The Synthetic Antimicrobial Peptide 19-2.5 Interacts With Heparanase and Heparan Sulfate in Murine Sepsis In Vivo and in Human Sepsis Ex Vivo

Introduction

Heparanase is an endo-β-glucuronidase that cleaves highly potent heparan sulfate (HS) from its proteoglycan, thereby triggering the inflammatory response in [1]. Thus, new anti-infective agents that interact with heparanase may be promising tools for sepsis therapy. As a novel anti-infective agent, peptide 19-2.5 (pep2.5) belongs to the class of synthetic anti-lipopolysaccharide peptides, however its activity is not restricted to Gram-negative bacterial infection [2, 3].

Objectives

To evaluate the interaction of pep2.5 with heparanase in murine sepsis in vivo and in human sepsis ex vivo.

Methods

First, we used a model of murine cecal ligature and puncture (CLP) sepsis to study the impact of pep2.5 on heparanase in vivo in 12 NMRI mice. Mice were treated with pep2.5 or NaCl 0.9%. Plasma was sampled 24h after CLP. Second, we investigated whether pep2.5 interacts with heparanase in human plasma samples ex vivo. We added pep2.5 (20µg/ml) to plasma of 18 septic shock patients according to the ACCP/SCCM definitions and to plasma of 10 healthy volunteers. Heparanase-levels, HS-levels and heparanase activity were measured using ELISA (AMS Biotechnology, Oxon, United Kingdom). All data are given as mean ± standard deviation. a t-test with Holm-Šídák correction was used and a p-value < 0.05 was considered significant.

Results

Mice subjected to CLP without treatment displayed higher heparanase levels in plasma compared to mice treated with pep2.5 (p < 0.0001). Treatment with pep2.5 resulted in lower heparanase activity (p < 0.0001) and reduced HS-levels (p < 0.0001), compared to untreated animals (Figure 1).

Figure 1

(A) Heparanase level in CLP-mice. (B) Heparanase level in human.*p < 0.05, **p < 0.005, ***p < 0.001.

Septic shock patients (78% male) were 70 ± 15 years old and healthy volunteers (50% male) were 67 ± 19 years old. Plasma heparanase levels, heparanase activity and HS-levels were significantly higher in individuals with septic shock than in healthy individuals (all p < 0.0001). the ex vivo addition of pep2.5 did not impact heparanase levels, however heparanase activity and HS-levels were decreased by adding pep2.5 to plasma of septic shock patients (all p < 0.05, Figure 1).

Conclusions

The synthetic antimicrobial peptide 19-2.5 interacts with heparanase in human and murine sepsis and reduces levels of highly potent HS. Thus, peptide 19-2.5 may have the potential for further development as a new anti-infective drug in sepsis therapy.

Grant Acknowledgment

This work was supported by an intramural grant to Dr. Lukas Martin (START 693900).

Figure 2

(C) Heparanase activity in CLP-mice. (D) Heparanase activity in human. *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 3

(E) Heparan sulfate level in CLP-mice. (F) Heparan sulfate level in human. *p < 0.05, **p < 0.005, ***p < 0.001.