Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF).

Vascular endothelial growth factor (VEGF) was originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. Some family members, VEGF C and D, are specifically involved in lymphangiogenesis. It now appears that VEGF also has autocrine functions acting as a survival factor for tumour cells protecting them from stresses such as hypoxia, chemotherapy and radiotherapy. The mechanisms of action of VEGF are still being investigated with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins which were not previously associated with angiogenesis. VEGF plays an important role in embryonic development and angiogenesis during wound healing and menstrual cycle in the healthy adult. VEGF is also important in a number of both malignant and non-malignant pathologies. As it plays a limited role in normal human physiology, VEGF is an attractive therapeutic target in diseases where VEGF plays a key role. It was originally thought that in pathological conditions such as cancer, VEGF functioned solely as an angiogenic factor, stimulating new vessel formation and increasing vascular permeability. It has since emerged it plays a multifunctional role where it can also have autocrine pro-survival effects and contribute to tumour cell chemoresistance. In this review we discuss the established role of VEGF in angiogenesis and the underlying mechanisms. We discuss its role as a survival factor and mechanisms whereby angiogenesis inhibition improves efficacy of chemotherapy regimes. Finally, we discuss the therapeutic implications of targeting angiogenesis and VEGF receptors, particularly in cancer therapy

Induction de l'angiogenèse par le VEGF : rôle des récepteurs Flk-1 et Flt-1

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Pre-eclampsia : The role of soluble VEGF receptor-1 and related anti-angiogenic factors beyond

