The Critical Role of RNA-Binding Protein RBPMS in Vascular Smooth Muscle Cells

Academic Background

Vascular Smooth Muscle Cells (VSMCs) are the primary structural components of large arteries. In healthy blood vessels, VSMCs exhibit a mature contractile phenotype, responsible for regulating vascular tone and blood flow. However, VSMCs possess phenotypic plasticity. In response to vascular wall injury or cardiovascular diseases (such as atherosclerosis and hypertension), they can dedifferentiate into a more proliferative and synthetic mesenchymal state. This phenotypic switch is accompanied by significant changes in the cellular transcriptome, including the loss of contractile markers. Although current definitions of the molecular networks governing VSMC phenotypes primarily focus on transcriptional-level marker expression, the role of post-transcriptional regulation (e.g., RNA splicing) in VSMC phenotypes remains underexplored.

RNA-binding proteins (RBPs) play a crucial role in post-transcriptional regulation processes such as RNA splicing. RBPMS (RNA Binding Protein with Multiple Splicing) is an RNA-binding protein, and previous studies have shown that it drives differentiation-associated splicing events in rat VSMCs. However, its role in human VSMCs remains unclear. This study aims to explore the splicing regulatory role of RBPMS in human embryonic stem cell-derived VSMCs (hESC-VSMCs) and reveal its impact on VSMC phenotypes.

Source of the Paper

This paper was co-authored by Aishwarya G. Jacob, Ilias Moutsopoulos, Alex Petchey, Rafael Kollyfas, Vincent R. Knight-Schrijver, Irina Mohorianu, Sanjay Sinha, and Christopher W.J. Smith from the Department of Biochemistry at the University of Cambridge. The research was published in 2024 in the journal Cardiovascular Research, with the article titled “RNA binding protein with multiple splicing (RBPMS) promotes contractile phenotype splicing in human embryonic stem cell–derived vascular smooth muscle cells.”

Research Process and Results

1. Establishment of the hESC-VSMCs Model and RBPMS Expression Analysis

The study first differentiated VSMCs from human embryonic stem cells (hESCs). VSMCs were derived via two pathways: neural crest (NC) or lateral plate mesoderm (LM). These cells exhibited partially differentiated splicing patterns, while RBPMS protein levels were low. The researchers hypothesized that RBPMS might be associated with mature VSMCs and is expressed at lower levels in hESC-VSMCs.

2. Impact of RBPMS Overexpression on Splicing Patterns

To investigate the role of RBPMS in human VSMCs, the researchers constructed an inducible RBPMS overexpression platform. By inserting an RBPMS-GFP fusion construct into the hESC genome, they successfully achieved RBPMS overexpression. Through flow cytometry sorting of high- and low-RBPMS-expressing cells and RNA sequencing analysis, it was found that RBPMS overexpression induced splicing patterns similar to those in mature tissue VSMCs. For example, RBPMS significantly increased splicing events in genes such as actin and vinculin, which are commonly observed in mature VSMCs.

3. Functional Synergy Between RBPMS and RBFox2

The researchers further discovered that RBPMS synergizes with another splicing factor, RBFox2, in regulating splicing events. By knocking down RBFox2, they found that RBPMS-driven splicing events partially depend on RBFox2. RNA sequencing data revealed that RBPMS and RBFox2 co-regulated multiple splicing events in genes related to cytoskeleton and contractile functions.

4. Impact of RBPMS on VSMC Phenotypes

The researchers also found that RBPMS overexpression significantly reduced the motility and proliferative capacity of hESC-VSMCs, consistent with the phenotype of mature VSMCs. Through live-cell imaging and EdU labeling experiments, they confirmed that RBPMS overexpression inhibited VSMC proliferation and reduced the proportion of cells entering the S phase.

Conclusions and Significance

This study demonstrates that RBPMS drives the formation of a mature contractile phenotype in human VSMCs by regulating RNA splicing events. RBPMS not only directly binds RNA to regulate splicing but also cooperates with RBFox2 to co-regulate multiple genes related to cytoskeleton and contractile functions. Additionally, RBPMS overexpression significantly reduces the motility and proliferative capacity of VSMCs, further supporting its critical role in maintaining the mature VSMC phenotype.

This research reveals a novel mechanism of RBPMS in VSMC phenotype regulation, providing new insights into the pathogenesis of cardiovascular diseases. The RBPMS-driven splicing regulatory network may serve as a potential target for future cardiovascular disease treatments.

Research Highlights

1. First Revelation of RBPMS Splicing Regulation in Human VSMCs: This study systematically investigated the function of RBPMS in human VSMCs for the first time, filling a research gap in this field.
2. Synergistic Role of RBPMS and RBFox2: The study found that RBPMS synergizes with RBFox2 in regulating splicing events, revealing the complexity of the splicing regulatory network.
3. Significant Impact of RBPMS on VSMC Phenotypes: RBPMS overexpression significantly reduces the motility and proliferative capacity of VSMCs, providing new experimental evidence for understanding VSMC phenotypic switching.

Other Valuable Information

The hESC-VSMCs model used in this study provides a powerful tool for investigating the differentiation and function of human VSMCs. Additionally, the RBPMS overexpression platform developed by the researchers offers new technical means for future studies on the role of RNA-binding proteins in VSMCs.

This study not only deepens the understanding of VSMC phenotype regulation mechanisms but also provides new research directions for the treatment of cardiovascular diseases.