Statins induce beneficial vascular effects. How statins induce beneficial vascular effects is yet to be determined. Here we examine Simvastatin and vascular endothelial growth factor (VEGF) acting through the phosphoinositide 3-kinase (PI3K) pathway in human coronary artery endothelial cells (HCAEC). While Simvastatin and VEGF both activated mediators in the PI3K pathway, the proteins and the rates of activation were not always consistent. This suggests that although Simvastatin and VEGF share a common PI3K pathway in HCAEC and similar vascular effects, the agonists diverge in the induction of cellular signaling cascades. Simvastatin also was shown to induce phosphoinositide 3, 4, 5-triphosphate (PIPS) organization and PI3K p110 gamma (y) perinuclear localization. Beneficial, non-lipid lowering effects of statins may occur through the PI3K pathway through activation of distinct mediators from those of VEGF. Better understanding of the pathways associated with statins is necessary for the discovery of better treatments for cardiovascular disease (CVD). / Department of Biology

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.

The delivery of growth factors has been attempted for a number of different therapies. The approach of delivering therapeutic growth factors in a safe and efficient manner is difficult and certain criteria should be met. These criteria include: binding the appropriate growth factors, maintaining their bioactivity, and delivering these proteins with controllable release kinetics for an extended period of time. These criteria encompass a set of guidelines that hope to mimic in vivo biological events such as neovascularization. The central goal of this thesis is to meet these criteria by introducing a novel delivery strategy for growth factors using a biocompatible polycation and heparin. It was hypothesized that a polycation could interact with heparin to form a complex with the potential to deliver bioactive growth factors with an adaptable release. This hypothesis was tested by examining the release kinetics of bFGF from the complex and investigating whether the released bFGF maintained its bioactivity. The [polycation:heparin:bFGF] complex was formed by mixing the components in water, resulting in a precipitate. This precipitate was able to deliver bFGF with controllable release kinetics and the bioactivity of the released bFGF was higher than bolus bFGF and comparable to heparin stabilized bFGF. This system is expected to have the ability to bind and deliver numerous heparin-binding growth factors. In conclusion, the delivery system developed in this research provides a novel mechanism for controlled release of growth factors. This delivery strategy has met the criteria listed earlier and this research has laid the foundation for a successful delivery vehicle. Further, a biocompatible polycation was synthesized, which is a critical component of the delivery system. This polycation exhibited in vitro and in vivo biocompatibility that was orders of magnitude higher than existing polycations and has the potential to be very useful in a variety of biomedical applications. This design principle is also expected to serve as a platform for the synthesis of other biocompatible polycations.