Pre-eclampsia is a pregnancy complication that affects about 5% of all pregnancies. It is known to be associated with alterations in angiogenesis -related factors, such as vascular endothelial growth factor (VEGF). An excess of antiangiogenic substances, especially the soluble receptor-1 of VEGF (sVEGFR-1), has been observed in maternal circulation after the onset of the disease, probably reflecting their increased placental production. Smoking reduces circulating concentrations of sVEGFR-1 in non-pregnant women, and in pregnant women it reduces the risk of pre-eclampsia. Soluble VEGFR-1 acts as a natural antagonist of VEGF and placental growth factor (PlGF) in human circulation, holding a promise for potential therapeutic use. In fact, it has been used as a model to generate a fusion protein, VEGF Trap , which has been found effective in anti-angiogenic treatment of certain tumors and ocular diseases. In the present study, we evaluated the potential use of maternal serum sVEGFR-1, Angiopoietin-2 (Ang-2) and endostatin, three central anti-angiogenic markers, in early prediction of subsequent pre-eclampsia. We also studied whether smoking affects circulating sVEGFR-1 concentrations in pregnant women or their first trimester placental secretion and expression in vitro. Last, in order to allow future discussion on the potential therapy based on sVEGFR-1, we determined the biological half-life of endogenous sVEGFR-1 in human circulation, and measured the concomitant changes in free VEGF concentrations. Blood or placental samples were collected from a total of 268 pregnant women between the years 2001 2007 in Helsinki University Central Hospital for the purposes above. The biomarkers were measured using commercially available enzyme-linked immunosorbent assays (ELISA). For the analyses of sVEGFR-1, Ang-2 and endostatin, a total of 3 240 pregnant women in the Helsinki area were admitted to blood sample collection during two routine ultrasoundscreening visits at 13.7 ± 0.5 (mean ± SD) and 19.2 ± 0.6 weeks of gestation. Of them, 49 women later developing pre-eclampsia were included in the study. Their disease was further classified as mild in 29 and severe in 20 patients. Isolated early-onset intrauterine growth retardation (IUGR) was diagnosed in 16 women with otherwise normal medical histories and uncomplicated pregnancies. Fifty-nine women remaining normotensive, non-proteinuric and finally giving birth to normal-weight infants were picked to serve as the control population of the study. Maternal serum concentrations of Ang-2, endostatin and sVEGFR-1, were increased already at 16 20 weeks of pregnancy, about 13 weeks before the clinical manifestation of preeclampsia. In addition, these biomarkers could be used to identify women at risk with a moderate precision. However, larger patient series are needed to determine whether these markers could be applied for clinical use to predict preeclampsia. Intrauterine growth retardation (IUGR), especially if noted at early stages of pregnancy and not secondary to any other pregnancy complication, has been suggested to be a form of preeclampsia compromising only the placental sufficiency and the fetus, but not affecting the maternal endothelium. In fact, IUGR and preeclampsia have been proposed to share a common vascular etiology in which factors regulating early placental angiogenesis are likely to play a central role. Thus, these factors have been suggested to be involved in the pathogenesis of IUGR. However, circulating sVEGFR-1, Ang-2 and endostatin concentrations were unaffected by subsequent IUGR at early second trimester. Furthermore, smoking was not associated with alterations in maternal circulating sVEGFR-1 or its placental production. The elimination of endogenous sVEGFR-1 after pregnancy was calculated from serial samples of eight pregnant women undergoing elective Caesarean section. As typical for proteins in human compartments, the elimination of sVEGFR-1 was biphasic, containing a rapid halflife of 3.4 h and a slow one of 29 h. The decline in sVEGFR-1 concentrations after mid-trimester legal termination of pregnancy was accompanied with a simultaneous increase in the serum levels of free VEGF so that within a few days after pregnancy VEGF dominated in the maternal circulation. Our study provides novel information on the kinetics of endogenous sVEGFR-1, which serves as a potential tool in the development of new strategies against diseases associated with angiogenic imbalance and alterations in VEGF signaling.Tässä väitöskirjatyössäni selvitimme Helsingin Naistenklinikalla vuosina 2001 2007 otetuista veri- ja istukkanäytteistä raskausmyrkytykselle ominaista istukan poikkeavaa verisuonikasvua ja verisuonen sisäpinnan, endoteelin, toimintahäiriötä. Tutkimusta