Angiogenesis modulating activity of nod factor-derived compounds and histidine-rich glycoprotein

Abstract

Angiogenesis, a complex and highly regulated process, is commonly referred to as the development of new blood vessels from pre-existing vessels. Either excessive or insufficient angiogenesis has been reported to be associated with a wide range of diseases. Therefore, targeting angiogenesis has enormous therapeutic potential, which has led to a plethora of studies on new pro- and anti-angiogenic agents. Previous studies have shown that certain Nod factor-derived compounds, lipo-chitooligosaccharides that are produced by Rhizobia and are involved in the establishment of a symbiotic relationship between Rhizobia and legumes, and the mammalian plasma protein histidine-rich glycoprotein (HRG), play an important role in modulating angiogenesis, either by enhancing or inhibiting the process. Thus, the aim of this study was to dissect the pro- or anti-angiogenic activity of these molecules using several in vitro angiogenesis assays, which portray multiple steps involved in this complex and highly regulated process including EC growth, proliferation, migration, tube formation and adhesion to ECM components such as fibronectin and vitronectin, as well as providing hints as to mechanisms of action underpinning their activity. The studies outlined in Chapter 3 have confirmed that some Nod factor-derived compounds can exhibit either pro- or anti-angiogenic activity, a finding evident from several in vitro angiogenesis assays. The most potent pro-angiogenic effects were seen with compounds T and U, Nod factors derivatives with a chitin pentamer backbone as well as compound 9, the deacetylated chitin disaccharide, GlcN-GlcNAc, derived from Nod factors. In contrast, structural variants of disaccharide 9, namely compounds 9a and 9c, that carry N-linked alkyl and alkyl-benzamide substitutions at the non-reducing terminus, showed anti-angiogenic activity. The mechanism of action of the anti-angiogenic compounds appears to be due to inhibition of integrin-mediated adhesion to ECM components. However less conclusive results were obtained to explain the mechanism of action of the pro-angiogenic compounds. Preliminary studies outlined in Chapter 4 investigated the effect of plasmin cleavage of HRG on the ability of the molecule to modulate angiogenesis based on several in vitro angiogenesis assays. The key finding was that DTT-reduced plasmin-cleaved HRG modestly but significantly enhanced rat aorta vessel outgrowths. In addition, plasmin-cleaved HRG slightly enhanced 3T3 fibroblast proliferation and all HRG preparations enhanced HUVEC tube formation. In contrast, no inhibitory activity was shown by intact HRG, plasmin-cleaved HRG and DTT-reduced plasmin-cleaved HRG in any of the in vitro angiogenesis assays used in this study. Collectively the results suggest that plasmin cleavage of HRG may predominantly release pro-angiogenic fragments from the molecule. In conclusion, the studies presented in this thesis have explored the potential of Nod factor-derived compounds as well as plasmin-cleaved HRG to modulate angiogenesis. As novel structures with probably novels mechanism of action, the anti- and pro-angiogenic compounds derived from the Nod factors may represent a new class of angiogenesis modifying drugs. Furthermore, the pro-angiogenic activity shown by HRG following plasmin cleavage has given some insight into the importance of plasmin proteolysis in regulating the ability of HRG to modulate angiogenesis. -- provided by Candidate.