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Abstract: In response to hypoxic conditions, tumours secrete pro-angiogenic factors that stimulate the development of new blood vessels. Given the critical role of angiogenesis in tumour progression, therapies counteracting cancer neovascularization have been developed. However, clinical studies have shown these therapies to be only temporarily effective; thus, further research is needed to enhance our understanding of anti-angiogenic signalling. Our research has focused on elucidating the anti-angiogenic response stimulated by thrombospondin-1 (TSP-1) binding to cell surface receptor CD36. While F-actin, Fyn (an Src-family kinase), and CD36 nanoclusters are essential components of TSP1-induced anti-angiogenic response, the interactions between these molecules have yet to be discovered. Given the small size of CD36 cytosolic domains, we hypothesize that additional proteins within CD36 nanoclusters are crucial for its function. By identifying and investigating the role of these proteins, we seek to decipher the set of interactions necessary for CD36 anti-angiogenic signalling and organization. Here we developed a proximity biotinylation technique to identify proteins in the vicinity of CD36 via mass spectrometry. Proximity biotinylation has revealed a list of candidate proteins potentially important in CD36 signalling and organization. shRNA knockdown of candidate proteins revealed integrin beta-1 (ITGB1) to be essential for CD36-Fyn signalling. Furthermore, proximity ligation assay and conditional colocalization analysis provide support for a CD36, ITGB1, and CD9 molecular complex, implicated in anti-angiogenic signalling. Our approach to investigating CD36 nanodomains has not only revealed potential targets to reduce tumour angiogenesis it has also provided a roadmap to investigate the organization of other membrane proteins within their native states.

Angiogenesis is the growth of new blood vessels from existing vasculature; it requires the breakdown of existing blood vessel walls followed by the migration and proliferation of endothelial cells to form the new vessels. It is a complex process that is regulated by many pro- and anti-angiogenic factors and the roles of some of these factors are still unclear. Angiogenesis is a key feature of many pathological conditions including cancer, polycystic ovary syndrome and endometriosis so is an area of great research interest. There are several methods currently available for the study of angiogenesis, both in vitro and in vivo, and whilst all of these methods have enhanced understanding of angiogenesis, they also have limitations. The ovary is an excellent model for the study of angiogenesis as it undergoes intense vascular morphogenesis in a cyclical manner. The female reproductive system is unique as no other healthy adult tissue undergoes spontaneous angiogenesis. The tissues in the ovary undergo constant remodelling during both folliculogenesis and the formation and regression of the corpus luteum. Blood vessels are recruited from the ovarian stroma at the preantral stage to form vascular sheaths, in the thecal layer, which surround the developing follicle and supply nutrients, hormones and allow gaseous exchange. As follicular development progresses to the antral stage, when gonadotrophin-dependence is established, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Previous research into ovarian angiogenesis has focussed on the corpus luteum but the mechanisms of the regulation of angiogenesis during folliculogenesis need further elucidation. The work in this thesis aims to develop and utilise an in vitro angiogenesis assay using the culture of intact preantral and early antral follicles to provide a new approach to the study of follicular angiogenesis. During the course of this thesis this assay was utilised to investigate the effect of various factors on follicular angiogenesis and ovarian function. The role of the putative anti-angiogenic factor thrombospondin-1 (TSP-1) in the regulation of physiological angiogenesis was investigated using the in vitro angiogenesis assay developed during the course of this thesis and the role of TSP-1 in normal ovarian function was investigated using the culture of isolated granulosa cells. The results suggest that TSP-1 is able to inhibit angiogenesis and that it has an extravascular role in the ovary, in vitro. These findings were extended to an in vivo angiogenesis model where follicular angiogenesis was assessed by quantitative immunohistochemistry for bromodeoxyuridine and the endothelial cell