varten etsimme varhais- ja keskiraskaudessa sikiön mahdollisten kehityshäiriöiden havaitsemiseksi järjestettävien ultraääniseulontatutkimusten yhteydessä kerätystä laajasta verinäytepankista sekä raskausmyrkytystä että yleisesti tunnetuista syistä riippumatonta ja varhaisessa vaiheessa raskautta ilmenevää sikiön kasvunhidastumaa poteneet naiset noin kahden vuoden aikaväliltä. Valitsimme löytyneelle aineistolle myös verrokkipotilaat. Tarkastelimme verisuonten uudiskasvua estävien sekä endoteelin toimintaa häiritsevien merkkiaineiksi tutkimuksen valitsemiemme tekijöiden pitoisuusvaihtelua raskausmyrkytyksen ennustajana ensimmäisen raskauskolmanneksen lopulla ja toisen kolmanneksen alussa otetuissa verinäytteissä. Lisäksi selvitimme, voisiko näitä merkkiaineita käyttää myöhemmin puhkeavan raskausmyrkytyksen ennustamiseen. Raskausmyrkytykseen sairastuu noin 5 % odottavista äideistä. Siihen on todettu liittyvän seikkoja, jotka muistuttavat metabolista oireyhtymää, tärkeintä sydän- ja verisuonisairauksille altistavaa oirekokonaisuutta. Raskausmyrkytyksen sairastaneilla naisilla onkin kaksinkertainen riski kuolla metabolisen oireyhtymän tunnusomaisiin sairauksiin, kuten sepelvaltimo- ja sokeritautiin, myöhemmin elämässään. Ennaltaehkäisevää tai parantavaa hoitoa ei raskausmyrkytykselle ole, ja sen syy on edelleen avoin. Vain istukan poistuminen johtaa lopulta oireiden helpottamiseen. Raskausmyrkytykseen sairastuneella istukan heikentyneeseen toimintaan liittyy kohdun verisuonten tavanomaista huonompi kehitys jo alkuraskaudessa. Lisäksi äidin endoteelisolujen toiminta myös muualla elimistössä on häiriintynyt. Täten väitöstutkimukseni kohdentuikin keskeisimpään verisuonten kasvua ja toimintaa säätelevistä tekijöistä, verisuonten endoteelikasvutekijään (VEGF), ja sen liukoiseen reseptoriin (liukoinen VEGFR-1), joka sitoo veressä kiertävää VEGF:ää. Verisuonten poikkeavan uudiskasvun yhteydessä on näiden tekijöiden tuotanto toisiinsa nähden epätasapainossa. Raskausmyrkytyksessä istukan erittämän liukoisen VEGFR-1:n tuotanto on havaittu kiihtyneeksi, ja sen pitoisuudet sekä lapsivedessä että äidin veressä ovat vastaavasti merkittävästi kohonneet terveisiin äiteihin verrattuna. Yhtenä väitöskirjatutkimukseni tärkeimmistä tavoitteista oli selvittää biokemiallisia ennustetekijöitä raskausmyrkytyksen kehittymiselle. Tällaisen työkalun löytyminen antaisi mahdollisuuden tunnistaa ne naiset, joita raskausmyrkytys jatkossa uhkaa. Tutkimuksen perusteella totesimmekin, että kohonnut liukoisen VEGFR-1:n pitoisuus äidin verenkierrossa ennustaa myöhempää raskausmyrkytystä jo toisen raskauskolmanneksen alussa, raskausviikoilla 16-20. Se on täten varteenotettava ehdokas etsittäessä merkkiainetta, jolla raskausmyrkytystä voitaisiin tehokkaasti ennustaa. Alkuraskauden verinäytteistä määritettiin myös muita endoteelisolujen toimintaan epäsuotuisasti vaikuttavia tekijöitä, kuten endostatiinin ja angiopoietiini-2:n pitoisuuksia. Raskausmyrkytysriskin tunnistava merkkiaine voisi osaltaan toimia lähtökohtana uusien diagnostisten menetelmien kehittämiselle. Sen lisäksi, että liukoisen VEGFR-1:n määrät olivat selkeästi suuremmat naisilla, joille myöhemmin kehittyi raskausmyrkytys, havaitsimme että myös verenkierron endostatiini- ja angiopoietiini-2 -taso oli heillä terveisiin naisiin nähden kohonnut. Yhdistämällä näistä merkkiaineista kaksi pystyttiin riskiä arvioimaan yhä paremmin. Tupakoivilla äideillä raskausmyrkytyksen todennäköisyys on aiempien tutkimusten mukaan merkittävästi tupakoimattomia naisia pienempi. Tutkimuksessamme tarkasteltiin siten tupakoinnin vaikutusta liukoisen VEGFR-1:n esiintymiseen raskaana olevien naisten veri- ja istukkanäytteissä, joskaan toteutetussa koeasetelmassa ei havaittu tilastollisesti merkitsevää eroa tupakoivien ja tupakoimattomien naisten välillä. Tutkimus auttaa kuitenkin osaltansa selvittämään yleisiä VEGF -kasvutekijäperheen välittämiä tupakoinnin verisuonivaikutuksia. Viimeisin osa tutkimusnäytteistä kerättiin aikasarjana joko suunniteltujen raskaudenkeskeytysten tai keisarinleikkausten yhteydessä liukoisen VEGFR-1:n biologisen puoliintumisajan määrittämistä varten. Istukan poistuessa myös liukoisen VEGFR-1:n lähde poistuu, jolloin sen puoliintumisaika voidaan märittää varsin luotettavasti. Totesimme, että tämä merkkiaine eliminoitui kaksivaiheisesti ja että sen puoliintumisajat olivat samankaltaisia kuin vertailuna sekä myös aiemmin istukkahormoonille (hCG) mitatut. Istukan poistuttua todettiin samalla myös vapaan VEGF:n pitoisuuden normalisoituvan kun se raskauden aikana oli liukoisen VEGFR-1:n vaikutuksesta lähes mittaamattoman matala. Näitä tuloksia voidaan mahdollisesti hyödyntää tutkittaessa jatkossa mm. verisuonisairauksien ja syövän syntyä

