Project title: Endothelial cell extracellular vesicles in cerebral small vessel disease

脑部小血管疾病(SVD)是血管性痴呆最常见的原因，也是阿尔茨海默病的原因之一，其最大的危险因素是年龄的增加。然而，尽管很常见，但还没有直接针对病理学的治疗方法。LACI-2试验(https://www.ed.ac.uk/usher/edinburgh-clinical-trials/our-studies/ukcrc-studies/laci)刚刚完成了两种用途改变的药物(西洛他唑和单硝酸异山梨酯)对SVD患者的疗效测试，并将很快报告。这些药物被选为稳定内皮细胞(EC)功能的药物，目前已知SVD的一个关键机制是EC调节失调。这导致内皮细胞和周围脑细胞之间的串扰改变，特别是白质的少突胶质细胞和髓鞘，以及改变血流和血脑屏障的完整性。我们已经在一只转基因大鼠(Atp11bKO)上建立了SVD模型，该模型显示了EC失调、白质异常、磁共振成像和与人类SVD3一致的行为变化。此模型中的ECS缺乏ATP11B，该基因中的SNP与散发性人类SVD2相关。人类脑白质的改变被用来诊断SVD，其程度的增加与认知功能的恶化有关，但我们还不清楚EC失调是如何引起脑白质改变的继发性影响的。由于ATP11B参与囊泡通过内质网的释放，我们假设这种串扰可能是通过从内皮细胞释放到周围大脑和血液的细胞外小泡(EV)。这个博士项目将解决这一假说，在大鼠EC培养和体内采用候选方法(已确定的候选对象)和公正的方法(蛋白质组学)，然后在可能的情况下将其转移到人类。目的1)使用候选方法(来自当前工作)和无偏倚的蛋白质组学方法，在Atp11bKO大鼠模型和对照组中确定不同年龄的大鼠脑内皮细胞EVS的数量和含量，并在体内的分离细胞和血清循环中循环。2)在用西洛他唑和ISMN治疗后，在Atp11bKO大鼠模型和对照组中重复这一方法，以反映Laci-2试验方案，3)分析已经收集的LACI-2试验患者血清中的EVS(使用SIMOA技术)以寻找关键候选对象，并与试验反应相关联，以确定是否可以在此基础上对患者进行分层。

Cerebral small vessel disease (SVD) is the commonest cause of vascular dementia, and also contributes to Alzheimer's disease, with its biggest risk factor being increasing age. However, although very common, there are no therapies that directly target the pathology1. The LACI-2 trial (https://www.ed.ac.uk/usher/edinburgh-clinical-trials/our-studies/ukcrc-studies/laci) has just finished testing two repurposed drugs (Cilostazol and isosorbide mononitrate (ISMN)) for effect in patients with SVD, and is due to report very soon. These drugs have been chosen as they stabilize endothelial cell (EC) function and it is now known that a key mechanism in SVD is EC dysregulation. This leads to changes of crosstalk between ECs and surrounding brain cells, particularly the oligodendrocytes and myelin of the white matter2, as well as altering blood flow and blood brain barrier integrity. We have generated a model of SVD in a transgenic rat (Atp11bKO) which shows EC dysregulation, white matter abnormalities, Magnetic resonance imaging and behavioural changes consistent with human SVD3. ECs in this model lack ATP11B and a SNP in this gene is associated with sporadic human SVD2. The white matter changes in human are used for diagnosis of SVD, and increased extent correlates with worse cognition, yet we are unclear about how EC dysregulation causes the secondary effect of white matter change. As ATP11B is involved in vesicular release through the endoplasmic reticulum3, we hypothesise that this crosstalk may be through extracellular vesicles (EVs) released from ECs to the surrounding brain, as well as into the blood.This PhD project will address this hypothesis, taking both a candidate approach (with already identified candidates4) and an unbiased approach (proteomics) in rat EC cultures and in vivo, and then moving this to humans where possible, to determine if there is a correlation between these levels and response in the LACI-2 trial.Aims1) Determine the number and content of rat brain EC EVs in the Atp11bKO rat model and controls at different ages in isolated cells and circulating in serum in vivo, using a candidate approach (from current work) and an unbiased approach using proteomics.2) Repeat this in the Atp11bKO rat model and controls after treatment with cilostazol and ISMN, to reflect the LACI-2 trial protocol, to identify if the cargo changes after treatment and can be correlated to outcome/hallmarks of SVD.3) Analyse EVs from already collected LACI-2 trial patient serum (using Simoa technology) for key candidates and correlate with trial response, to determine if patients can be stratified on this basis.