The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

Abstract

Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which angiogenesis represents a critical early event of synovial inflammation. The present study aimed to reveal the potential molecular mechanisms of SAA/TLR4 induction of angiogenesis through NETs in RA. Materials and methods: Firstly, immunohistochemistry and immunofluorescence were used to determinate TLR4 and NETs expression in synovial tissue, respectively. ELISA was used to detect the content of SAA, MPO and NE in serum and synovial fluid of patients. DNA quantification was done by fluorescence. DNA fluorescence staining was used to compare NETs formation in RA and HC sera, and to investigate the mechanism of NETs formation induced by SAA stimulation. PicoGreen DNA testing was used to characterize the DNA in the supernatants. Also, DNA fluorescence staining to explore whether NETs formation induced by SAA was dependent or independent on NADPH oxidase pathway. MTT assay, Wound healing assay, Tube formation assay were performed to analyze human veins umbilical cells (HUVECs) proliferation, migration, and tube vessels formation, respectively under NETs or NETs + DNase stimulants. Results: Firstly, we demonstrated that TLR4 was predominantly and widely expressed in synovial tissues with elevated serum levels of SAA, compared to osteoarthritis (OA) patients, and the similar results were observed for NETs formation. Afterwards, in a series of in vitro experiments, we reported an increased MPO and NE levels, and a relatively decreased DNA level in the sera of RA patients. Set apart, the levels of MPO and NE in RA were correlated to the disease activity. Moreover, an increased spontaneous NETs formation was observed in RA patients, enhanced under SAA stimulation and regulated by TLR4 activation. And the total DNA expressed in RA patients was partly composed of NET-DNA. Also, SAA induced NETs formation dependent on NADPH pathway. Finally, our results indicated that extracted SAA-induced NETs promoted endothelial cells (ECs) migration, proliferation, and vascular tube formation. Conclusion: Our current study highlighted the role of SAA/TLR4 interaction in the induction of angiogenesis through formed NETs. Therefore, this study offers new perspectives in the understanding of RA pathogenicity and its management.