Targeting CD44v6, a co-receptor for Met and VEGFR-2 in endothelial cells, inhibits tumour angiogenesis

Isoformen der Transmembran-Glykoproteinfamilie CD44, die die Sequenz des varianten Exons v6 enthalten, können als Ko-Rezeptoren für die Rezeptortyrosinkinase Met in Epithelzellen dienen. Diese Arbeit zeigt, dass die Funktion von CD44v6 als Ko-Rezeptor auch auf Endothelzellen (ECs, endothelial cells) und auf eine andere Rezeptortyrosinkinase, nämlich VEGFR-2, übertragen werden kann. Da der VEGFR-2 einer der wichtigsten Rezeptoren in der Angiogenese ist, zeigen diese Ergebnisse, dass auch CD44v6 im Prozess der Angiogenese eine bedeutende Rolle spielt. Sowohl für VEGFR-2 als auch für Met wurde gezeigt, dass CD44v6 nicht nur für die Rezeptoraktivierung benötigt wird, eine Aufgabe, die die Ektodomäne von CD44 übernimmt, sondern auch für die intrazelluläre Signalweiterleitung. Die zytoplasmatische Domäne von CD44v6 kann ERM-Proteine (Ezrin, Radixin, Moesin) binden, welche wiederum an das Aktinzytoskelett gekoppelt sind. Dies ist eine Voraussetzung für die Aktivierung weiterer Bestandteile der Signalkaskade. Interessanterweise kann die Ko-Rezeptorfunktion von CD44v6 sowohl durch ein kleines Peptid als auch durch einen Antikörper, die beide gegen CD44v6 gerichtet sind, blockiert werden. Sie verhindern die Aktivierung von Met und VEGFR-2 durch die entsprechenden Liganden HGF und VEGF-A. Als Folge davon beeinträchtigen sie das physiologische Verhalten der Endothelzellen, wie zum Beispiel die ligand-gerichtete Migration, das Aussprießen aus einem Zellverbund sowie die Bildung von gefäßähnlichen Strukturen. Die Behandlung mit dem CD44v6 Antikörper oder dem CD44v6 Peptid reduziert auch die Vaskularisierung von Tumoren in zwei orthotopen Tumor-Modellen in Mäusen. Sowohl der Antikörper als auch das Peptid reduzierten die Gefäßdichte in einem Tumor der Brust signifikant. Deutlich stärkere Effekte konnten in einem Pankreas-Karzinom-Modell erzielt werden. Hier waren nicht nur die Gefäßdichte, sondern auch die Gefäßgröße und das Tumorvolumen deutlich reduziert. Zusammenfassend zeigt diese Arbeit, dass die Blockierung von CD44v6 ein interessanter Ansatz ist, um pathologische Angiogenese wie zum Beispiel bei der Tumorentwicklung zu hemmen. Die gezielte Ausschaltung eines einzigen Proteins, CD44v6, führt zur Inaktivierung von zwei Rezeptortyrosinkinasen. Deshalb ist das CD44v6 Peptid ein vielversprechendes Mittel, das in einer antiangiogenen Therapie eingesetzt werden könnte

