Browsing by Subject "lymphangiogenesis"

Vascular endothelial growth factor C (VEGF-C) is the most studied of the growth factors that control the growth of lymphatic vessels (lymphangiogenesis) and belongs to the same VEGF family as VEGF-A, which controls the growth of blood vessels. The growth of blood vessels and lymphatic vessels is centrally related to the pathophysiology of several cancers that form solid tumours and wet macular degeneration. Unlike VEGF-A, VEGF-C is not currently (2023) a target molecule of any approved drugs, but in clinical trials in the indications mentioned above, combining a VEGF C inhibitor with VEGF-A inhibitors has provided better results than VEGF-A inhibitor monotherapy. The study's objective was converting a phage display library containing single-chain antibody variable fragments (scFvs) screened against VEGF-C into full IgG class antibodies. The scFvs had shown a binding affinity towards the human, mouse, or both VEGF-C variants. The DNA sequences of the best binders of the library had previously been cloned into pLK06H plasmids. The scFvs comprise the variable region of the light and heavy chain (VL and VH) but do not contain the constant regions of the antibody (CL and CHx). Using single-chain antibody fragments as drugs is limited because, in most indications, better stability of whole antibodies, lower immunogenicity, and a longer half-life enabling less frequent dosing is desirable. In addition, the Fc part of whole antibodies often mediates the drug effect, such as complement activation, and whole antibodies are also used as research tools. Secondly, the study aimed to investigate how changing the antibody format affects the binding affinity. To produce whole antibodies, original DNA sequences of pVitro-trastuzumab-IgGk1 plasmid encoding VH and VL regions were replaced with new VH and VL sequences from the phage display library. Several recombinant DNA technology methods were utilised, but the most crucial method was the commercially available NEBuilder HiFi DNA Assembly, which enabled the seamless joining of several DNA fragments into a recombinant DNA molecule in a single-tube reaction. The cloning workflow proved uncertain, as only one constructed antibody production plasmid was sufficiently amplified and expressed in bacterial and mammalian cell cultures. Suboptimal overlapping of DNA fragments and insufficient competence of the bacterial strain used in the transformation were probable bottlenecks. Therefore, as such, the method is not suitable for use on a large scale to convert single-chain antibody fragments into whole antibodies. Also, binding tests were not performed. However, the work done and the antibody production plasmid built is a good basis for further optimisation of the method. In the optimisation, attention should be paid, especially to the quality of the DNA primers and the competence of the bacterial cell line. Also, alternative cloning methods, such as restriction enzymes and ligases, could be used instead of the NEBuilder HiFi DNA Assembly.

The lymphatic system is a network of vessels that permeate a substantial part of the whole body. It plays an essential role in fluid homeostasis by the drainage of interstitial fluid from the blood capillaries, after which the fluid, now called lymph, is transported through the vessel network and back to the blood circulation. The lymphatic system also plays an important role in the transportation of immune cells and in activation and maintenance of the immune system. Due to these crucial functions, there is a growing interest in exploiting the lymphatic system in the treatment of many immunological and inflammatory diseases. In many cases, an ideal treatment method would be to induce lymphatic growth (lymphangiogenesis) to boost immunological functions, facilitate resolution of inflammation and reduce the harm from lymphatic vascular abnormalities. However, there is a gap in knowledge in how to induce lymphangiogenesis in a controlled manner, with the major lymphangiogenic growth factor, vascular endothelial factor C (VEGF-C), tending to create disorganized lymphatic networks. The purpose of this thesis is to investigate factors influencing lymphangiogenesis, in an attempt to find ways to control it. Vaahtomeri research group has preliminary results showing a role of planar cell polarity (PCP) in control of dermal lymphatic vessel sprouting (the initial step for the formation of new lymphatic branches) and lymphatic network expansion. The focus of Vaahtomeri research group has been the core PCP protein Van Gogh-like protein 2 (VANGL2), which together with the other core PCP proteins is known to play an important role in the collective cell polarization and morphogenesis in many tissue types. The role of VANGL2 has previously been studied in the lymphatic system, and so far, VANGL2 has been implicated in both lymphatic valve morphogenesis and in flow-induced control of lymphatic endothelial cell (LEC) polarization. However, there still remains a gap in knowledge in what role VANGL2 plays in lymphangiogenesis and the lymphatic network as a whole. In this thesis, I investigated the role of VANGL2 in lymphangiogenesis, firstly by the use of an in vivo lymph node experiment, which offered a robust model to investigate the role of VANGL2 in the mature lymph node lymphatic network. In the experiment, I induced growth of the lymph node lymphatic network by means of an immunization reaction, and then I compared the lymphatic networks of Vangl2-deleted and control mice. Despite some minor differences between the Vangl2-deleted and control lymphatic networks, this experiment did not show a role for VANGL2 in the mature lymph node lymphatic network. Secondly, I investigated the potential mechanistic role of VANGL2 in control of dermal lymphatic vessel sprouting in growth conditions. This experiment showed a specific role for VANGL2 in sprouting of the lymphatic network, thus providing valuable research in understanding how lymphangiogenesis is regulated. Altogether, the results presented in this thesis work as a steppingstone for finding new treatments relating to the safe induction of lymphangiogenesis.