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Ghezzo, Francesca (2008) Peptidi derivati da fattori endogeni ad attività pro- ed anti-angiogenica: sintesi, caratterizzazione conformazionale e saggi biologici in vitro e in vivo. [Tesi di dottorato]

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Abstract (inglese)

Angiogenesis, the formation of new capillaries, is among the key events in some physiological processes, like organ growth and development, wound healing and reproduction. However, an impairment of this process is also a hallmark of different pathologies, such as chronic inflammation, ischemic diseases, tumor growth and metastasis. Angiogenesis is a complex process involving extensive interplay among cells, soluble factors and extracellular matrix.
Physiologically, the body controls angiogenesis through a series of "on" and "off" regulatory switches, maintaining a perfect balance of positive and negative angiogenesis modulators. Normally, endothelium exists in a quiescent state ("turned off"); angiogenic stimuli, including hypoxia, inflammation, and mechanical factors, activate endothelial cells (ECs), by initiating the autocrine or paracrine production and release of growth factors or cytokine. Uncontrolled growth of vessels, due to a balance breakdown, may lead to angiogenesis-dependent diseases. So, the regulation of both angiogenesis stimulators and inhibitors, can represent a promising target for therapy and drug development.
Among endogenous modulators, the chemokine Stromal Derived-Factor 1 (SDF-1), and the small polypeptide Thymosin ß4 (Tß4), were found to play a crucial role in vascular biology, stimulating proliferation, migration and differentiation of ECs. The above mentioned events are also mediated by cell adhesion molecule. Compound containing adhesive sequences, such as RGD motif and heparin-binding domain, can be used either in tissue engineering as adhesion factors, or as anti-angiogenic agents, through a competition with extracellular matrix components for the binding to cell surface.
This work focused on i) design and solid phase synthesis of peptide sequences contained in SDF-1, Tß4 and ECM protein; ii) conformational studies and iii) evaluation in vitro and in vivo of their angiogenic effects on human umbilical vein endothelial cells (HUVECs) culture and chorioallantoic membrane (CAM).
Tß4 is a member of the ß-thymosins, molecules with hormone-like properties, and is the most abundant G-actin sequestering peptide in human blood platelets and white cells. Philp et al (2003), suggested that Tß4 exerts its pro-angiogenic activity in vitro e in vivo through the sequence motif 17LKKTET22, involved in actin binding. Starting from these considerations it was studied the behavior of three bio-active peptides representing the central part, the N and C terminus of Tß4, and characterized of containing the binding actin site. The structural analysis (NMR and Circular Dichroism), demonstrates that each peptide reproduces the conformation adopted in native protein and exerts both in vitro and in vivo pro-angiogenic effects, comparable to that induced by Fibroblast Growth Factor 2 (FGF-2). These results confirm the significant role of central binding actin site in the induction of neo-vessel formation and suggest in vitro a positively modulation by the N terminus part of Tß4.
Stromal Derived Factor-1 (SDF-1) is an ?-chemokine that binds to G-protein-coupled CXCR4 receptor. The SDF-1/CXCR-4 axis plays an important role in the regulation of stem/progenitor cell trafficking, in normal development of the nervous system, and in hematopoietic system. Moreover, SDF-1 favours the formation of metastasis by increasing tumor cell motility (Libura et al. 2002), and plays an important role in the development of the vascular system by exerting a strong pro-angiogenic effect both in vitro and in vivo. SDF-1 is constitutively expressed in stromal cells, ECs, dendritic cells, and other cells. It is present mainly in two splice forms alpha and beta, which have identical amino acid sequences except for the presence of four additional amino acids at the carboxy terminus of SDF-1ß. The biological significance of the existence of 2 splice forms of SDF-1 has remained unclear. SDF-1 consists of three structural regions: N-terminus, a central beta sheet core, and C-terminus. Structure-function analysis of chemokine identified the NH2-terminal amino acids as critical to CXCR4 binding and activation, and suggested that C-terminus of protein, is one of interaction sites with glycosaminoglycans (GAGs) and may exert a modulation to biological function of SDF-1. This research verified that the C-terminus could be responsible of the pro-angiogenic effects of SDF-1. The designed peptides correspond to the C-terminus sequences of the two natural isoforms of splicing, mutated analog peptides of SDF-1ßT C-terminal region, and fusion forms containing both N- and C-terminus. The collected data indicated that C-terminal region is functional implicated in pro-angiogenic SDF-1 activity, but only the wild-type sequence were able to induce both in vitro and in vivo an angiogenic response.
In 1971, Folkman proposed that tumour growth and metastasis are angiogenesis-dependent, and hence, blocking angiogenesis could be a strategy to arrest tumour growth. This possibility stimulated an intensive search for anti-angiogenic molecules. Cell adhesion to macromolecules of the extracellular matrix is needed to allow EC proliferation and migration inside the tumor. EC adhesion can be mediated by i) RGD-dipendent integrin receptors or ii) heparin sulphate proteoglycans (HSPGs), which recognized heparin-binding sites on extracellular matrix proteins. Since the loss of anchorage can induce pro-apoptotic signals leading to an inhibition of angiogenic process, RGD-peptides and vitronectin heparin-binding domain (HVP) were assayed as soluble agents to evaluate the effects on angiogenesis. Experimental evidences indicate that some designed peptides, i.e. (GRGDSP)4K and HVP, may represent promising anti-angiogenic agents whose effects seems to be related to an inhibition of both EC morphogenesis and FGF-2-induced angiogenic effects.

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Tipo di EPrint:Tesi di dottorato
Relatore:Grandi, Claudio
Dottorato (corsi e scuole):Ciclo 20 > Scuole per il 20simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:29 Gennaio 2008
Anno di Pubblicazione:29 Gennaio 2008
Parole chiave (italiano / inglese):Angiogenesi, Peptidi, Timosina-beta4, SDF-1, Motivi di Adesione, RGD
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/06 Anatomia comparata e citologia
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:611
Depositato il:25 Nov 2008
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