How does Vascular Smooth Muscle-specific master splicing regulator - Rbpms - contribute to vascular performance in vivo?
Lead Research Organisation:
University of Cambridge
Department Name: Wellcome Trust - MRC Cam Stem Cell Inst
Abstract
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.
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.
Technical Summary
Vascular Smooth Muscle Cells (VSMCs) populating the medial layer of healthy arteries represent their most contractile and differentiated phenotypic state. In response to injury and in several cardiovascular diseases including atherosclerosis, they dedifferentiate into more proliferative states by virtue of phenotypic plasticity. Contractile VSMCs express a unique network of alternatively spliced (AS) isoforms of genes pertaining to contractile function which are downregulated during phenotype switching alongside characteristic VSMC identity markers. It is not fully understood how VSMC-AS contributes to contractility, VSMC phenotype and overall vascular function. We have uncovered a master regulator - RNA Binding Protein Multiple Splicing (RBPMS) that controls at least 20% of the VSMC-AS network in partially differentiated cultured VSMCs. However, RBPMS is only fully expressed in differentiated VSMCs in vivo, and Rbpms -/-mice are inviable.
We therefore propose to conditionally knockout Rbpms in vivo in adult mouse VSMCs and establish: A) how Rbpms impacts vascular performance; B) how the Rbpms-directed VSMC-AS network correlates with cardiovascular function and with VSMC phenotypes at single cell resolution using novel single cell sequencing platform VASA-seq, and; C) how injury response kinetics and the associated phenotype switching are impacted by the loss of RBPMS and its regulated VSMC-AS network - with single cell level mapping of AS profiles concurrent with the transcriptional signatures using a combination of 10x genomics and VASA-seq.
Ours is a timely interdisciplinary study that aims to bridge the gap in our current understanding of VSMC biology by dissecting the previously unappreciated post-transcriptional component - AS - and seeking to understand how AS patterns influence VSMC functionality and phenotypic state.
We therefore propose to conditionally knockout Rbpms in vivo in adult mouse VSMCs and establish: A) how Rbpms impacts vascular performance; B) how the Rbpms-directed VSMC-AS network correlates with cardiovascular function and with VSMC phenotypes at single cell resolution using novel single cell sequencing platform VASA-seq, and; C) how injury response kinetics and the associated phenotype switching are impacted by the loss of RBPMS and its regulated VSMC-AS network - with single cell level mapping of AS profiles concurrent with the transcriptional signatures using a combination of 10x genomics and VASA-seq.
Ours is a timely interdisciplinary study that aims to bridge the gap in our current understanding of VSMC biology by dissecting the previously unappreciated post-transcriptional component - AS - and seeking to understand how AS patterns influence VSMC functionality and phenotypic state.
