Dissertations / Theses on the topic 'Neovascularization Regulation'

Choroidal neovascularization (CNV) is a leading threat to severe vision loss, particularly in patients with age-related macular degeneration (AMD). In CNV, newly formed blood vessels sprout from the choroid to the sub-retinal space, where leakage and bleeding of the abnormal vessels lead to photoreceptor death and subsequent vision loss. It is believed that CNV is mediated by growth factors (e.g. vascular endothelial growth factor {VEGF}) produced by the retinal pigment epithelium (RPE) under pathological states (e.g. hypoxia). Current treatments for CNV aiming at countering VEGF only help decrease leakage and inhibit formation of CNV, but none of them is curative and the recurrence rate remains high. In order to find other more powerful potential therapeutic targets, the regulations of VEGF signaling in the pathophysiology of CNV is the focus of numerous translational investigations. Previously, Hypoxia-inducible factor-1 (HIF-1), a crucial transcriptional factor in response to hypoxia, is identified as the master transcriptional factor controlling VEGF expression in the RPE promoting CNV. Alpha-enolase (ENO1), a key glycolytic enzyme, is known to be over expressed in several types of carcinomas also under the regulation of HIF-1. ENO1 has been reported to be closely associated with cancer progression, angiogenesis, and venous invasion. The molecular events of ENO1 in the pathogenesis of promoting angiogenesis are of interest but still barely understood. Recently, the association of ENO1 antibodies with retina has been seen in patients with AMD. We hypothesize that ENO1 expression in the RPE may play a role in the development of CNV, participating in the regulation of VEGF. Hypoxia is an important pathological condition in the formation of CNV. Here, we first determined ENO1 expression and cell death in a human RPE cell line, ARPE-19, under cobalt (II) chloride (CoCl2)-induced hypoxia or anoxia (95% N2, 5% CO2). To further investigate the regulation of ENO1 in CNV, HIF-1α-diminished RPE cells were generated using small interfering RNA (siRNA) and the change of ENO1 expression in response to hypoxic injury was determined. Upon 24 hr of treatment with CoCl2-induced hypoxia or anoxia, the expression of ENO1 and VEGF increased significantly along with HIF-1α in ARPE-19 cells, both of which could in turn be significantly down-regulated by HIF-1α siRNA. Interestingly, cell death remained low in ARPE-19 cells, even after 24 hr of CoCl2-induced hypoxia or anoxia. To further study the role of ENO1 in CNV, we started by investigating the relationship between ENO1 and VEGF. SiRNA was used to knock down the expression of ENO1 in ARPE-19 cells. Upon transfection with the siRNA, ENO1 expression was successfully down-regulated when treated with CoCl2-induced hypoxia. However, VEGF secretions from the ENO1-diminished ARPE-19 cells under CoCl2-induced hypoxia remained unchanged. Double knockdown of ENO1 together with HIF-1α by siRNA also did not help to further suppress VEGF secretion in the hypoxic ARPE-19 cells. Hence, ENO1 was demonstrated to be activated and up-regulated by HIF-1 in RPE cells responding to hypoxia, suggesting a potential role of ENO1 in favoring the formation of CNV, but not through influencing VEGF secretion.
published_or_final_version
Ophthalmology
Master
Master of Philosophy