The control and manipulation of angiogenesis in the primate ovarian follicle

The ovary is one of the most plastic tissues in the body undergoing constant serial remodelling throughout its reproductive lifetime, during both folliculogenesis and the formation and regression of the corpus luteum. The process of follicle growth and selection is intimately associated with the de novo establishment of vasculature supporting the developing follicles. Blood vessels are recruited from the ovarian stroma to form vascular sheaths surrounding each developing follicle supplying steroid hormone precursors, oxygen and nutrients to the expanding follicle. During folliculogenesis only the theca of the developing follicle becomes vascularised, the granulosa cells remaining avascular until ovulation at which point the basement membrane that has been separating the granulosa and theca breaks down. After ovulation the granulosa cells become heavily vascularised during the process of luteinisation and the formation of the corpus luteum. Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, requires the degradation of the established vessels followed by endothelial cell proliferation and finally stabilisation of the new vessels. Recently, techniques to quantify angiogenesis, identify putative molecular regulators, and inhibit them in vivo have become available. The work in this thesis applies these advances to the following questions:1) What is the effect of the inhibition of the gonadotrophins, using a gonadotrophin releasing hormone (GnRH) antagonist, on follicular angiogenesis? The hypothesis being tested was that follicular angiogenesis would be dependent on follicle stimulating hormone (FSH) / luteinising hormone (LH) and be severely inhibited by GnRH antagonist treatment. In vivo follicular angiogenesis was assessed by quantitative immunocytochemistry of bromodeoxyuridine and the endothelial cell marker CD31. The effect of treatment on follicular development and angiogenesis at the molecular level was assessed by in situ hybridisation of mRNA for vascular endothelial growth factor (VEGF) and aromatase. The results suggest that while VEGF expression in the preovulatory follicle is under gonadotrophic control, it is not dependent on normal gonadotrophin secretion in tertiary follicles, indicating that there are other paracrine factors regulating VEGF expression in the developing ovarian follicle. The second chapter extends the findings by determining granulosa cell response to FSH stimulation with respect to induction of the VEGF and aromatase genes.2) What is the effect of inhibition of vascular endothelial growth factor, using the antagonist, VEGF trap R1R2, on follicular angiogenesis, follicular development, ovulation and the establishment of the corpus luteum (CL)? The hypothesis being tested was that VEGF is essential for increasing permeability and the growth of the selected follicles. The immunocytochemical techniques used in the first study were again employed. The effect of treatment on the molecular regulation of angiogenesis was assessed by in situ hybridisation of mRNA for VEGF and its two receptors. In vivo inhibition of VEGF caused dramatic reductions in angiogenesis and in VEGF receptor expression but did not reliably prevent dominant follicle growth or ovulation once dominant follicle selection had occurred.3) Is a novel factor, endocrine gland vascular endothelial growth factor (EGVEGF) expressed in our animal model? The hypothesis being tested was that EGVEGF is an additional angiogenic factor that is expressed in the marmoset and the human ovary. This was assessed by in situ hybridisation in various marmoset tissues as well as in the human corpus luteum. Findings demonstrated that EG-VEGF is expressed in the granulosa lutein cells in the human corpus luteum while the marmoset ovary does not appear to express EG-VEGF.This thesis has improved our understanding of the gonadotrophic control of follicular angiogenesis and the role VEGF plays in the latter stages of folliculogenesis

