How does Vascular Smooth Muscle-specific master splicing regulator - Rbpms - contribute to vascular performance in vivo?

血管平滑肌特异性主剪接调节因子 Rbpms 如何促进体内血管性能？

• 批准号: MR/X018776/1
• 负责人: Sanjay Sinha
• 金额: $ 90.27万
• 依托单位: University of Cambridge
• 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%2FX018776%2F1
• 关键词: How; does; Vascular; Smooth; Muscle

Vascular smooth muscle cells (VSMCs) are an important cell type lining blood vessel walls. In a normal, mature, healthy state, these cells contract and help to maintain the blood pressure and proper blood flow. However, in some cardiovascular diseases (e.g. atherosclerosis) these cells respond by changing to immature forms that no longer function normally but contribute to disease progression. What we don't fully understand is how this change, called "phenotype switching" occurs in disease and injury and what are the molecular mechanisms causing this. Specifically, we are studying a molecular process called "alternative splicing". This is a method by which the cell can make many different types of proteins starting from the same gene by making different messenger RNAs from which to produce the proteins. Previously, we have shown that VSMCs from healthy arteries produce a unique set of alternative spliced messenger RNAs that give rise to specific protein forms essential for the cells to work. We have identified a protein called RBPMS that we predict to be necessary for alternative splicing to work properly in mature VSMCs. We also know that when the cells undergo phenotype switching, they no longer produce RBPMS or any of the alternatively spliced RNA forms. We also have some fundamental information about how RBPMS affects the cardiovascular system. Our preliminary studies showed that RBPMS makes cultured, immature, VSMCs divide less and move less. These features are similar to mature cells from healthy blood vessels. Also, if we temporarily remove RBPMS in adult mouse blood vessels, then their blood pressure seems to drop, and their vessel walls enlarge and become damaged.If we understand how RBPMS and alternative splicing contribute to normal vascular smooth muscle function, we can decipher how to target them in diseases where they are inactivated. Our hypothesis is that RBPMS and the alternative splicing in mature VSMCs are important for normal working of the blood vessel and are a key factor affecting phenotype switching. To test these, we will use "genetically modified" mice, specially engineered so that we can inactivate RBPMS gene in the VSMCs only. We need to do this because we want to investigate how RBPMS works in blood vessels without affecting other organs. To characterise the unique alternative splice RNAs in VSMCs, we will use a novel technology - "VASA-seq". which allows us to chart individual cells and categorise how mature they are.We will first study normal blood vessels to see how the circulatory system works once we inactivate RBPMS. We will measure blood pressure and examine whether the walls of the vessels change in structure when RBPMS is lost. We will the use a pioneering technology called VASA-seq to see how these changes are linked to alternative splicing in individual VSMCs where RBPMS is inactivated. Second, we will study injured blood vessels that show phenotype switching. We will measure the response to the injury all the way to healing (3 weeks) by examining the vessel wall structure, comparing the response in normal and RBPMS-inactivated mice. With VASA-seq, we will track how alternative splicing changes in the VSMCs during this time. This will establish how RBPMS affects injured cells and phenotype switching process.Once complete, our study will show how RBPMS and alternative splicing are important molecular mechanisms responsible for normal working of vascular smooth muscle cells. It will give us in-depth insight into phenotype switching process and how RBPMS behaves in this context. Once we know how normal vessels and injured or diseased vessels work, we might be able to control this process. By understanding how alternative splicing is controlled, it has already been possible to develop life-saving treatments for Spinal Muscular Atrophy. We hope that our research might also reveal effective new ways to treat diseases of the cardiovascular system and save many lives.

血管平滑肌细胞(VSMCs)是一种重要的血管壁细胞类型。在正常、成熟、健康的状态下，这些细胞收缩并帮助维持血压和正常的血液流动。然而，在一些心血管疾病(如动脉粥样硬化)中，这些细胞通过改变为不成熟的形式做出反应，这些形式不再正常功能，但有助于疾病的进展。我们还不完全了解的是，这种变化，即所谓的“表型转换”，是如何在疾病和损伤中发生的，以及导致这种变化的分子机制是什么。具体地说，我们正在研究一种叫做“选择性剪接”的分子过程。这是一种细胞可以通过制造不同的信使RNA来产生蛋白质的方法，通过这种方法，细胞可以从同一基因开始制造许多不同类型的蛋白质。此前，我们已经证明，来自健康动脉的VSMCs会产生一组独特的选择性剪接信使RNA，这些信使RNA会产生细胞工作所必需的特定蛋白质形式。我们已经确定了一种名为RBPMS的蛋白质，我们预测它是成熟VSMC正常工作的替代剪接所必需的。我们还知道，当细胞经历表型转换时，它们不再产生RBPM或任何选择性剪接的RNA形式。我们也有一些关于RBPMS如何影响心血管系统的基本信息。我们的初步研究表明，RBPMS使培养的、未成熟的VSMC分裂较少，运动较少。这些特征类似于来自健康血管的成熟细胞。此外，如果我们暂时移除成年小鼠血管中的RBPMS，它们的血压似乎会下降，血管壁扩大并受损。如果我们了解RBPMS和选择性剪接如何有助于正常的血管平滑肌功能，我们就可以破译如何在它们被灭活的疾病中靶向它们。我们的假设是，RBPMS和成熟VSMCs中的选择性剪接对于血管的正常工作是重要的，是影响表型转换的关键因素。为了测试这些基因，我们将使用经过特殊设计的“转基因”小鼠，这样我们就可以使血管平滑肌细胞中的RBPMS基因失活。我们需要这样做，因为我们想要研究RBPMS如何在不影响其他器官的情况下在血管中发挥作用。为了描述VSMC中独特的选择性剪接RNA，我们将使用一种新技术-“Vasa-seq”。这使我们能够绘制单个细胞的图表，并对它们的成熟程度进行分类。我们将首先研究正常的血管，看看一旦我们使RBPMS失活，循环系统是如何工作的。我们将测量血压，并检查当RBPMS丢失时，血管壁是否发生结构变化。我们将使用一种名为VASA-SEQ的开创性技术来了解这些变化如何与RBPMS失活的单个VSMC中的替代剪接联系起来。其次，我们将研究表现出表型转换的受损血管。我们将通过检查血管壁结构，比较正常小鼠和RBPMS灭活小鼠的反应，来测量在愈合的整个过程中对损伤的反应。使用VASA-SEQ，我们将跟踪这段时间内VSMC中替代剪接的变化。这将确定RBPMS如何影响受损细胞和表型转换过程。一旦完成，我们的研究将表明RBPMS和选择性剪接是负责血管平滑肌细胞正常工作的重要分子机制。它将使我们深入了解表型转换过程以及RBPMS在这一背景下的行为方式。一旦我们了解了正常血管和受伤或患病的血管是如何工作的，我们就可能能够控制这一过程。通过了解选择性剪接是如何控制的，已经有可能开发出拯救脊髓肌萎缩的治疗方法。我们希望我们的研究还可以揭示治疗心血管系统疾病的有效新方法，拯救许多人的生命。

项目成果

Differentiation and quality control of smooth muscle cells from human pluripotent stem cells via the neural crest lineage

通过神经嵴谱系从人多能干细胞中分化平滑肌细胞并进行质量控制

• DOI: 10.1101/2023.05.31.543049
• 发表时间: 2023
• 作者: Holt P