Signalling mechanisms associated with cyclin-dependent kinase 5 activities during angiogenesis

Ali, Kamela (2017) Signalling mechanisms associated with cyclin-dependent kinase 5 activities during angiogenesis. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Stroke is one of the major causes of death and disability in developing countries. It takes place when the blood supply to a fraction of the brain is abruptly interrupted or severely reduced by, for instance, a blood clot. This is known as an ischaemic stroke. A number of studies indicate that deregulation of a set of cell cycle kinases has been implicated in neural death following an ischaemic insult and in neurodegenerative disorders. Overall the cyclin-dependent kinase 5 (Cdk5) and its two activators; the p35/p25 proteins, have been highlighted as critical players in neural survival, and potential mediators of angiogenesis. Hence, a detailed understanding of the mechanisms involving them in the pathogenesis of stroke can provide a platform for therapeutic intervention and potentially enable adoption of strategies to prevent the disease. The aim of the study was to identify key regulatory factors associated with Cdk5 signalling pathway during angiogenesis in a human brain microvascular endothelial cell line (hBMECs). Two cell clones of hBMECs were generated by stable transfection; overexpression of wild-type human Cdk5 and CIP (Cdk5 inhibitory peptide). Cdk5 and p35 protein co-localisation were detected by Immunofluorescence analysis. The findings suggest that the ratio of p35/p25 is altered in favour of p25 by hypoxia. It was able to cleave p35 into a p25, which reduce the migration. The overexpression of Cdk5 increased the ratios of p35/p25 under hypoxic condition. Activation of p35/Cdk5 signalling occurred at the expense of p25-Cdk5, therefore, may have a protective role against endothelial cells apoptosis in hypoxia and positively contribute to preserving cell motility and the proper spatial and temporal control of cytoskeletal dynamics, which is essential for sprout formation (angiogenesis). Here it is shown that the protective roles of CIP are mediated through the down-regulation of HSP-70 and active caspase-3 and up-regulation of the phospho-extracellular signal-regulated kinase (p-ERK). CIP was able to protect hBMECs against apoptosis, and to allow angiogenesis to continue effectively during hypoxia. Therefore, it may be considered a potential future protector therapeutic after stroke and other brain injury.