury in the rat. The effect in the intestine and in two major remote organs (liver and lungs) was evaluated. Furthermore, the NF-κB activation was studied using both a commercially available ELISA and the “gold standard” EMSA analysis, for studying NF-κB activation, thereby comparing methods.