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
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