SMAD-Specific E3 Ubiquitin Ligase 2 Promotes Angiogenesis by Facilitating PTX3 Degradation in Mesenchymal Stem Cells of Ankylosing Spondylitis Patients

Abstract

Background: Although emerging evidence suggests that abnormal angiogenesis is a clinical symptom of ankylosing spondylitis (AS) and might be used as a diagnostic marker to predict disease progression, the cause of ectopic angiogenesis in AS patients is yet unexplored. Methods: Abnormal angiogenesis in AS patients was assessed by anti-CD31 immunohistochemistry. After culturing with mesenchymal stem cells from AS patients (ASMSCs) or healthy donors (HDMSCs), the human umbilical vein endothelial cells (HUVECs) were assayed using a Cell Counting Kit-8 (CCK-8) to evaluate cell proliferation, and a wound healing assay was used to investigate cell migration. In addition, a tube formation assay was used to determine angiogenesis efficiency. RT-qPCR and western blotting were used to quantify the expression of SMAD-specific E3 ubiquitin protein ligase 2 (Smurf2). Proteome profilers were used to identify differential regulatory factors between ASMSCs and HDMSCs. Co-immunoprecipitation, immunofluorescence and mass spectrometry analyses were performed to investigate Smurf2/PTX3 interactions. Dual-luciferase reporter assays and chromatin immunoprecipitation were used to identify the ATF4-specific binding site of the Smurf2 promoter for the activation of transcription. Findings: Compared to HDMSCs, which abnormally promote endothelial cell angiogenesis, we found that the elevated Smurf2 in MSCs is the major cause of abnormal angiogenesis in AS patients. Downregulation of Smurf2 in ASMSCs precludes angiogenesis, whereas overexpression of Smurf2 in HDMSCs promotesangiogenesis. By acting as an E3 ubiquitin ligase, Smurf2 can regulate the abundance of PTX3, which has been shown to be an angiogenesis repressor, in MSCs. Moreover, the Smurf2 transcription is regulated by ATF4 induced ER stress. Interpretation: These results identify a novel role for Smurf2,which negatively regulate of PTX3 stability, and promote angiogenesis in ASMSCs. Funding: This study was financially supported by grants from the National Natural Science Foundation of China (81871750), the Science and Technology Project of GuangDong Province (2018A030313232, 2017A020215070), the Medical Science and Technology Research Project of GuangDong Province (A2018292), and the Key Realm R&D Programme of GuangDong Province (2019B020236001). Declaration of Interest: The authors have no potential conflicts of interest to declare. Ethical Approval: This study was approved by the Ethics Committee of the Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, China.