The IGF system is a crucial regulator of normal growth and development, however dysregulation of the system on multiple levels is associated with the incidence of a wide variety of malignancies including the breast, thyroid, lung, and colon, making the IGF system an important anti-cancer therapeutic target. Due to its role in mediating cellular proliferation, protection from apoptosis, and metastasis, traditional focus has been set on examining the role of the type 1 IGF receptor [IGF1R] in cancer. However there is mounting evidence to suggest the insulin receptor [IR] may also be involved in the potentiation and pathogenesis of cancers. The observation that IGF-II is overexpressed, compared to normal tissues, by cancers suggests signaling via target receptors by this ligand has important implications on cancer pathogenesis. Indeed, both the IGF1R and IR have been demonstrated to be up-regulated in a variety of malignancies. In regards to IR isoform, the IGF-II binding IR-A is preferentially expressed by a number of cancer cell types. Together with the observation that an autocrine proliferative loop exists between IGF-II and the IR-A in malignant thyrocytes and cultured breast cancer cells, suggests signaling via the IR-A may play a role in cancer cell growth and survival. However, very few studies on the IR-A have been conducted in cells co-expressing the IGF1R. This is mainly due to the difficulties associated with discrimination between signaling arising from IGF1R homodimers, IR-A homodimers, and IGF1R/IR-A hybrid receptors. It is not known how the IR-A interacts, and functions in conjunction with the other receptors of the IGF system to signal biologically relevant outcomes, especially in terms of anti-cancer therapeutics that aim to block and down-regulate the IGF1R. Current anti-cancer therapies targeting the IGF system have concentrated on blocking IGF signaling via the IGF1R, due mostly to the functional properties of the receptor, but also in part due to the metabolic consequences associated with blockade and inhibition of the IR. This individual targeting of the IGF1R potentially leaves a pathway by which IGF-II secreted by the tumour can circumvent current IGF1R based therapies. Consequently, this thesis investigated whether the IR-A could compensate for the targeted loss of the IGF1R and how the IR-A interacts with the IGF1R in cells co-expressing these two receptors. In addition, the individual ability of the IR isoforms to signal biological outcomes in response to IGF stimulation was assessed. The main experimental techniques used throughout this body of work included; assessment of protein expression and activation by Western blot, siRNA mediated gene silencing, and measures of cell proliferation, survival, and migration. The key areas of investigation included: 1. Investigation of the individual ability of the IR isoforms to signal biological outcomes in response to IGF stimulation 2. Identification of an appropriate cell line model in which to investigate the interactions between the IR-A and IGF1R 3. Optimisation of siRNA mediated knock-down of the IR-A and IGF1R in SW480 colorectal adenocarcinoma cells 4. Determination of the biological role of the IR-A in SW480 cells co-expressing the IGF1R The key findings from this work included: 1. The IR-A could not compensate for IGF1R depletion in SW480 cells 2. Dual silencing of the IR-A and IGF1R indicated signaling via the IGF1R was dominant to signaling via the IR-A in SW480 cells 3. Signaling via IR-A/IGF1R hybrid receptors may not be as potent as signaling via IGF1R homodimers 4. IGF-I at physiological concentrations can stimulate biological responses via both isoforms of the IR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337339 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN) while IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects including IGF binding protein-5 (IGFBP-5) production, IGF type-1 receptor autophosphorylation and cell migration. Thus, this study examined the hypothesis that a link between IGF-I and VN must occur and may be mediated through IGFBPs. Studies using competitive binding assays with VN and [125I]-labelled IGFs in the absence and presence of IGFBPs revealed IGFBP-4, IGFBP-5 and non-glycoyslated IGFBP-3 significantly enhance binding of IGF-I to VN, while IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogues indicate that glycosylation status of IGFBP-3 and the heparin-binding domains of IGFBP-3 and IGFBP-5 are important in this interaction. The functional significance of IGFs binding to VN on cell migration in MCF-7 breast carcinoma cells was examined and cell migration was found to be enhanced when VN was pre-bound to IGF-I in the presence of IGFBP-3, -4 and -5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding analogue, des(1- 3)IGF-I. Additionally, higher doses of IGFs in the presence of VN also could stimulate cell migration. Together, these data indicated the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells. Future directions for this work include investigations into the mechanisms underlying formation of the trimeric complex and the associated signalling pathways involved.

