Smad1/5 is acetylated in the dorsal aortae of the mouse embryo driving early arterial gene expression

Academic Background

During embryonic development, arteriovenous differentiation (AV differentiation) is a critical step in ensuring proper blood vessel formation and maturation. Defects in arterial or venous identity can lead to inappropriate fusion of vessels, resulting in so-called arteriovenous malformations (AVMs). Currently, the mechanism behind AVM formation remains unclear, and treatment options are limited. In mammals, AV differentiation is initiated before the onset of blood flow in the embryo, but this “pre-flow mechanism” is still poorly understood. This study aims to uncover the role of the SMAD1/5 signaling pathway in pre-flow arterial identity and unexpectedly discovered a novel regulatory mechanism for SMAD1/5 signaling.

Source of the Paper

This paper was co-authored by Margo Daems, Ljuba C. Ponomarev, Rita Simoes-Faria, Max Nobis, Colinda L.G.J. Scheele, Aernout Luttun, Bart Ghesquière, An Zwijsen, and Elizabeth A.V. Jones. The research team is affiliated with the Center for Molecular and Vascular Biology at KU Leuven in Belgium, the VIB Center for Cancer Biology, the Laboratory of Oncology, and the Department of Cardiology at Maastricht University in the Netherlands. The paper was accepted on June 22, 2024, and published online on September 10, 2024, in the journal Cardiovascular Research.

Research Process

1. Research Objectives

The primary goal of this study is to reveal the role of the SMAD1/5 signaling pathway in the expression of arterial genes before the onset of blood flow in the embryo and to explore its regulatory mechanisms.

2. Experimental Design

The study used mouse embryo models and employed various experimental techniques to analyze the role of the SMAD1/5 signaling pathway in arterial differentiation. The specific experimental process is as follows:

a) Embryo Culture and Treatment

Researchers isolated embryos from mice and cultured them in vitro. The embryos were divided into different experimental groups and subjected to various treatments, including the use of Notch1-Fc protein, TGFβ1 neutralizing antibodies, ALK1 neutralizing antibodies, ALK5 inhibitors, and more.

b) Gene Expression Analysis

Using whole-mount in situ hybridization (WMISH), the researchers detected the expression of early arterial genes (such as Hey1, Gja4, and Dll4). Additionally, real-time quantitative PCR (RT-qPCR) and Western blot techniques were used for quantitative analysis of gene and protein expression.

c) Protein Acetylation Detection

The researchers employed proximity ligation assay (PLA) to detect the acetylation status of SMAD1/5 proteins. Immunohistochemical staining was further used to validate the acetylation of SMAD1/5 proteins in the endothelial cells of the embryonic dorsal aorta.

d) Metabolic Pathway Inhibition Experiments

To investigate the role of acetyl-CoA in SMAD1/5 acetylation, the researchers used the pyruvate dehydrogenase (PDH) inhibitor CPI-613 and the deacetylase inhibitor trichostatin A (TSA) to observe their effects on arterial gene expression.

3. Main Results

a) Notch Signaling Is Inactive Before Blood Flow

The study found that although Notch1 is expressed in pre-flow embryos, it is not activated and is not necessary for the expression of early arterial genes (Hey1 and Gja4). Using the NCX1 knockout model, the researchers further confirmed the important role of shear stress in maintaining arterial identity.

b) SMAD1/5 Signaling Drives Early Arterial Gene Expression

The study revealed that SMAD1/5 signaling is activated in pre-flow embryos and is mediated through the ALK1/ALK5/TGFβRII receptor complex. TGFβ1 is a necessary ligand for activating SMAD1/5 signaling. Additionally, the acetylation of SMAD1/5 proteins makes them more sensitive to TGFβ1 stimulation.

c) Acetyl-CoA Production Is Crucial for Early Arterial Gene Expression

By inhibiting acetyl-CoA production, the researchers found that the expression of early arterial genes (Hey1 and Gja4) was blocked. Stabilizing acetylation, however, restored the expression of these genes.

d) Hypoxia Suppresses Arterial Gene Expression

Under hypoxic conditions, the expression of early arterial genes was suppressed, while the use of TSA restored their expression, indicating that hypoxia affects arterial gene expression by inhibiting SMAD1/5 acetylation.

4. Conclusion

This study highlights the critical role of the SMAD1/5 signaling pathway in the expression of arterial genes before the onset of blood flow in the embryo and identifies a novel regulatory mechanism involving protein acetylation to enhance SMAD1/5 sensitivity to TGFβ1. This discovery provides new insights into the molecular mechanisms of arterial differentiation and offers potential therapeutic targets for treating vascular diseases such as arteriovenous malformations.

5. Research Highlights

• Key Finding: The SMAD1/5 signaling pathway regulates early arterial gene expression through acetylation in pre-flow embryos.
• Novelty: This is the first study to reveal the critical role of SMAD1/5 protein acetylation in arterial differentiation.
• Application Value: Provides new ideas for treating vascular diseases such as arteriovenous malformations.

6. Other Valuable Information

The study also found that SMAD1/5 acetylation is suppressed under hypoxic conditions, leading to the inhibition of early arterial gene expression. This discovery offers a new perspective on understanding the impact of hypoxia on vascular development.

Summary

Through a series of meticulously designed experiments, this study reveals the critical role of the SMAD1/5 signaling pathway in the expression of arterial genes before the onset of blood flow in the embryo and identifies a novel regulatory mechanism. This discovery not only deepens our understanding of arterial differentiation but also provides new potential therapeutic targets for treating vascular diseases.