Class IA PI3K (PI3K) functions have been widely investigated over the last two decades. PI3K signalling is located at the crossroads of many cell surface receptors sending signals to coordinate multiple cellular functions such as cell growth, survival, motility, and metabolism. Fine-tune regulation of PI3K signalling in cells is needed to ensure the functionality of tissues and organs. However, it is still not clear how or even which PI3K isoforms are concerted into precise morphogenic events. On the other hand, PI3K activity plays central roles in several cellular processes critical for cancer progression. Hence, PI3K pathway inhibition is considered an important target for therapeutic intervention in cancer, and progress in the clinical area is being monitored by many clinical trials with PI3K inhibitors. We were interested in investigating the role of PI3K activity in endothelial cells during the process of angiogenesis. Although ECs express all class I PI3K isoforms, only inactivation of the catalytic subunit p110α in endothelial cells (not p110β or p110δ inactivation) leads to vascular defects in the embryo (Graupera et al. 2008). This indicates that p110α activity in ECs is required in a cell- autonomous manner to ensure proper vascular development and remodelling during the embryogenesis. However, progress in the understanding of how p110α-PI3K signalling regulates the different steps of vascular morphogenesis has been hampered by embryonic lethality that both the constitutive and endothelial specific p110α mutant mice exhibit. By using a tamoxifen-inducible endothelial Cre line in mouse and genetic and pharmacological approaches in zebrafish embryos, we have found that p110α signalling is required to maintain vessel stability. The lack of p110α activity leads to endothelial tubular structures composed of single cells that show an elongated shape with multiple protrusions and no lumen. These ECs fail in the elongation of the inter-endothelial contacts during the sprout outgrowth. Furthermore, I found that p110α is involve in the initial steps of fusion and is necessary for proper establishment of a new connection. Finally, I identifyed that p110α negatively controls actomyosin contractility independently of Rho/ROCK signalling pathway and that this control could be exerted through the regulation of MLC phosphatase activity by the impact on mRIP and/or MYPT proteins.
La señalización PI3K de clase IA se requiere de una manera autónoma en células endoteliales para el correcto crecimiento de los vasos sanguíneos. Aunque las células endoteliales expresan todas las isoformas de la clase IA de PI3Ks, sólo la subunidad catalítica p110α es necesaria para la angiogenesis fisiológica. Sin embargo, poco se sabe sobre el papel de p110α -PI3K en las diferentes etapas de la morfogénesis vascular. Mediante la generación de una línea inducible Cre endotelial de ratón y la inactivación genética y farmacológica de la proteína en embriones de pez cebra, hemos encontrado que la señalización a través de la proteína p110α es necesaria para mantener la estabilidad del los vasos sanguíneos. La falta de la actividad de la proteína p110α da lugar a la formación de una vasculatura aberrante formada por estructuras endoteliales muy delgadas compuestas por células individuales que emiten múltiples protrusiones y carecen de lumen. Durante la elongación del nuevo brote vascular las células endoteliales no puede elongar la superficie de adhesión entre células endoteliales y por tanto no pueden sufrir reordenamientos necesarios para el crecimiento del nuevo vaso. También hemos visto que la proteína p110α está implicada en la estabilización de los nuevos contactos durante el proceso de anastomosis vascular y su inactivación da lugar a inestabilidad vascular y la aparición de desconexiones en entre vasos. La falta de la proteína p110α se asocia con un aumento en la formación de los cables de actina cortical e hiperfosforilacion de la cadena ligera de la miosina. Por tanto, identificamos que la ruta de señalización p110α-PI3K controla negativamente la contractilidad de las fibras de actomiosina de forma independiente a la via de señalización Rho-ROCK y que este control podría ser ejercido a través de la regulación de la actividad de la fosfatasa MLC a través de las proteínas mRIP y/o MYPT1.