marker CD31. The extravascular role for TSP-1 was also further investigated in vivo and was assessed by quantitative immunohistochemistry for activated caspase-3. The results confirmed the findings of the in vitro study, indicating that TSP-1 has antiangiogenic action and acts to clear non-dominant follicles from the ovary through the induction of atresia. Vascular endothelial growth factor (VEGF) is the main factor involved in stimulating angiogenesis and many advances have been made into elucidating the role, and the mechanisms of action, of VEGF on angiogenesis. Angiopoietin-1 (Ang-1) is considered to be one of the main factors acting in concert with VEGF to stabilise new blood vessels and its role in angiogenesis has been the subject of much discussion and controversy. This thesis investigates the effects of Ang-1 on follicular angiogenesis and development, using the in vitro angiogenesis assay, granulosa cell culture and RNA knockdown experiments. The results have shown that Ang-1 can induce follicular angiogenesis at high doses and that at low doses stimulates prosurvival pathways and inhibits apoptotic mediators. This thesis describes a novel in vitro culture system for the study of angiogenesis in ovarian follicles. Using this system the effects of various factors on follicular angiogenesis and on follicle development and survival have been investigated. Investigations into the mechanisms of action of these factors have also been performed. These studies have improved understanding of the regulation of follicular angiogenesis and have indicated extravascular roles for angiogenic factors in the ovary. Since angiogenesis is a key feature of many pathological conditions, the ability to manipulate angiogenesis and to investigate and quantify the effects of proor anti-angiogenic compounds may have important clinical implications.

Tumors are heterogeneous systems, whose growth is influenced by intrinsic properties of malignant cells, external systemic factors (i.e. immune, neural, endocrine, etc.), and the dynamic interactions between tumor cells and their microenvironment. Given the inherent complexity of cancers, combined with the continual evolution of tumors and the development of treatment resistance, a precision medicine approach may not provide an optimal clinical response. Exploring a new paradigm that focuses on regulating cancer as a system may not only control tumor progression but also address the extraordinary challenges of tumor heterogeneity and disease recurrence in order to improve clinical outcomes. As a group of discrete, growth-restricted tumor colonies that regulate their own growth and secrete a large number of tumor-inhibitory signals, RENCA macrobeads function as a biological-system, providing the opportunity for a systems-therapeutic approach to cancer management. Previous work has demonstrated that RENCA macrobeads restrict the growth of various cancer cells both in vitro as well as in preclinical and clinical studies; however, the molecular mechanism(s) of this inhibition is unknown. In this study, we demonstrated that factors secreted by RENCA macrobeads significantly altered the transcript levels of multiple MEF2 isoforms in targeted tumor cells. Suppression of various MEF2 isoforms markedly reduced the growth inhibitory effect of RENCA macrobeads and abrogated macrobead induced S-phase arrest. Importantly, we identified an essential role for the MEF2D isoform in mediating RENCA macrobead-induced inhibition. In addition, the cell-surface receptor, EGFR, was shown to be involved in the anti-proliferative response to RENCA macrobeads. Growth inhibition was more robust in cells overexpressing EGFR and was associated with cell accumulation in S-phase. In cell lines with reduced EGFR kinase activity or low-levels of cell-surface receptor, we demonstrated that RENCA macrobeads inhibited growth, although to a lesser degree and exhibited G2/M arrest, supporting the notion that factors secreted by RENCA macrobeads regulate multiple cell cycle checkpoints. Lastly, we identified three proteins in conditioned media of RENCA macrobeads (RTN4, TSP1, TIMP2) that partially contribute to growth regulation of external tumor cells with functional EGFR activity. Moreover, we identified a novel role for these proteins in modulating MEF2 activity and regulating MEF2 expression, particularly the MEF2D isoform.Overall, these studies support a mechanism by which RENCA macrobeads, at least partially, regulate tumor growth external to the macrobead. These findings could identify patients most likely to benefit from RENCA macrobead therapy.

