Investigating notch/Dll4 signalling in retinal microvascular cells in health and disease

Endothelial cells, which form the inner lining of all blood vessels, maintain various critical functions to enable the efficient transport of oxygen and nutrients to all metabolising cells of the body. Finely tuned regulation of endothelial function is important for the establishment and maintenance of an efficient and functional vascular transport system. As such, endothelial dysfunction can result in the development of tissue ischaemia and has been highlighted as a major step in the development of various degenerative diseases that affect the micro and macro vasculature including a variety of retinopathies such as Retinopathy of Prematurity (ROP). A vast repertoire of signalling pathways have been identified in regulating the functional maintenance of endothelial cells. Two such pathways known to have crucial regulatory roles governing endothelial function are the Notch and endothelial nitric oxide synthase (eNOS) signalling pathways: currently, the interaction between these pathways is poorly described. In part 1 we developed a heterologous system to investigate the effect of eNOS on Notch activation by overexpressing eNOS in HEK 293 cells; cells which are free of endogenous eNOS. eNOS overexpression resulted in increased cell-cell aggregation as well as increased N-Cadherin and Notch1 expression, the latter of which correlated with increased availability of the Notch intracellular domain (NICD). The results from chapter1 implicate eNOS in promoting an epithelial-like phenotype with increased cell-cell adhesion which was associated with enhanced Notch1-Dll4 signalling. In the presence of induced Dll4 expression the epithelial-like phenotype was further enhanced culminating in excessive cell-cell aggregation and the lack of any observable effect with γ-secretase inhibition indicated that augmented Dll4/Notch1 binding enhances cell aggregation. This provides evidence of the presence of Dll4 and Notch1 expression within adjacent cells leading to heterotypic cell-cell binding/adhesion. In part 2 of the study we investigated the functional role of constitutively expressed Dll4 on Notch1 activation in mature primary retinal microvascular endothelial cells (RMECs). We demonstrated using lentiviral-mediated shRNA knockdown that Dll4 has important roles in mediating cell-cell junction and that removal of Dll4 induces an enlarged senescent-like phenotype. These findings were linked to decreased Notch1 activation evidenced by reduced NICD levels indicating Notch 1 as the receptor mediating these effects. In retinal pericytes, the Notch1 receptor also showed evidence of regulating smooth muscle properties through increased Notch 3 signalling. Overall, these findings suggest a previously unknown role for the Notch1 isoform in retinal pericytes (RP) and endothelial cells (EC) in facilitating EC-P interaction. In part 3 using hyperoxia to model the early pathology of ROP we investigated the effect of hyperoxia on EC and on Notch signalling in particular. We demonstrated hyperoxia to have detrimental effects upon endothelial functions such as decreased proliferation with increased senescence and apoptosis while inducing a phenotypic switch to an elongated, spindle-shaped morphology with evidential decreased cell-cell adhesion as shown from epifluorescent and phase contrast images. We also presented evidence of increased Notch signalling under hyperoxia which included increased Notch1 cleavage, upregulation of target genes and activation of a fluorescent Notch1 reporter. Such target genes included the arterial markers Dll4 and Hey2, thus suggesting an arterial phenotype switch under hyperoxia. Collectively, the results generated in this thesis provide new insights into the complexity of the Dll4 and Notch1 signalling pathway in particular the differential signalling dynamics observed in the presence of eNOS and identified functional roles of Dll4 in mature retinal EC. The current study also identified detrimental effects of hyperoxia on endothelial function including decreased proliferation coinciding with increased apoptosis and senescence, all of which correlated with increased Notch signalling. The results provide insight into the pathology of ROP on a molecular and cellular level in particular the role of Notch1.