Modulation of hyaluronan metabolism and its role in endothelial functions.

My doctorate dissertation has been performed in the laboratory of biochemistry of the Department of Biochemical, Experimental and Clinical Science of the Faculty of Medicine and Surgery (University of Insubria, Varese, Italy), following the 20th program for PhD studies in “Biotechnology”. My thesis work is focused on the biochemical interactions between endothelial cells and the components of extracellular matrix. In particular, it studies the metabolism of hyaluronic acid (or hyaluronan), one of the most abundant glycosaminoclycan in extracellular matrix, and the modulation of endothelial functions induced by this molecule. All cellular functions are regulated by precise interactions between the cells themselves and the molecules in extracellular space. The hyaluronic acid is not only a scaffold for tissue, but it is acquiring the role of a regulator molecule in several cellular functions, such as differentiation, proliferation and migration. Endothelial cells line the inner surface of all blood and lymph vessels. Interactions between endothelial cells and their extracellular matrix are involved in neovascularisation process, not only for tissue development, but also in tumorigenesis and cancer diffusion. Endothelial cells form a barrier between blood and vessel wall for regulating exchanges of substances, blood clotting and vascular tone. Thus, they are involved during inflammatory processes taking contacts with the cells of innate immune system under influences of secreted cytokines and growth factors. For these reasons briefly described above, I have investigated “the modulation of hyaluronan metabolism and its role in endothelial functions”. For this purpose, the studies have been performed by modern detection methods and new techniques were developed. In the first part of the Results, a quantification analysis of HA amount in endothelial cells is reported by using PAGEFS (PolyAcrylammide Gel Elechtrophoresis of Fluorophore-labeled Saccharides), HPLC (High Performance Light Cromathography) techniques and immunofluorescence analysis. Having knowledge of hyaluronan amount in primary endothelial cell lines and in immortalized cell line of endothelial origin, I investigate the expression of genes related to hyaluronan metabolism, such as hyaluronan synthases, hyaluronidase and receptors. In the second part of the PhD work, a new non radioactive method is assed to investigate the activity of hyaluronan synthases, the only enzymes able to synthesise the hyaluronic acid, and to address the question of their activity during intracellular protein trafficking in eukaryotic cells. This new method is applied to evaluate the effects of different compounds on hyaluronan synthases and to find that these enzymes can be modulate by post-translational modifications. The last part of the research is focused of hyaluronan role in two vital pathways: angiogenesis and inflammation. For angiogenic process I evidence a predominant activity of one isoform of hyaluronan synthase (HAS3) that produces hyaluronan of low molecular weight. A biochemical pathway of the trasductional signal is partially identified as responsible for hyaluronan stimulatory effect on angiogenesis. It has also been considered the involvement in tumor development. During inflammatory process I investigate the mechanism and the peculiar structures that hyaluronan is able to build for leukocyte recruitment. A part of these results are published or submitted to international journals; these papers are reported in the Dissertations.