In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

PV-1: Leukocyte trafficking molecule and vascular marker

The distinction between lymphatic vessels and blood vessels is a crucial factor in many studies in immunology, vascular biology and cancer biology. They both share several characteristics and perform related, though different functions. They are equally important for the performance of the human immune system with the continuous recirculation of leukocytes from the tissues via lymphatics to the blood vessels and back into the tissue presenting the link between both systems. This study was undertaken to elucidate the differences in the gene expression between primary blood- and lymphatic endothelial cells as well as the two immortalized cell lines HMEC-1 (human microvascular endothelial cell line 1) and TIME (telomerase immortalized microvascular endothelial cell line). Furthermore, we wanted to investigate the mystery surrounding the identity of the antigen recognized by the prototype blood vascular marker PAL-E. In the last step we wanted to study whether the PAL-E antigen would be involved in the process of leukocyte migration from the bloodstream into the surrounding tissue. Our results clearly show that the gene expression in primary blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and the cell lines HMEC-1 and TIME is plastic. Comparison of a large set of BEC- and LEC datasets allowed us to assemble a catalog of new, stable BEC- or LEC specific markers, which we verified in independent experiments. Additionally, several lines of evidence demonstrated that PAL-E recognizes plasmalemma vesicle associated protein 1 (PV-1), which can form complexes with vimentin and neuropilin-1. Finally, numerous in vitro and in vivo experiments identify the first function of the protein PV-1 during leukocyte trafficking, where it acts as regulatory molecule.

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.