Hepatic cirrhosis is a largely diffused pathology caused by alcohol abuse and hepatitis C in developed countries, whereas hepatitis B is the cause in most parts of Asia and sub-Saharan Africa. It's characterized by development of regenerative nodules delimited by fibrous septa. Regenerative nodules are composed by proliferating hepatocytes, entrapped by deposition of extracellular matrix, and have been shown to be involved in growth factor production, in particular VEGF, which is responsible of angiogenic induction that culminates with formation of a dense network of blood vessels into fibrous septa. The newly formed blood vessels of fibrous septa connect the vessels of the portal region with terminal hepatic veins, determining an alternative route of the blood flow that, in this way, is redirected into the systemic circulation bypassing the liver. At pre-hepatic level, the cirrhosisrelated portal hypertension induces development of new blood vessels that shunt the portal blood into the systemic circulation. As consequence of both intrahepatic and pre-hepatic angiogenesis, the liver cannot metabolize several blood components such as drugs, nutrients, toxins, and bacteria, with obvious deleterious effects. Despite angiogenesis is one of the main complications of liver cirrhosis and VEGF has a pivotal role in the control of blood vessels formation, the molecular mechanisms that govern VEGF expression during angiogenesis and hepatic cirrhosis are poorly understood. In order to address whether CPEB family of proteins may have a function in the regulation of angiogenesis in liver diseases, we analysed the expression of CPEBl and CPEB4 in liver of patients affected by hepatic cirrhosis. The expression of CPEBl and CPEB4 was increased in regenerative nodules, compared with basal levels of expression detected in healthy hepatic parenchyma. Interestingly, also VEGF expression was higher in regenerative nodules. The numerous fibrous septa that characterized cirrhotic livers resulted highly vascularized, and the endothelium of newly formed blood vessels expressed high levels of CPEB1, CPEB4 and VEGF. The description of CPEB1, CPEB4 and VEGF expression in healthy and cirrhotic conditions represented a first cue of CPEB-mediated translational regulation of VEGF mRNA during angiogenesis. To prove the accuracy of this hypothesis we implemented two animal models that allowed the study of intrahepatic and prehepatic angiogenesis correlated with liver cirrhosis. CBDL experiments performed in rats enabled to recapitulate the histopathological conditions observed in human hepatic cirrhosis. Cirrhotic rat livers showed deep histological perturbations, with high proliferation of blood vessels and biliary ducts. Compared with control healthy samples, the expression of CPEB1, CPEB4 and VEGF in pathological liver was strongly increased. These proteins localized at level of both, blood vessels and biliary ducts. Partial portal vein ligation (PPVL) experiments performed in rats enabled to show an important correlation between CPEBl and CPEB4 expression with VEGF synthesis and consequent high vascularization of mesentery. In angiogenic condition, both pre-existing and newly formed blood vessels expressed CPEB1, CPEB4 and VEGF at level of endothelium, smooth muscle and adventitia, tissues that playa pivotal role in the development of the vascular net. To better define the mechanistic relevance of these correlations, we characterized the endothelial cell line HSV. In this in vitro model we modulated the levels of CPEBl and CPEB4, and showed a direct involvement of this two proteins in the translational regulation of VEGF mRNA. Taking advance of the ability of HSV cells to form blood vessel like structure in an in vitro angiogenesis assay, we were able to show that CPEBl and CPEB4 are required for VEGF synthesis and secretion, which in turn are essential to create the correct microenvironment necessary to activate the cells and induce the formation of a dense network of vascular structures on Matrigel. Our results suggest that CPEBl drives the nuclear cleavage of VEGF 3'UTR while CPEB4 is responsible of its cytoplasmic polyadenylation.
"Regulación de la angiogénesis a través del control traduccional mediato par las proteínas CPEB " En muchas enfermedades hepáticas cr6nicas, la angiogénesis es una importante característica patológica y juega un papel crucial en la progresión de la fibrogénesis hepática a cirrosis, y en la aparición y agravamiento de la hipertensión portal, la cual determina las principales complicaciones de la enfermedad. A pesar de que es evidente que VEGF es el principal efector de la angiogénesis patológica, los mecanismos moleculares que gobiernan la activación post-transcripcional de su síntesis durante la cirrosis hepática son en gran parte desconocidos. En este trabajo se muestra que la síntesis de VEGF está regulada a través de funciones secuenciales y no redundantes de dos miembros de la familia de las proteínas CPEB:. CPEB1 y CPEB4. Por 10 tanto, CPEB1 promueve el procesamiento alternativo de ambos los pre-ARNm de CPEB4 y VEGF, acortando las 3'UTRs y excluyendo elementos de inhibición de la traducción de los transcritos maduros. Como resultado de este procesamiento alternativo, CPEB4 se sobreexpresa, y polyadenyla el ARNm de VEGF, aumentando aún más su traducción. Entonces, se requieren tanto CPEB1 como CPEB4 para la síntesis de VEGF y la consecuente angiogénesis. Por tanto, todas las proteínas se sobreexpresan de forma secuencial en pacientes y en modelos animales de cirrosis hepática e hipertensión portal, y ambos ratones knock-out para CPEB1 y CPEB4 no lograron activar la angiogénesis tras la inducción de hipertensión portal. A través del análisis de la angiogénesis en ensayos in vitro, las muestras de humanos y modelos animales, nuestros resultados ponen de relieve el papel crucial de CPEBs en la neovascularización patol6gica, en el marco de la hipertensión portal y cirrosis, e identifican CPEBs como potenciales nuevas dianas moleculares para el tratamiento de la enfermedad hepática cr6nica y otras enfermedades dependientes de la neovascularización, como el cáncer.