Members of the insulin-like growth factor (IGF) family are mitogenic growth factors which have been shown to play critical roles in both normal growth and development, and tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by diverse interactions between many molecules, including interactions with the extracellular matrix (ECM). Recent observations have demonstrated that IGFs can associate with the ECM protein vitronectin (VN) and this interaction can modulate IGF-stimulated biological functions. It has been demonstrated previously that IGF-II can bind directly to VN, while IGF-I associates with VN indirectly via the involvement of IGF-binding proteins (IGFBPs) -2, -3, -4 and -5. As the IGF system plays important roles in both normal breast development and in the transformation and progression of breast cancer, this study aimed to describe the effects of substrate-bound IGF-I:IGFBP:VN complexes on breast cell functions and to dissect the mechanisms underlying these responses. The studies reported in this thesis demonstrate that substrate-bound IGF-I:IGFBP:VN complexes, containing IGFBP-3 and IGFBP-5, are potent stimulators of proliferation and migration in the "normal", non-tumourigenic MCF-10A breast epithelial and MCF-7 breast carcinoma cell lines. Interestingly, substrate-bound IGF-I:IGFBP:VN complexes were less effective in increasing the migration of the metastatic MDA-MB-231 breast cancer cell line. This, however, is due to these cells expressing the αvβ3 integrin which can support a highly migratory phenotype independent of IGF-I-stimulation. Taken together this suggests a particularly important role for these complexes in stimulating a highly migratory phenotype in pre-invasive or poorly metastatic breast cells. Studies using IGF-I analogues were also undertaken to establish if there was a requirement for ternary complex formation and the type-1-IGF receptor (IGF-1R) in the enhanced migration responses observed. These studies determined IGF-I:IGFBP:VN-stimulated migration to be dependent upon both heterotrimeric IGF-I:IGFBP:VN complex formation and activation of the IGF-1R. Furthermore, the enhanced cellular migration was abolished upon incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. In addition, analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway, while simultaneously producing a sustained activation of the PI3-K/AKT pathway. Optimal intracellular signaling required activation of both the IGF-1R and VN-binding integrins, as antibody mediated inhibition of either receptor led to substantial decreases in both ERK/MAPK and PI3-K/AKT pathway activation. Furthermore, experiments using pharmacological inhibitors of these pathways determined a pivotal role for PI3-K/AKT activation in substrate-bound IGF-I:IGFBP:VN-stimulated cell migration. In order to confirm an important role for the PI3-K/AKT pathway in these responses, wild-type and activated-AKT was transiently overexpressed in MCF-10A cells. Overexpression of both wild-type and activated-AKT further enhanced cellular migration in response to substrate-bound IGF-I:IGFBP:VN complexes. However, these responses still required co-activation of the IGF-1R and VN-binding integrins. In an attempt to obtain a global view of the possible molecular mechanisms underpinning IGF-I:IGFBP:VN-stimulated cell migration, oligonucleotide microarrays were used to screen for candidate genes important for the observed migratory responses. The microarray studies identified 165 genes which were differentially expressed in cells migrating in response to substrate-bound IGF-I:IGFBP:VN complexes. Gene ontology and functional analysis revealed many of these genes to be significantly associated with biological functions relevant to cancer transformation and progression, including cell growth and proliferation, cell death and cellular movement. In regard to cell migration, a number of the genes identified have previously reported roles in cellular movement, migration and metastasis, which may provide future targets to augment IGF-I:IGFBP:VN-stimulated cell migration. Taken together, the studies reported throughout this thesis have provided the first mechanistic insights into the action of IGF-I:IGFBP:VN complexes and add further evidence to support the involvement of VN-binding integrins and their co-operativity with the IGF-IR in the promotion of tumour cell migration. Importantly, identifying the molecular mechanisms by which IGF:VN complexes enhance breast cell function will lead to not only a better understanding of this critical interaction, but also aid in developing diagnostic tests and therapeutics directed at treating breast cancer.

The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient’s healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker ..- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP ... These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP .. in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

The management of significant bone defects, delayed and non-union of fractures can be extremely challenging. Development of specific treatment is hindered by an absence of information regarding the molecular events which regulate these processes. In this thesis, a bilateral cancellous bone defect model of the femur and tibia was developed in a rodent and the spatiotemporal profile of TGF-β, BMP 2 and 7, Smads 1, 4 and 5 characterised. Next, the capability of acid solution to augment healing was tested in both a bone defect and in a closed femoral fracture model. Finally, a long term oestrogen deficiency (OVX) rat model of postmenopausal osteoporosis was characterised and the spatiotemporal profiles of IGF-1, IGFR-1, MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, BMP-2, BMP-4, BMP-7, TGF-β, Smad4, Smad7, VEGF, Flt-1, Ihh and FGF-2 were compared in femoral osteotomies between OVX and Sham groups. The bilateral cancellous defect model was successfully created with a number of advantages with which to recommend its use in future studies. TGF-β, BMP 2 and 7, Smads 1, 4 and 5 had characteristic spatiotemporal profiles during cancellous bone defect healing suggesting that they have a regulatory role. The results of the acid study were inconclusive and problems with substance delivery and maintenance at the desired site need to be addressed in the future to fully test this hypothesis. No significant differences were detected on histology or three-point mechanical testing between the fracture calluses of acid and control groups. In the final study, OVX rats after six months had significantly increased weight and decreased bone mineral density compared to their sham counterparts. A histological delay in osteotomy healing was observed in the OVX group but no significant differences on tensile testing were seen between OVX and Sham groups up to six weeks. Immunohistochemistry revealed that delayed healing may be due to the down-regulation of IGF-1, BMP-2, 4, and 7 and the up-regulation of MMP-3 in OVX compared to Sham groups. In conclusion, the results of this thesis give some insight into the molecular biology of bone defects and osteoporotic fractures. This information may also be useful in the development of specific treatments aimed at augmenting healing in bone defects and osteoporotic fractures.

Includes bibliographical references. / 1 v. (various foliations) : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Indicates that retarded fetal growth in sheep, associated with restricted supply of substrates to the fetus, is accompanied by reduced concentrations of insulin-like growth factor I in fetal blood and its decreased production in several major fetal tissues. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1995