Studies on the mechanism of action of an anti-edema peptide

Abstract

Pulmonary edema is a life-threatening pathological condition caused by excessive extravascular fluid accumulation in the lung tissue. In 11 different animal models the Tip peptide, a cyclic peptide derived from the lectin-like tip domain of the human tumor necrosis factor α (TNF-α), was shown to up-regulate the rate of lung liquid clearance (LLC), contributing to the resolution of pulmonary edema.<br />One aim of this thesis was to establish an in vitro activity assay that reflects the Tip peptide s anti-pulmonary edema action and is useful for studies on its mechanism of action. For this purpose, the three human lung epithelial cell lines A549, H441 and Calu-3 were characterized with respect to their suitability for in vitro studies on anti-edema drug candidates. The Calu-3 cell line met all requirements for in vitro analysis of transepithelial ion and fluid transports in regard to expression of ion channels and transporters involved in LLC (e.g. the epithelial sodium channel ENaC, the chloride channel CFTR and the Na,K-ATPase), expression of the water channel aquaporin-5, formation of tight and polarized monolayers, and the capability of transepithelial fluid transport (dome formation) within confluent monolayers. Therefore, Calu-3 cells were used to establish two complementary in vitro assays for pulmonary edema resorption:<br /><br />(1) Bioelectrical properties of polarized cell monolayers were studied by using the Transepithelial Electrical Resistance (TEER) technology ( TEER assay ).<br />(2) The ability of Calu-3 cells for spontaneous dome formation within confluent monolayers allowed the quantitative examination of transepithelial fluid transport ( dome assay ).<br /><br />TEER assay experiments proved that the Tip peptide influences transepithelial conductance of Calu-3 cell monolayers in a polarized and dose dependent manner. This activity requires sodium ions in the assay buffer. Dome assay experiments confirmed that dome formation by lung epithelial cells is a sodium dependent process and that it is induced by the Tip peptide. The effect of the Tip peptide in TEER and dome assays was (partly) inhibitable by the oligosaccharide N,N -diacetylchitobiose. However, a lectin-like binding of this sugar by the Tip peptide could not be detected. Further investigations revealed that the action of the Tip peptide on TEER and dome formation of Calu-3 cells was independent of β-adrenergic receptor activation and cAMP up-regulation. Effects onTEER and dome formation may partly be caused by acetate ions in the peptide preparation. Protein interaction studies led to the identification of nine potential receptor proteins for the Tip peptide, one of which is the transmembrane receptor tyrosine kinase ErbB2. Testing of 40 potential anti-pulmonary edema peptides in the TEER and dome assays revealed that<br /><br />(1) the activity of the Tip peptide was maintained with conservative amino acid exchanges within the TPEGAE core motif.<br />(2) circularization of active peptides enhanced their activity.<br />(3) trimerization of the TPEGAE core motif (or variants thereof) with flexible polyglycine linker sequences increased the activity.<br /><br />In conclusion, this thesis reveals that complementary dome assay and TEER assay experiments with Calu-3 cells are valuable in vitro test systems for the screening of anti-pulmonary edema compounds that directly act on the lung epithelium and stimulate active fluid resorption. It supports the hypothesis that the Tip peptide activates LLC by the direct or indirect activation of apical localized sodium channels of pulmonary epithelial cells and suggests that acetate ions and/or ErbB2 signaling may contribute to the Tip peptide s activity.