In response to nutritional variation, white adipose tissue (WAT) undergoes a physiological remodelling that involves qualitative and quantitative changes in resident cells and is coordinated with angiogenesis. In a condition of chronic over nutrition WAT expansion is associated to insufficient vascularisation which in turn leads to local hypoxia, inflammation and adipocytes death (hallmark of obesity). Currently, enhanced WAT angiogenesis is believed to be a promising intervention to ameliorate obesity associated metabolic dysfunctions. However, we still lack understanding of the cell intrinsic function of endothelial cells in WAT remodelling. Here we take advantage of our mouse model of PTEN (a dual lipid/protein phosphatase that counterbalance the activity of PI3K) deletion in ECs to promote vessel growth, in a cell autonomous manner. To this end, we crossed Ptenflox/flox mice with PdgfbiCreERT2 transgenic mice that express a tamoxifen-inducible Cre recombinase in ECs; 4-hydroxytamoxifen was administered in vivo at postnatal day 1 (P1) and P2 to activate Cre expression. Increased ECs proliferation, induced by PTEN loss, promotes vascular hyperplasia exclusively in WAT and leads to a progressive loss of WAT mass. PTEN null ECs undergo a metabolic switch towards an oxidative metabolism; in vivo inhibition of - oxidation is sufficient to revert both vascular hyperplasia and loss of WAT mass. Enhanced adipose vascularisation prevents from high fat diet induced WAT hypertrophy, limits body weight gain and improves glucose tolerance. Taken together our results suggest that, under obesogenic stimuli, more functional ECs prevent unhealthy WAT expansion and consequently the onset of obesity related comorbidity.

Notre travail a permis tout d’abord d’investiguer les effets des nucléotides extracellulaires sur les cellules

dendritiques (DCs) qui sont des cellules présentatrices d’antigènes capables d’initier et de réguler la

réponse immunitaire. Afin d’avoir une vue globale de l’action des nucléotides extracellulaires sur les DCs,

un profil d’expression génique de l’ATPgS – dérivé stable de l’ATP - a été réalisé par microarray dans les

cellules dendritiques dérivées de monocytes (MoDCs).

Notre groupe a préalablement montré que malgré que l’ATP est considéré comme un signal de danger, il

confère des propriétés immunosuppressives aux DCs (Marteau et al, 2005). Nous nous sommes focalisés

sur des régulations géniques pouvant être mises en relation avec un action anti-inflammatoire de l’ATP.

Nous avons ainsi démontré que l’ATP était capable d’inhiber la sécrétion des chimiokines MCP-1 et MIP-

1a initiée par l’action du LPS, ce qui a pour conséquence de diminuer la capacité des DCs à recruter des

monocytes ou d’autres DCs. Ce travail a fait l’objet d’une publication en tant que premier auteur

(Horckmans et al, 2005).

Un grand nombre d’autres gènes régulés liés à la réponse immune et à l’inflammation a été identifiée

dans le profil microarray de l’ATPgS. Nous avions notamment pu identifier une augmentation de la

sécrétion de VEGF-A en réponse à l’ATP, amplifiée en présence de LPS. Cette régulation est extrêmement

intéressante au vu de l’action immunosuppressive du VEGF sur les DCs. Par ailleurs, cette régulation

pourrait constituer un lien entre les DCs et l’angiogénese. Ce travail a fait l’objet d’une publication en tant

que premier co-auteur dans la revue Journal of Immunology (Bles et al, 2007).

