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

脑血管病（SVD）是血管性痴呆的最常见原因，也是阿尔茨海默病的诱因，其最大的危险因素是年龄的增长。然而，虽然很常见，但没有直接针对该病理的治疗方法。LACI-2试验（https://www.ed.ac.uk/usher/edinburgh-clinical-trials/our-studies/ukcrc-studies/laci）刚刚完成了两种重新用途的药物（西洛他唑和单硝酸异山杉酯（ISMN））对SVD患者的效果的测试，并将很快发布报告。选择这些药物是因为它们能稳定内皮细胞（EC）的功能，现在已经知道SVD的一个关键机制是EC失调。这导致内皮细胞与周围脑细胞，特别是少突胶质细胞和白质髓磷脂之间的串扰发生变化，并改变血流量和血脑屏障的完整性。我们在转基因大鼠（Atp11bKO）中建立了SVD模型，该模型显示EC失调，白质异常，磁共振成像和行为变化与人类SVD3一致。该模型中的ECs缺乏ATP11B，该基因的SNP与散发性人类SVD2相关。人类白质变化用于SVD的诊断，其升高程度与认知能力下降相关，但EC失调如何引起白质变化的继发效应尚不清楚。由于ATP11B通过内质网参与囊泡释放3，我们假设这种串扰可能通过细胞外囊泡（EVs）从ECs释放到周围的大脑，以及进入血液。该博士项目将解决这一假设，在大鼠EC培养和体内采用候选方法（已经确定的候选方法4）和无偏倚方法（蛋白质组学），然后在可能的情况下将其转移到人类身上，以确定这些水平与LACI-2试验中的反应之间是否存在相关性。目的1)采用候选方法（来自当前工作）和无偏倚的蛋白质组学方法，确定Atp11bKO大鼠模型和不同年龄的对照组中分离细胞和体内血清循环的大鼠脑EC ev的数量和含量。2)在使用西洛他唑和ISMN治疗后的Atp11bKO大鼠模型和对照组中重复此操作，以反映LACI-2试验方案，确定治疗后货物是否发生变化，是否与svd的结局/特征相关。3)分析已收集的LACI-2试验患者血清中的ev（使用Simoa技术），以确定关键候选物并与试验反应相关，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.