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如何控制大脑血管的渗漏或渗透性，以便我们能够找到治疗这些血管渗漏疾病的新方法。血管在我们的身体周围运输液体和细胞。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通过血管的能力或渗透性，以防止泄漏。脑内的血管是独特的，因为它们是身体内所有血管中泄漏最少的，因此在循环血液和大脑之间形成屏障，称为血脑屏障（BBB）。血脑屏障保护大脑免受血液中可能损害大脑或可能导致大脑感染的物质的伤害，同时允许重要的营养物质到达大脑。在许多疾病中，如糖尿病和中风，脑内血管通透性增加会导致损伤或液体积聚，从而引起肿胀。这可能导致器官衰竭甚至死亡。血管渗透性由体内的信号控制，通过改变血管细胞彼此粘附的紧密程度，或通过控制物质穿过血管壁的容易程度，使血管变得更多或更少泄漏。100多年来，BBB泄漏被认为主要是由血管细胞相互紧密粘附的能力控制的，减少了从血液进入大脑的物质。最近，已经清楚的是，为了形成BBB，脑内的血管细胞积极地使用信号，该信号阻止物质通过血管细胞壁进入脑。在中风的动物模型中，物质通过血管细胞壁的运输增加，这是屏障破坏的早期迹象。这在血管细胞的粘性恶化和出血进入大脑之前出现。减少血管渗漏的信号还没有得到很好的理解，也没有有效减少人类血管渗漏的治疗方法。我们使用斑马鱼来了解血管渗漏是如何控制的，因为我们可以用荧光标记血管。这使我们能够观察生物体中的血管泄漏。重要的是，斑马鱼和人类共享许多控制血管渗漏的信号和机制。我们已经制造出了脑内血管渗漏的斑马鱼。利用这些斑马鱼，我们发现了一种新的信号（Calcrlb/Ramp 2a），它通过使物质更难穿过血管壁进入大脑来减少血管渗漏。在人类中，Ramp 2和Calcrl的差异与中风风险增加有关。我们希望准确了解Ramp 2a/Calcrlb如何防止血管渗漏，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血管渗漏的新方法。使用我们的斑马鱼减少Calcrlb/Ramp 2a信号（这些信号通常防止血管变得泄漏），我们将麻醉斑马鱼胚胎，将少量荧光染料注入血管，或荧光标记血管，并使用我们的光片显微镜测量泄漏量。我们使用的染料非常大，不能从正常血管中泄漏。我们还将用药物治疗我们的斑马鱼Calcrlb/Ramp 2a突变胚胎，这些药物可以降低物质通过血管壁的能力，并测量之后血管的泄漏程度。我们还可以标记我们的Ramp 2a/Calcrlb胚胎中血管细胞连接在一起的地方，并通过使用非常高放大率的电子显微镜，看看这些区域在血管渗漏时是否异常。我们还将确定在我们的Calcrlb/Ramp 2a突变体中脑血管中哪些信号增加和减少。这些实验将确切地告诉我们Calcrlb/Ramp 2a信号如何防止脑血管渗漏。