A biologically inspired lung-on-a-chip device for the study of protein-induced lung inflammation.

This study reports a biomimetic microsystem that reconstitutes the lung microenvironment for monitoring the role of eosinophil cationic protein (ECP) in lung inflammation. ECP induces the airway epithelial cell expression of CXCL-12, which in turn stimulates the migration of fibrocytes towards the epithelium. This two-layered microfluidic system provides a feasible platform for perfusion culture, and was used in this study to reveal that the CXCL12-CXCR4 axis mediates ECP induced fibrocyte extravasation in lung inflammation. This 'lung-on-a-chip' microdevice serves as a dynamic transwell system by introducing a flow that can reconstitute the blood vessel-tissue interface for in vitro assays, enhancing pre-clinical studies. We made an attempt to develop a new microfluidic model which could not only simulate the transwell for studying cell migration, but could also study the migration in the presence of a flow mimicking the physiological conditions in the body. As blood vessels are the integral part of our body, this model gives an opportunity to study more realistic in vitro models of organs where the blood vessel i.e. flow based migration is involved.

Functional characterization of ECP-heparin interaction: a novel molecular model.

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.

In silico prediction and in vitro characterization of multifunctional human RNase3.

Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, (34)RWRCK(38) (HBR1), (75)RSRFR(79) (HBR2), and (101)RPGRR(105) (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members.