The role of c-Rel in endothelial mechanobiology and atherosclerosis

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Summary - Atherosclerosis develops preferentially at branches and bends of arteries exposed to low shear stress. Evans' group recently demonstrated that the transcription factor c-Rel was expressed preferentially at the low shear site, suggesting a potential role in regulating atherogenesis. The function of c-Rel in endothelial cells (EC) is poorly described but it is known to promote anti-apoptotic signalling in epithelial and other cell types. This Studentship will investigate the function of c-Rel in EC exposed to flow using in vitro systems and murine models. The influence of c-Rel on lesion patterning will be studied by deleting c-Rel from EC in hypercholesterolemic mice. The study brings together Evans' knowledge of endothelial mechanobiology with Oakley's expertise in c-Rel signalling.Hypothesis - It is hypothesised that c-Rel protects against lesion formation by limiting EC apoptosis and dysfunction at low shear sites.Experimental aims1. Does c-Rel reduce injury and dysfucntion of cultured EC exposed to flow? Cultured EC will be exposed to low or high shear using systems established in Evans' lab (parallel plate and orbital flow). The expression of c-Rel will be silenced using siRNA or enhanced by delivery of an expression plasmid containing c-Rel cDNA (available in Oakley's lab). Apoptosis will be assessed by cleavage of Caspase-3 and TUNEL and senescence (known to be induced by low shear) will be studied by measuring SA-b-Gal, p53 and p16. We predict that c-Rel silencing will enhance apoptosis and senescence in sheared EC and overexpression of c-Rel will have the opposite effect.2. Does c-Rel protect against EC dysfucntion at low shear sites in vivo? Wild-type mice will be compared with c-Rel knockout mice (available in Oakley's lab). En face staining for cleaved caspase-3 or TUNEL (apoptosis) and SA-b-Gal/p53/p16 (senescence) will be studied in EC at low (inner curvature of aortic arch) and high (outer curvature) shear sites. It is predicted that c-Rel deletion will enhance EC apoptosis and senescence at low shear sites.3. Does c-Rel regulate the spatial localisation of atherosclerosis? c-Rel knockout mice will be studied. Hypercholesterolemia will be induced using an adenovirus containing PCSK9 (recently established in Sheffield by Evans). After 6-12 weeks, lesion size will be assessed by oil red O staining of the aorta and aortic root. Plaque cellular composition will be studied by immunohistochemistry. It is predicted that c-Rel deletion will alter the patterning of atherosclerosis by enhancing lesion formation at low shear sites.Value -The work will identify c-Rel as a potential therapeutic target in early atherosclerosis.