TARGETED INHIBITION OF SP1 TRANSCRIPTION FACTOR AND ANTI-ANGIOGENESIS OF HUMAN PANCREATIC CANCER

Transcription factor Specificity Protein 1(Sp1) is reported to be essential for vascular endothelial growth factor (VEGF) constitutive expression in human pancreatic adenocarcinoma. The definitive role of Sp1 in angiogenesis, the impact of anti-angiogenic therapy on the Sp1/ VEGF signaling and the Sp1 signaling alteration on the anti-angiogenic therapy effect are unclear. The understanding of Sp1 regulation on VEGF and their interactions has significant clinical implications. Sp1 and VEGF expression and microvessel density (MVD) were analyzed using pancreatic cancer patients specimens through immunohistochemistry staining. The impact of Sp1 expression alternation on angiogenesis and tumor progression in nude mice were determined by knockdown Sp1 with small-interfering RNA (siRNA). Sp1 protein expression was correlated with the MVD (P \u3c 0.001) and VEGF expression (P \u3c 0.05). In mouse models, tumor progression and metastasis were inhibited after knockdown of Sp1 expression. The antitumor activity was correlated with the down-regulation of Sp1 downstream angiogenic factors caused by Sp1 knockdown. Sp1 and its downstream angiogenic genes expression were suppressed by mithramycin treatment both in vitro and in vivo. Moreover, mithramycin treatment reduced MVD in vivo. This was consistent with the down-regulation of VEGF, PDGF, and EGFR. Human xenograft pancreatic tumor growth was suppressed by Bevacizumab treatment. Both western blot and immunohistochemistry staining revealed that Sp1 and its downstream angiogenic genes expression were up-regulated by Bevacizumab treatment. Bevacizumab and mithramycin combination treatment synergistically suppressed tumor growth in vivo. This is correlated with the down-regulation of Sp1 and its downstream angiogenic genes expression. Bevacizumab treatment may trigger a positive feedback to up-regulate angiogenic factors through Sp1 trans-activation and this mechanism can be diminished by mithramycin treatment. Combination treatment of mithramycin and tolfenamic acid which has been shown to facilitate Sp1 protein degradation had synergistic cell growth inhibition effect in vitro. In vivo, metronomic low-dose combined treatment of mithramycin and tolfenamic acid produced tumor suppression in mouse model. Gene expression analysis showed that the combination treatment synergistic down-regulated Sp1 and its downstream angiogenic molecule VEGF. In summary, experimental results and clinical research suggested that Sp1 signaling is very important for angiogenesis of pancreatic cancer. Mithramycin treatment down-regulated Sp1 protein through interpose its transcription and decreased Sp1 downstream angiogenic genes. These down-regulations were correlated with the antitumor activity. Synergistic down-regulation of Sp1 and decreased expression of its downstream angiogenic molecules in turn significant reduced the angiogenic potential of pancreatic cancer cells and is an effective anti-angiogenesis strategy. Therefore, this study showed that Sp1 is a key factor of angiogenesis and manipulation of Sp1/VEGF signaling has clinical implication in anti-angiogenic therapy of pancreatic cancer

Vascular endothelial growth factor-eluting, polymer-coated, coronary stents: an in vitro and in vivo evaluation

INTRODUCTION: Percutaneous intervention (PCI) is complicated by restenosis, stent thrombosis and delayed endothelial recovery at the PCI site. One approach to reduce these complications is to deliver potent agents directly to the PCI site. This local drug delivery can be achieved by absorbing drugs into a polymer coating applied to the stent itself. Vascular Endothelial Growth Factor (VEGF) has been shown to accelerate the recovery of endothelium over a stent, reducing intimal hyperplasia and thrombosis. It has not previously been delivered bound onto the stent itself. VEGF-eluting stents were tested in vitro and in a rabbit model.ORIGINAL HYPOTHESIS: Polymer coated stents may absorb and gradually release Vascular Endothelial Growth Factor. This released VEGF may reduce the stent complications of thrombosis and neointimal hyperplasia, primarily by accelerating the recovery of denuded endothelium. This was tested in vitro and in an animal model.METHODS: Studies were performed to determine optimum stent loading with VEGF, which was radiolabelled. Loaded stents were perfused and the drug release kinetics measured. VEGF's potential to stimulate growth was assessed in endothelial cell culture. The VEGF-eluting stents were placed in rabbit iliac arteries and their effects on flow through the artery and acute platelet deposition were determined. Other animals were used to show longer-term effects on endothelial recovery and stent thrombosis (at 7 days) and intimal hyperplasia (at 28 days).RESULTS: 21,7pg of VEGF was absorbed. This was released with a bi-exponential release curve with 20% remaining at 9 days. In arterial tissue, 11% of the VEGF was detectable in the tissue at 24hr. VEGF-eluting stents stimulated endothelial cell growth by 11% over 5 days, with effects that were sustained beyond the initial rapid VEGF release. The animal studies showed a trend (p=0.07) towards reduced platelet deposition early after PCI, with reduced thrombus formation at 7 days (Omg in VEGF stents vs. 12.5mg in controls). No benefit of VEGF stents was seen on the re-endothelialisation process or on intimal hyperplasia.CONCLUSIONS: VEGF can be delivered by polymer-coated stents. Prolonged drug release to the injured vessel wall can be shown. However, VEGF did not fulfil the potential suggested by previous work and by the cell culture experiments when tested in vivo. It did appear to reduce thrombus formation and so may have potential benefits in clinical practice as a stent-based therapy