En conclusion, nos données nous ont ainsi permis de montrer que les nucléotides adényliques peuvent

avoir par leur action sur les cellules dendritiques une action anti-inflammatoire voire pro-angiogénique,

en inhibant le recrutement leucocytaire et une action immunosuppressive en stimulant la sécrétion de

VEGF.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Background: Our laboratory identified ELTD1, an orphan GPCR belonging to the adhesion GPCR family (aGPCR), as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. ELTD1 is normally expressed in both endothelial cells and vascular smooth muscle cells and expression is significantly increased in the tumour vasculature. The aim of this project was to analyse ELTD1's function in endothelial cells and its role in breast cancer. Method: 62 sequenced vertebrate genomes were interrogated for ELTD1 conservation and domain alterations. A phylogenetic timetree was assembled to establish time estimates for ELTD1's evolution. After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) and validated with qPCR and confocal microscopy. ELTD1's signalling was investigated by applying the aGPCR ‘Stinger/tethered-agonist Hypothesis'. For this, truncated forms of ELTD1 and peptides analogous to the proposed tethered agonist region were designed. FRET-based 2^nd messenger (Cisbio IP-1;cAMP) and luciferase-reporter assays (NFAT; NFÎoB; SRE; SRF-RE; CREB) were performed to establish canonical GPCR activation. To further investigate ELTD1's role in endothelial cells, ELTD1 was stably overexpressed in HUVECS. Functional angiogenesis assays and mRNA array profiling were then performed. To investigate ELTD1 in breast cancer, a panel of cell lines representative of all molecular subtypes were screened using qPCR. Furthermore, an exploratory pilot study was performed on matched primary and regional nodal secondary breast cancers (n=43) which were stained for ELTD1 expression. Staining intensity was then scored and compared with relapse free survival and overall survival. Results: ELTD1 arose 435 million years ago (mya) in bony fish and is present in all subsequent vertebrates. ELTD1 has 3 evolutionary variants of which 2 are most common: one variant with 3 EGFs and a variant with 2 EGFs. Additionally, ELTD1 may be ancestral to members of aGPCR family 2. HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of the mitochondrial citrate transporter SLC25A1, and ACLY which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. A review of lipid droplet (fatty acid and cholesterol) accumulation by confocal microscopy and flow cytometry (FACS) revealed no changes with ELTD1 silencing. Silencing was also shown to affect the Notch pathway (downregulating the Notch ligand JAG1 and target gene HES2; upregulating the Notch ligand DLL4) and inducing KIT, a mediator of haematopoietic (HSC) and endothelial stem cell (ESC) maintenance. Signalling experiments revealed that unlike other aGPCRs, ELTD1 does not couple to any canonical GPCR pathways (Gαi, Gαs, Gαq, Gα12/13). ELTD1 overexpression in HUVECS revealed that ELTD1 induces an endothelial tip cell phenotype by promoting sprouting and capillary formation, inhibiting lumen anastomoses in mature vessels and lowering proliferation rate. There was no effect on wound healing or adhesion to angiogenesis associated matrix components. Gene expression changes following ELTD1 overexpression included upregulation of angiogenesis associated ANTRX1 as well as JAG1 and downregulation of migration associated CCL15 as well as KIT and DLL4. In breast cancer, none of the representative breast cancer cell lines screened expressed ELTD1. ELTD1 breast cancer immunohistochemistry revealed higher levels of vascular ELTD1 staining intensity within the tumour stroma contrasted to normal stroma and expression within tumour epithelial cells. Additionally, ELTD1 expression in tumour vessels was differentially expressed between the primary breast cancer microenvironment and that of the matched regional node. Due to the small size of the pilot study population, survival comparisons between the various subgroups did not yield significant results. Conclusion: ELTD1 is a novel regulator of endothelial metabolism through its suppression of ACLY and the related citrate transporter SLC25A1. ELTD1 also represses KIT, which is known to mediate haematopoietic and endothelial progenitors stem cell maintenance, a possible mechanism through which endothelial cells maintain terminal endothelial differentiation. ELTD1 does not signal like other adhesion GPCRS with CTF and FL forms of ELTD1 not signalling canonically. Additionally, ELTD1 regulates various functions of endothelial cell behaviour and function, inducing an endothelial tip cell phenotype and is highly evolutionarily conserved. Lastly, ELTD1 is differentially expressed in tumour vessels between primary breast cancer and regional nodal metastases and is also expressed in a small subset of breast cancer cells in vivo despite no cancer cell lines expressing ELTD1. The pilot study investigating ELTD1 in the primary breast cancer and regional involved nodes will be followed up with a larger study including the investigation of ELTD1 in distant metastases.