Limb and digit loss poses a significant problem across the animal kingdom. Ambystoma mexicanum, commonly known as the axolotl, however, is one species that has achieved a remarkable ability to bypass the misfortune associated with a lost limb. Viewed as a model organism in regenerative studies, the axolotl retains extraordinary regenerative properties well into adulthood that humans severely lack. While the basics of regeneration have been described, much about the molecular processes of regeneration is still largely unknown. Thrombospondin-1 (TSP-1), an angiogenesis inhibitor, has been identified as a potential factor to play a significant role in the regrowth of limbs. Vascularization of tissues is vital to the survival of biological structures, and TSP-1 has been shown to play a regulatory role in the development and remodeling of tissue vasculature. Here, we study the effect of a loss-of-function mutation in the tsp-1 gene on the process of limb regeneration in the axolotl. Our studies reveal that tsp-1 -/- animals lag in regeneration time, developing smaller blastemas in the first three weeks of regeneration. We show that the loss of TSP-1, however, is not deleterious to the overall process of regeneration as late stage blastemas of the -/- animals catch up in size and development to the wild type animals after three weeks. Our data suggests that while TSP-1 may be important during the initial stages, it may not be required for proper regeneration.

CD36 is a multiligand scavenger receptor expressed in many cell types, including microvascular endothelial cells (MVEC). Binding of thrombospondin-1 to CD36 inhibits angiogenesis by inducing apoptosis of growth factor stimulated MVEC and also by inhibiting growth factor signaling at the receptor level. CD36 phosphorylation on its extracellular domain inhibits binding of thrombospondin-1. The mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells are not known. Here we determined structure-function relationships of CD36 phosphorylation related to thrombospondin-1 peptide binding in vitro and explored mechanisms regulating phosphorylation by protein kinase C (PKC) in CD36 transfected melanoma cells. Treatment of CD36-transfected melanoma cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, induced substantial CD36 phosphorylation and decreased ligand-mediated recruitment of Src-family proteins to CD36. PMA treatment did not induce detectable extracellular or cell surface-associated kinase activity, and both cycloheximide and brefeldin A blocked CD36 phosphorylation. New protein synthesis and trafficking through the Golgi were required for PMA-induced CD36 phosphorylation, suggesting that phosphorylation probably occurs intracellularly. These studies suggest a novel in vivo pathway for CD36 phosphorylation that modulates cellular affinity for thrombospondin-related proteins to blunt vascular cell signaling. Thrombospondin-1 inhibits vascular endothelial growth factor (VEGF) signaling at the receptor level in MVEC and CD36 has been suggested to be involved in this inhibition, however, the mechanisms are not known. Here we showed CD36 is required for the inhibition by silencing CD36 expression in MVEC by siRNA or genetic deletion of cd36 in mice. Immunoprecipitation experiments showed that CD36 is required for thrombospondin-induced association of a phosphatase, SHP-1, with VEGFR2 in MVEC. Phosphatase activity was increased in precipitated VEGFR2 complexes from thrombospondin treated cells, consistent with thrombospondin-1-induced SHP-1 association with the VEGFR2 complex. By using an in vitro endothelial tube formation assay, we showed that thrombospondin-1 inhibited VEGF-induced tube formation of MVEC on matrigel, while CD36 silenced cells and SHP-1 inhibitor treated cells were less responsive to thrombospondin-1. These studies suggest a mechanism by which thrombospondin-1 inhibits VEGF signaling at the receptor level. Together the results, we provide new understanding of the role of CD36 in regulation of angiogenesis.

