Targeting of Formyl Peptide Receptor 2 for in vivo imaging of acute vascular inflammation

© The author(s). Inflammatory conditions are associated with a variety of diseases and can significantly contribute to their pathophysiology. Neutrophils are recognised as key players in driving vascular inflammation and promoting inflammation resolution. As a result, neutrophils, and specifically their surface formyl peptide receptors (FPRs), are attractive targets for non-invasive visualization of inflammatory disease states and studying mechanistic details of the process. Methods: A small-molecule Formyl Peptide Receptor 2 (FPR2/ALX)-targeted compound was combined with two rhodamine-derived fluorescent tags to form firstly, a targeted probe (Rho-pip-C1) and secondly a targeted, pH-responsive probe (Rho-NH-C1) for in vivo applications. We tested internalization, toxicity and functional interactions with neutrophils in vitro for both compounds, as well as the fluorescence switching response of Rho-NH-C1 to neutrophil activation. Finally, in vivo imaging (fluorescent intravital microscopy [IVM]) and therapeutic efficacy studies were performed in an inflammatory mouse model. Results: In vitro studies showed that the compounds bound to human neutrophils via FPR2/ALX without causing internalisation at relevant concentrations. Additionally, the compounds did not cause toxicity or affect neutrophil functional responses (e.g. chemotaxis or transmigration). In vivo studies using IVM showed Rho-pip-C1 bound to activated neutrophils in a model of vascular inflammation. The pH-sensitive (“switchable”) version termed Rho-NH-C1 validated these findings, showing fluorescent activity only in inflammatory conditions. Conclusions: These results indicate a viable design of fluorescent probes that have the ability to detect inflammatory events by targeting activated neutrophils.British Pharmacological Society; Wilkinson Trust; EPSRC; German Research Foundation

VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

Blood vessel tubulogenesis requires the formation of stable cell-to-cell contacts and the establishment of apicobasal polarity of vascular endothelial cells. Cell polarity is regulated by highly conserved cell polarity protein complexes such as the Par3-aPKC-Par6 complex and the CRB3-Pals1-PATJ complex, which are expressed by many different cell types and regulate various aspects of cell polarity. Here we describe a functional interaction of VE-cadherin with the cell polarity protein Pals1. Pals1 directly interacts with VE-cadherin through a membrane-proximal motif in the cytoplasmic domain of VE-cadherin. VE-cadherin clusters Pals1 at cell-cell junctions. Mutating the Pals1-binding motif in VE-cadherin abrogates the ability of VE-cadherin to regulate apicobasal polarity and vascular lumen formation. In a similar way, deletion of the Par3-binding motif at the C-terminus of VE-cadherin impairs apicobasal polarity and vascular lumen formation. Our findings indicate that the biological activity of VE-cadherin in regulating endothelial polarity and vascular lumen formation is mediated through its interaction with the two cell polarity proteins Pals1 and Par3