Introduction Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, is a pre-requisite for tumour growth and metastasis. Tumour-angiogenesis is regulated by various pro- and anti-angiogenic factors released by both endothelial and tumour cells, as well as by the micro-environment. Numerous studies have implicated various systems in the acquisition of the angiogenic phenotype. The present study sought to investigate the role of the kallikrein-kinin system (KKS) in tumour-angiogenesis. The kallikreins consist of two serine proteases, plasma and tissue kallikrein (TK), involved in the release of kinin peptides by enzymatic cleavage of kininogens. Stimulation of the cognate bradykinin receptors (BKR), B1R and B2R, mediates the mitogenic and vasoactive properties of kinins. In addition, TK activates matrix metallo-proteinases (MMPs) involved in extracellular matrix (ECM) degradation. The expression profiles of TK and kinins have been found to be dys-regulated in numerous human cancers, and several studies have demonstrated the involvement of the KKS in growth and metastasis of prostate tumours. Further, previous in vitro models in our laboratory have established an association between the KKS and prostate tumour-angiogenesis. In those studies it was postulated that the up-regulated TK (produced by endothelial and tumour cells) stimulated endothelial cell proliferation. Thus, the aim of the present study was to define the effects of the KKS and seek a direct correlation with angiogenesis using in vitro models with tumour conditioned medium (CM), kinin receptor agonists and antagonists. Methods Ethical approval for this project was granted by the Biomedical Research Ethics Committee, University of KwaZulu-Natal (reference number BE152/08). Micro-vascular endothelial cells represent a suitable in vitro angiogenic model and dermal micro-vascular endothelial cells (dMVECs) were obtained commercially for this purpose. The tumour model used in this study was an immortalised prostate cancer (DU145) cell line. The CM model involves the treatment of one cell line with the metabolites of another. In the angiogenic model, dMVECs were exposed to increasing concentrations of DU145 CM. Stimulation was further augmented with BKR agonists. Specific BKR antagonists were used to test the specificity of stimulation. In addition, vascular endothelial growth factor (VEGF) was tested as a positive proliferation control. The potential of these agents to induce proliferation and migration was determined using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and a modified Boyden chamber assay, respectively. Previous studies investigating the pro-angiogenic effects of CM differed, in many respects, in terms of their models and methodologies. In an attempt to fully explore the pro-mitogenic effects of CM on endothelial cells, various modifications, as well as alternate endothelial and tumour cell types, were employed in the present study. The mitogenic and migratory effect of BKR agonists and antagonists on DU145 cells was also assessed. Further, the tumour model was expanded to investigate the autocrine potential of the KKS, by investigating the effect of DU145 CM on DU145 migration. Results In the angiogenic model, although the addition of DU145 CM elicited a statistically significant increase in micro-vascular endothelial cell proliferation, this increase was very small (<10%) and not dose-dependent. Pre-incubation of dMVECs with a B1R or B2R antagonist did not influence this small effect of CM on proliferation. In addition, neither B1R nor B2R agonists, at any concentration, produced any significant proliferative effect on endothelial cells. In contrast to these findings VEGF, a well-known mitogen, was able to stimulate proliferation of dMVECs. Migration assays revealed that DU145 CM failed to stimulate endothelial cell motility. Further, neither BKR agonist displayed any chemo-attractant potential in those assays. The most important finding was in the tumour model, where stimulation with a B1R agonist significantly enhanced proliferation and especially migration of DU145 cells. In addition, pre-treatment with a B1R antagonist abolished both these effects. B2R agonists could not produce the same positive effect as the B1R agonist on growth and migration of prostate tumour cells. DU145 CM did not prove to be a migratory stimulus for DU145 cells at any concentration. Discussion Previous studies in our laboratory have shown prostate-tumour CM to promote proliferation of endothelial cells and have postulated that TK up-regulation may be the reason for this. However, the present study could not reproduce this effect of CM. Further, BKR antagonists had no notable or consistent effect on the minimal promotion of proliferation that had been produced by DU145 CM. In addition, selective BKR agonists failed to induce proliferation or migration of endothelial cells, key events in the angiogenic cascade. Although in contrast to some studies, the present study was unable to implicate the KKS in angiogenesis, tumour neo-vascularisation is a consequence of several angiogenic factors functioning together as opposed to a single, isolated factor. For example, we were able to demonstrate a positive mitogenic effect of VEGF on endothelial cells and it may be this as well as other factors in the CM that are responsible for the small proliferation we observed. Up-regulation of kallikreins and kinins in tumours may enhance fundamental events in tumourigenesis in an autocrine manner, and bradykinin (BK) has previously been shown to promote tumour growth in mouse models. Our study supported the involvement of the KKS in tumourigenesis. Although CM from DU145 cells did not self-stimulate the migration of these cells, a B1R agonist enhanced both proliferation and migration, an effect that was also abrogated by the relevant antagonist, indicating a role for kinins. In contrast to the findings of another study, stimulation of the B2R failed to significantly promote tumour growth or motility. However, this is not an unexpected finding because it is thought that the ubiquitous B2R mediates physiological effects in the prostate while the inducible B1R plays a role in prostate cancer pathology. In summary, this study lends support to the ongoing exploration of BKR antagonists as possible candidates in the development of alternate approaches to cancer therapy. This may be particularly beneficial to hormone-independent tumours, such as those of the prostate, for which there exists few effective treatment options.
Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

