Defining the roles of endothelial adhesion receptors during mammary gland development, functional differentiation, and cancer

Angiogenesis is a fundamental process entailing the generation of nascent vasculature from pre- existing blood vessels. Driven by endothelial cells (ECs) in response to pro-angiogenic factors, the angiogenic cascade results in the upregulation of multiple essential proteins in these cells; including integrin-β3, integrin-α5 and neuropilin-1. All three molecules are attractive anti-angiogenic targets. Despite an array of studies supporting their druggability, attempts at targeting them in patients have failed. With breast cancer (BC) being the second most common cancer globally, gaining an understanding of how angiogenesis affects the development of the mammary gland throughout its lifecycle will provide insight into why current anti-angiogenics are ineffective. I hypothesized that the ablation of these three molecules to impede angiogenesis during different stages of the mammary life cycle would offer the opportunity to ascertain how angiogenesis influences mammary epithelial morphogenesis. I developed model systems in which I can deplete the EC expression of integrin-β3, integrin-α5 and neuropilin-1, simultaneously, and then study the effects of this depletion on the mammary gland in vivo, or on mammary ECs in vitro. My findings show that impairing the angiogenesis that is mediated by these three proteins during pubertal development of the gland has minor effects on mammary ductal branching morphogenesis. Similarly, there is little to no effect on mammary alveolar development during gestation and early lactation. However, I uncovered changes in placental development that may help to explain these findings. In contrast to what happens during the physiological life cycle of the mammary gland, I observed a significant reduction in BC growth when all three targets were depleted, suggesting pathological development of the breast is dependent on angiogenesis driven by integrin-β3, integrin-α5 and neuropilin-1. Lastly, the in vitro study of mammary ECs provide insight into potential alternative signalling pathways through which the mammary gland may achieve angiogenesis, specifically, via progesterone

The role of macrophages in the formation and repair of myelin

Foetal rat brain aggregate cultures resemble the developing brain providing a unique system to investigate myelinogenesis, demyelination and repair. Supplementing aggregate cultures with macrophages accelerated cellular organisation and increased myelin deposition over time without affecting activity of the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Pro-inflammatory cytokines and anti-myelin oligodendrocyte glycoprotein (MOG) antibodies induced demyelination in myelinated aggregate cultures while oligodendrocytes were spared. Demyelination was associated with increased levels of a myelin basic protein (MBP) degradation peptide indicating proteolysis of myelin. MBP continued to accumulate following removal of demyelinating agents while peptide levels declined. Myelinogenesis in the aggregates was associated with patterns of growth factor mRNA expression comparable with those of the developing brain. The mRNA levels of platelet-derived growth factor-A (PDGF-A), a potent mitogen for oligodendrocyte progenitors, rose rapidly while fibroblast growth factor-2 (FGF-2) and ciliary neurotrophic factor (CNTF) mRNA increased gradually as MBP accumulated. The peak of transforming growth factor-β1 (TGF-β1) and neurotrophin-3 (NT-3) mRNA expression coincided with the appearance of MBP mRNA, while that of insulin-like growth factor-I (IGF-I) was more closely associated with the detection of MBP protein. Enhanced myelination in macrophage-enriched cultures was associated with reduced expression of CNTF and increased levels of TGF-β1 and FGF-2 mRNA both of which promote oligodendrocyte development in vitro. Demyelination induced a distinct pattern of expression of many myelination-associated growth factors. A rapid rise in CNTF mRNA in standard cultures closely followed by increases in FGF-2 and IGF-I was in contrast to the delayed induction of PDGF-A mRNA. In macrophage-enriched aggregates the rise in IGF-I mRNA following demyelination preceded that in standard cultures suggesting that macrophage- enrichment instigates a faster IGF-I response during remyelination. Since macrophage-rich demyelinating multiple sclerosis lesions also display signs of remyelination, macrophages, as a source of growth factors, have the potential to promote myelination and repair