Identification of ACTA2 as a Key Contributor to Venous Malformation.

Song Wang,Zifu Zhou,Hongwen Li,Renrong Lv,Jian Zhang,Guangqi Xu,Yu Wang,Jing Li,Ran Huo,Jianhai Bi

doi:10.3389/fcell.2021.755409

Abstract

Objectives: Proteomics and high connotation functional gene screening (HCS) were used to screen key functional genes that play important roles in the pathogenesis of venous malformation. Furthermore, this study was conducted to analyze and explore their possible functions, establish a gene mutation zebrafish model, and perform a preliminary study to explore their possible pathogenic mechanisms in venous malformation.Methods: Pathological and normal tissues from patients with disseminated venous malformation were selected for Tandem Mass Tag (TMT) proteomics analysis to identify proteins that were differentially expressed. Based on bioinformatics analysis, 20 proteins with significant differential expression were selected for HCS to find key driver genes and characterize the expression of these genes in patients with venous malformations. In vitro experiments were then performed using human microvascular endothelial cells (HMEC-1). A gene mutant zebrafish model was also constructed for in vivo experiments to explore gene functions and pathogenic mechanisms.Results: The TMT results showed a total of 71 proteins that were differentially expressed as required, with five of them upregulated and 66 downregulated. Based on bioinformatics and proteomics results, five highly expressed genes and 15 poorly expressed genes were selected for functional screening by RNAi technology. HCS screening identified ACTA2 as the driver gene. Quantitative polymerase chain reaction (qPCR) and western blot were used to detect the expression of ACTA2 in the pathological tissues of patients with venous malformations and in control tissues, and the experimental results showed a significantly lower expression of ACTA2 in venous malformation tissues (P < 0.05). Cell assays on the human microvascular endothelial cells (HMEC-1) model showed that cell proliferation, migration, invasion, and angiogenic ability were all significantly increased in the ACTA2 over-expression group (P < 0.05), and that overexpression of ACTA2 could improve the inhibitory effect on vascular endothelial cell proliferation. We constructed an ACTA2-knockdown zebrafish model and found that the knockdown of ACTA2 resulted in defective vascular development, disruption of vascular integrity, and malformation of micro vein development in zebrafish. Further qPCR assays revealed that the knockdown of ACTA2 inhibited the Dll4/notch1 signaling pathway, Ephrin-B2 signaling pathway, and vascular integrity-related molecules and activated the Hedgehog signaling pathway.Conclusion: This study revealed that ACTA2 deficiency is an important factor in the pathogenesis of venous malformation, resulting in the disruption of vascular integrity and malformed vascular development. ACTA2 can be used as a potential biomarker for the treatment and prognosis of venous malformations.

Highlights

Venous malformation (VM) is a common congenital vascular malformations comprising dilated and tortuous veins of variable size with a sparse and disorganized arrangement of vascular smooth muscle cells, which gradually expand and tortuously form clusters with growth and development (Dompmartin et al, 2010; Wassef et al, 2015). It can occur in all parts of the body, with the oral and maxillofacial areas, head and neck, and extremities being the main sites of onset (Judith et al, 2014)
Based on the mode of inheritance, VM can be divided into disseminated VM and familial venous malformations (VMCM), of which the familial type is more complex and difficult to treat
Clustering analysis was performed on these proteins, and the results suggested that there was significant differential expression of proteins in venous malformation tissues compared to normal tissues, where red represents upregulated molecules and blue represents down-regulated molecules (Figure 1B)

Summary

Introduction

Venous malformation (VM) is a common congenital vascular malformations comprising dilated and tortuous veins of variable size with a sparse and disorganized arrangement of vascular smooth muscle cells, which gradually expand and tortuously form clusters with growth and development (Dompmartin et al, 2010; Wassef et al, 2015). It can occur in all parts of the body, with the oral and maxillofacial areas, head and neck, and extremities being the main sites of onset (Judith et al, 2014). Several studies have found that many VMCM patients have specific genetic mutations, namely TIE2-R849W (Calvert et al, 1999; Shu et al, 2012)

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