Polymeric Nanoparticles for the Controlled Administration of Bioactive Protein Agents

Controlled drug delivery technology represents one of the most rapidly advancing areas of science in which several fields, such as chemistry, pharmaceutical technology, and medicine are contributing to human health care. For many years, fundamental and applied investigations have focused on the development of pharmaceutical formulates allowing for maximization of the therapeutic efficacy and minimization of the adverse effects of the drugs of interest. The research activities reported in the present thesis are the results of experimental work performed at Laboratory of Polymeric Materials for Biomedical and Environmental Applications (BIOLab) of the Department of Chemistry and Industrial Chemistry of the University of Pisa and in part at Laboratory of Microbiology, School of Life Sciences, Tsinghua University (Beijing, China). The main goal of this research project was to produce Growth Factors (GFs) loaded NPs in order to obtain nanocarriers for the administration of these proteins in the treatment of ischemic diseases. Ischemic diseases remain among the most prominent health problems in the western countries. Defects in blood supply compromise tissues and organs functions causing several pathologic conditions, such as ischemic limbs and chronic ulcers. Therapeutic angiogenesis is a promising strategy for the treatment of these diseases. It is based on the utilization of angiogenic related GFs to enhance the natural healing processes in ischemic tissues. Two recombinant GFs, Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) were selected for the preparations of NPs. Moreover, since it is known that platelets are a great natural source of angiogenic related GFs, the possibility to encapsulate Platelet Lysate (PL), as natural source of GFs was investigated. In these research activities two polymers, with different characteristics (biodegradable and bioerodible) were employed. Poly (lactic-co-glycolic acid) [PLGA] and Poly (methacryloylglycylglycine-OH-co-hydroxypropylmethacrylamide) [Poly (Gly-co-HPMA)] are polymeric matrices selected to carry out this project, in order to obtain nanosystems that allow respectively a fast (e.g. few days) and a slow (e.g. several weeks) drug release. Moreover, a proteic model drug, Human Serum Albumin (HSA) was encapsulated with the intention of giving a better understanding of the suitability and versatility of the methods for the encapsulation of proteic drugs. In order to improve the maintenance of bioactivity of the encapsulated GFs, Heparin sodium salt (Hp) was used as stabilizing agent in all of the NPs formulations. NPs morphology, size, surface charge and encapsulation efficiency were determined, as well as the evaluation of drug release profiles. In the perspective of a practical application in the pharmaceutical field, the NPs were submitted to in vitro biological evaluation in order to investigate the cytocompatibility and bioactivity of the prepared nanosystems. Moreover, during six months visiting period spent at Tsinghua University the research activities was aimed at the preparation of NPs by using an innovative biosurfactant, the protein Phasin P (PhaP). PhaP is small bacteria amphiphilic protein, located on the surface of polyhydroxyalkanoates (PHAs), with the capability to non-specifically bind hydrophobic polymers. Two different polymeric materials, PLGA, and Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were used for the NPs formulation. In order to prepare NPs endowed with targeting moieties, magnetite (Fe3O4) was loaded in the nanosystems as a physical targeting agent. This technology allows the possibility to drive NPs to a specific tissue in the body by the applications of simple external magnetic field. The prepared NPs were characterized in term of size, Z-potential and morphology