Ischaemia/reperfusion (IR) is the commonest cause of acute kidney injury which is a major cause of morbidity and an important contributing factor to mortality in patients with a wide range of medical and surgical conditions. IR often occurs in an unpredictable manner but in certain situations, most notably organ transplantation its occurrence can be pre-empted thus allowing consideration of preventative therapies. IR injury is an inflammatory process involving complex interactions between resident and infiltrating cells of both innate and cognate arms of the immune system. The immediate sequelae of renal IR are necrosis of tubular epithelial cells associated with an acute decline in renal function. Chronic kidney injury may follow and is preceded by endothelial cell injury and loss of peritubular capillaries. The mechanisms linking acute and chronic injury, or more specifically acute inflammatory tubular damage and long-term endothelial cell injury, are not well understood. The tissue factor (TF) initiated coagulation pathway has been shown to contribute to renal IR injury. Signalling by TF and the coagulation protease thrombin leads to chemokine upregulation, neutrophil infiltration and kidney injury after IR. These effects are primarily dependent on activation of the thrombin receptor protease activated receptor (PAR)-1. TF and thrombin may promote survival and growth of endothelial cells; therefore it is possible that inhibition of TF or PAR-1 signalling could have deleterious effects on endothelial cells after IR. The primary aims of the studies described in this thesis were two-fold:- 1. To characterise the expression of factors contributing to endothelial cell survival or apoptosis after renal IR and determine whether the TF-thrombin-PAR-1 system is involved in their regulation. 2. To determine whether signalling by the cytoplasmic domain of TF, which does not promote thrombin production but may signal directly, is involved in renal IR injury. Results A murine model of 25 min of warm renal ischaemia was used to explore the regulation of angiogenic factors. Differential regulation of the three predominant isoforms of vascular endothelial growth factor (VEGF) was detected in kidneys during the first 24 h after ischaemia. The expression of VEGF164 and 188 was suppressed after IR. Despite this down-regulation of the main isoforms, the level of VEGF protein in kidneys was not altered by IR. Circulating levels of VEGF were increased suggesting that VEGF was generated at extra-renal sites after IR. Angiopoietin-1 (Ang-1) was also down-regulated in kidneys after IR. The expression of VEGF receptors, angiopoietin-2 and angiopoietin receptor were not significantly altered after IR. Thus, angiogenic factors were down-regulated in kidneys after IR, potentially predisposing endothelial cells to apoptosis. Thrombin inhibition did not significantly alter this pattern of expression. However, thrombin inhibition or PAR-1 deletion prevented up-regulation of the anti-angiogenic factor thrombospondin-1 and the pro-apoptotic caspase-3, suggesting that PAR-1 signalling could potentially contribute to endothelial cell injury after IR. Mice with deletion of the cytoplasmic domain of TF generated on a mixed genetic background (TFCT/CT) developed more severe renal impairment and tubular injury after IR than wild type strain control mice. The expression of the chemokine KC was greater in TFCT/CT mice than wild-type and this was associated with a higher number of infiltrating neutrophils in kidneys. PAR-1 was up-regulated to a greater degree in TFCT/CT mice and pharmacologic inhibition of PAR-1 abrogated their more severe injury, suggesting that the cytoplasmic domain of TF may inhibit PAR-1 signalling. PAR-2 deletion did not alter renal IR injury. The effect of the TF cytoplasmic domain on renal injury appeared to be strain dependent, because the renal impairment of fully backcrossed mice with TF cytoplasmic domain deletion was not different to wild-type mice after IR.

Objetive: Thrombospondin-1 serum levels is correlate with pelvic pain in patients with ovarian endometriosis. Patients: Thrombospondin-1 serum levels were prospectively analysed in 51 patients (group A asymptomatic patients or patients presenting mild dysmenorrhea and women comprised group B severe dysmenorrhea and/or chronic pelvic pain and/or dyspareunia) who underwent surgery for cystic ovarian endometriosis to asses whether a correlation exists among thrombospondin-1 serum levels and pelvic pain. Results: From 56 patients, five cases were ultimateley excluded, because the histological diagnosis was other than cystic ovarian endometriosis (2 teratomas and 3 haemorragic cysts). The mean thrombospondin-1 serum levels in group A was 256,69 pg/ml_+37,07 and in group B was 291,41 pg/ml + 35,59. Conclusion: Pain symptoms in ovarian endometriosis is not correlated with thrombospondin-1 serum levels.