Acute administration of recombinant Angiopoietin-1 ameliorates multiple-organ dysfunction syndrome and improves survival in murine sepsis

Abstract

Introduction

Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis.

Methods

Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1 μg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8 h thereafter. Sham-operated animals served as time-matched controls.

Results

Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model.

Conclusions

In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept.

Introduction

Septic multiple-organ dysfunction syndrome (MODS) is caused by widespread endothelial-cell activation and subsequent vascular barrier breakdown due to exaggerated systemic inflammation in response to a pathogen. It is characterized by an increased expression of pro-inflammatory cytokines, systemic capillary leakage with tissue edema, recruitment and transmigration of leukocytes, and vasodilation refractory to vasopressors [1], [2]. MODS is the leading cause of death in non-coronary intensive care units and mortality estimates for sepsis accompanied by shock and MODS range as high as 85% [2], [3]. Despite advances in understanding of the pathophysiological alterations the mainstay of treatment remains nonspecific supportive care [4]. In particular, specific anti-mediator strategies proved essentially ineffective, probably due to the redundancy, heterogeneity and complexity of the hosts´ (disproportionate) immune response [2]. Thus, an important goal in pre-clinical research is to identify and validate novel therapeutic approaches to prevent sepsis-associated syndromes such as acute lung injury, acute respiratory distress syndrome (ALI/ARDS) and acute kidney injury (AKI). Recently, the endothelial-specific Angiopoietin-Tie2 ligand–receptor system has been recognized as a major signalling pathway that controls vascular inflammation and permeability, key features in the pathogenesis of ALI/ARDS and AKI, in a non-redundant manner.

Angiopoietins are angiogenic factors essential for vascular development, maturation, and inflammation [5], [6], [7]. As circulating or matrix-bound molecules, Angiopoietin-1 (Ang-1) and its context-dependent antagonist Angiopoietin-2 (Ang-2) bind to the extracellular domain of the tyrosine kinase receptor Tie2 that is predominantly expressed on endothelial cells [8], [9]. Produced by vascular smooth-muscle cells and precursor pericytes, constitutive Ang-1 expression and low-level Tie2 phosphorylation probably represent a control pathway that maintains vessel integrity, suppresses inflammatory gene expression, and prevents transmigration of leukocytes [10], [11]. The importance and non-redundancy of Ang-1/Tie2 signalling is illustrated by either Ang-1^−/− and Tie2^−/− knockout mice which die in utero owing to severe vascular remodelling defects causing perturbed vascular integrity [6], [5]. Ang-2 is expressed in endothelial cells, where it is stored in granules, the so-called Weibel–Palade bodies [12]. The release of Ang-2 upon activation of the endothelium with for instance thrombin, histamine, or hypoxia disrupts the constitutive Ang-1/Tie2 signalling by preventing Ang-1 from binding to the receptor [9]. Consequently, loss of Tie2 signalling facilitates endothelial inflammation, ultimately leading to capillary leakage and endothelial barrier breakdown [13], [14].

In human endotoxemia and sepsis, circulating Ang-1 levels remain unchanged, or even decrease, whereas Ang-2 is rapidly released by the activated endothelium [15], [16], [17]. Hence, the balance between circulating Ang-1 and Ang-2 is shifted in favor of the more dynamic player Ang-2. Elevated Ang-2 levels in plasma from critically ill patients seem to correlate with the extent of pulmonary vascular leak in ALI/ARDS [18], the severity of AKI [19], and independently predict outcome [13], [16], [18], [19], [20], [21], [22]. In mice, injection of recombinant Ang-2 alone is sufficient to provoke pulmonary vascular leak and congestion [13].

Consistent with the opposing roles of Ang-1 and Ang-2 on the non-redundant Tie2 receptor pathway, mice receiving adenoviral constructs encoding either native or engineered Ang-1 (AdAng-1) are largely protected from vascular barrier breakdown and show improved survival during endotoxemia [23], [24], [25], [26]. Thus, the application of Ang-1 may have potential as an endothelium-targeted therapeutic agent in patients with septic shock and MODS [27], [28], [29]. However, as human gene therapy (with AdAng-1) is not yet feasible, we particularly wanted to test if intravenously administered recombinant human Ang-1 (rhAng-1) is protective in a murine cecal ligation and puncture (CLP) model of sepsis.