Understanding suppression of transcytosis in formation of the blood-brain barrier (BBB) and how Calcrl/Ramp2 signalling limits BBB permeability

了解血脑屏障 (BBB) 形成过程中转胞吞作用的抑制以及 Calcrl/Ramp2 信号如何限制 BBB 通透性

• 批准号: MR/X008215/1
• 负责人: Robert Neil Wilkinson
• 金额: $ 79.7万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX008215%2F1
• 关键词: Understanding; suppression; transcytosis; formation; blood

The aim of this research is to understand how the leakiness, or permeability, of blood vessels in the brain are controlled so that we can identify new ways to treat diseases where these vessels become leaky. Blood vessels transport fluids and cells around our body. To do this effectively, the ability of substances, including liquids and cells, to pass through blood vessels, or permeability, must be tightly controlled to prevent leakage. Blood vessels within the brain are unique because they are the least leaky of all vessels within the body and so form a barrier between the circulating blood and the brain known as the blood-brain barrier (BBB). The BBB protects the brain from substances in the blood which could damage it, or could cause brain infection, while allowing important nutrients to reach the brain. In many diseases such as diabetes and stroke, increased blood vessel permeability within the brain causes damage or build-up of fluid that induces swelling. This can lead to organ failure and even death. Blood vessel permeability is controlled by signals within the body, which make vessels more, or less leaky by altering how tightly blood vessel cells stick to each other, or by controlling how easily substances can pass through blood vessel walls. For over 100 years, BBB leakiness was thought to be mainly controlled by the ability of blood vessel cells to stick tightly to each other, reducing substances passing from the blood into the brain. More recently, it has become clear that to form the BBB, blood vessel cells within the brain actively use signals which stop substances passing through vessel cell walls into the brain. In animal models of stroke, transport of substances through vessel cell walls is increased and is an early sign of barrier breakdown. This arises before stickiness of blood vessel cells deteriorates and bleeding into the brain occurs. The signals which reduce vessel leakiness are not well understood and there are no treatments to effectively reduce this in humans.We use zebrafish to understand how blood vessel leakiness is controlled since we can label blood vessels fluorescently. This allows us to observe vessel leakiness in a living organism. Importantly, zebrafish and humans share many of the signals and mechanisms which control blood vessel leakiness. We have made zebrafish that have leaky blood vessels within the brain. Using these zebrafish we have identified a new signal (Calcrlb/Ramp2a) which reduces vessel leakiness by making it harder for substances to pass through blood vessel walls into the brain. In humans, differences in Ramp2 and Calcrl are linked with increased risk of stroke. We want to understand precisely how Ramp2a/Calcrlb prevent vessels becoming leaky because this may allow us to identify new ways of treating vessel leakiness in people with diseases such as stroke, diabetes, or vascular dementia. Using our zebrafish with reduced Calcrlb/Ramp2a signals (these signals normally prevent blood vessels from becoming leaky), we will anaesthetise zebrafish embryos, inject small amounts of fluorescent dye into their blood vessels, or fluorescently label their blood vessels, and measure the amount of leakiness using our lightsheet microscope. The dyes we use are very large and cannot leak from normal blood vessels. We will also treat our zebrafish Calcrlb/Ramp2a mutant embryos with drugs that reduce the ability of substances to pass through blood vessel walls and measure how leaky the blood vessels are afterwards. We can also label the places where blood vessel cells are joined together in our Ramp2a/Calcrlb embryos and by using a very high magnification electron microscope, see if these areas are abnormal when the blood vessels become leaky. We will also identify which signals are increased and reduced in brain blood vessels in our Calcrlb/Ramp2a mutants. These experiments will tell us exactly how Calcrlb/Ramp2a signals prevent brain blood vessels from becoming leaky.

这项研究的目的是了解大脑中血管的泄漏或渗透性如何受到控制，以便我们可以确定这些血管泄漏的新方法来治疗疾病。血管在我们体内运输液体和细胞。为了有效地做到这一点，必须严格控制包括液体和细胞在内的物质（包括液体和细胞）通过血管或渗透率的能力，以防止泄漏。大脑内的血管是独一无二的，因为它们是体内所有血管的最小泄漏，因此在循环血和被称为血脑屏障（BBB）的大脑之间形成障碍。 BBB可保护大脑免受血液中可能损害其损害或可能引起大脑感染的物质，同时使重要的营养成分到达大脑。在许多疾病（例如糖尿病和中风）中，大脑内的血管渗透性增加会导致造成肿胀的液体损害或积累。这可能导致器官衰竭，甚至导致死亡。血管渗透性受体内信号的控制，通过改变血管细胞彼此粘附的紧密性或控制物质如何轻松地通过血管壁，使血管更加漏水，或者减少漏水。 100多年来，人们认为BBB泄漏主要由血管细胞彼此紧密粘附的能力控制，从而减少了从血液进入大脑的物质。最近，很明显，要形成BBB，大脑中的血管细胞会主动使用信号，以阻止物质通过血管细胞壁进入大脑。在中风的动物模型中，物质通过血管细胞壁的运输增加，是障碍物崩溃的早期迹象。这是在血管细胞的粘性变质和出血到大脑之前就产生的。减少血管泄漏的信号尚不清楚，并且没有治疗方法可以有效地减少人类。我们使用斑马鱼来了解如何控制血管泄漏，因为我们可以将血管荧光标记。这使我们可以观察到生物体中的血管泄漏。重要的是，斑马鱼和人类共享控制血管泄漏的许多信号和机制。我们制作的斑马鱼在大脑内有血管漏水。使用这些斑马鱼，我们已经确定了一个新信号（CalCRLB/RAMP2A），该信号通过使物质更难通过血管壁进入大脑而难以减少血管泄漏。在人类中，RAMP2和CALCRL的差异与中风的风险增加有关。我们想精确地了解RAMP2A/CALCRLB如何防止船只泄漏，因为这可能使我们能够确定治疗中风，糖尿病或血管性痴呆等疾病患者的血管泄漏的方法。使用我们的斑马鱼和降低的Calcrlb/ramp2a信号（这些信号通常可以防止血管渗漏），我们将使斑马鱼的胚胎减少斑马鱼的胚胎，将少量的荧光染料注入血管中，或使用荧光标记血管，并使用我们的光线镜来测量漏水的量。我们使用的染料非常大，无法从正常血管中泄漏。我们还将使用药物来治疗斑马鱼Calcrlb/ramp2a突变体胚胎，从而降低物质通过血管壁的能力并测量后来血管漏水的能力。我们还可以将血管细胞在我们的RAMP2A/CalCRLB胚胎中连接在一起的地方并使用很高的放大倍数电子显微镜，查看这些区域是否在血管漏出时是否异常。我们还将确定在CalCRLB/RAMP2A突变体中，脑血管中有更多信号并减少了哪些信号。这些实验将准确地告诉我们CalCRLB/RAMP2A信号如何防止脑血管泄漏。