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.

Notch signaling controls normal and pathological angiogenesis through transcriptional regulation of a wide network of target genes. Despite intensive studies of the endothelial Notch function, a comprehensive list of Notch-regulated genes, especially direct transcriptional targets, has not been assembled in endothelial cells (ECs). Here we uncovered novel EC Notch targets that are rapidly regulated by Notch signaling using several unbiased in vivo and in vitro screening approaches that captured genes regulated within 6 hours or less of Notch signal activation. We used a gamma-secretase inhibitor in neonates to profile Notch targets in the brain endothelium using the RiboTag technique, allowing for isolation of endothelial specific mRNA from a complex tissue without disrupting cell-cell contact. We used two types of primary cultured endothelial cells to define ligand-specific Notch targets by tethered-ligand stimulation. The identified Notch targets were validated by determining their regulation within one to two hours of EGTA-mediated Notch activation. By comparing significantly regulated genes in each of the screens, we assembled a comprehensive database of potential Notch targets in endothelial cells. Of particular interest, we uncovered G protein pathway related genes as potential novel Notch targets. We focused on a novel candidate target passing selection criteria after all screens, a small GTPase RND1. RND1(Rho GTPase1) regulates cytoskeleton arrangement through Rho and Ras signaling. RND1 was validated as an endothelial Notch target in multiple endothelial cell types. In Human Umbilical Vein Endothelial Cells (HUVECs) we established angiogenic activity for RND1 that included regulation of cell migration towards VEGF and function in sprouting angiogenesis. We established that Notch and RND1 suppressed Ras activation but had no effects on Rho activation in HUVECs. These results demonstrate that RND1 expression is regulated by Notch signaling in endothelium and suggest that RND1 functions downstream of Notch in sprouting angiogenesis, revealing an unexplored role of endothelial Notch in regulating G protein pathways.

Wang, Chengdong.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 113-134).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.