The role of c-Rel in endothelial mechanobiology and atherosclerosis

总结：动脉粥样硬化优先发生在低剪切应力下的动脉分支和弯曲处。Evans的研究小组最近发现转录因子c-Rel优先在低剪切位点表达，这表明它在调节动脉粥样硬化中具有潜在的作用。c-Rel在内皮细胞（EC）中的功能描述很少，但已知它可以促进上皮细胞和其他细胞类型的抗凋亡信号传导。本研究将使用体外系统和小鼠模型研究c-Rel在暴露于血流中的EC中的功能。c-Rel对病变模式的影响将通过从高胆固醇血症小鼠EC中删除c-Rel来研究。该研究将Evans的内皮力学生物学知识与Oakley在c-Rel信号方面的专业知识结合在一起。假设-假设c-Rel通过限制EC的凋亡和低剪切位点的功能障碍来防止病变的形成。实验aims1。c-Rel是否能减少暴露于血流中的培养EC的损伤和功能障碍？培养的EC将使用Evans实验室建立的系统（平行板和轨道流）暴露于低剪切或高剪切下。c-Rel的表达将使用siRNA沉默或通过传递含有c-Rel cDNA的表达质粒（可在Oakley的实验室中获得）来增强。凋亡将通过Caspase-3和TUNEL的切割来评估，衰老（已知由低剪切诱导）将通过测量SA-b-Gal， p53和p16来研究。我们预测c-Rel沉默会促进剪切EC细胞的凋亡和衰老，而过表达c-Rel则会产生相反的效果。c-Rel在体内低剪切位点保护EC功能障碍吗？野生型小鼠将与c-Rel敲除小鼠（可在Oakley的实验室中获得）进行比较。将在EC低（主动脉弓内弯曲）和高（外弯曲）剪切部位研究劈裂caspase-3或TUNEL（凋亡）和SA-b-Gal/p53/p16（衰老）的表面染色。预测c-Rel缺失会促进EC低剪切位点的凋亡和衰老。c-Rel是否调节动脉粥样硬化的空间定位？将对c-Rel敲除小鼠进行研究。将使用含有PCSK9的腺病毒诱导高胆固醇血症（最近由Evans在谢菲尔德建立）。6-12周后，通过主动脉及主动脉根部油红O染色评估病变大小。斑块细胞组成将通过免疫组织化学进行研究。据预测，c-Rel缺失将通过增强低剪切位点的病变形成来改变动脉粥样硬化的模式。价值：这项工作将确定c-Rel作为早期动脉粥样硬化的潜在治疗靶点。

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.