S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis.

Chien-Chung Huang,Shan-Chi Liu,Tzu-Ting Tseng,Shih-Wei Wang,Sung-Lin Hu,Chun-Hao Tsai,Yat-Yin Law,Chih-Hsin Tang,Yen-You Lin

doi:10.3390/cells10082168

Abstract

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. Vascular endothelial growth factor (VEGF) stimulation of endothelial progenitor cells (EPCs) facilitates angiogenesis and the progression of RA. Phosphorylation of sphingosine kinase 1 (SphK1) produces sphingosine-1-phosphate (S1P), which increases inflammatory cytokine production, although the role of S1P in RA angiogenesis is unclear. In this study, we evaluated the impact of S1P treatment on VEGF-dependent angiogenesis in osteoblast-like cells (MG-63 cells) and the significance of SphK1 short hairpin RNA (shRNA) on S1P production in an in vivo model. We found significantly higher levels of S1P and VEGF expression in synovial fluid from RA patients compared with those with osteoarthritis by ELISA analysis. Treating MG-63 cells with S1P increased VEGF production, while focal adhesion kinase (FAK) and Src siRNAs and inhibitors decreased VEGF production in S1P-treated MG-63 cells. Conditioned medium from S1P-treated osteoblasts significantly increased EPC tube formation and migration by inhibiting miR-16-5p synthesis via proto-oncogene tyrosine-protein kinase src (c-Src) and FAK signaling in chick chorioallantoic membrane (CAM) and Matrigel plug assays. Infection with SphK1 shRNA reduced angiogenesis, articular swelling and cartilage erosion in the ankle joints of mice with collagen-induced arthritis (CIA). S1P appears to have therapeutic potential in RA treatment.

Highlights

Angiogenesis is a critical driver of rheumatoid arthritis (RA) development [1], as are endothelial progenitor cells (EPCs) [2,3], which contain the cell surface markers CD34, CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), which stimulate postnatal vasculogenesis [4] and angiogenic function [5]
Higher levels of S1P and Vascular endothelial growth factor (VEGF) expression were identified in samples of RA synovial fluid than in OA samples (Figure 1A,B), suggesting that S1P and VEGF are more critical in RA than in OA
S1P Facilitates VEGF-Dependent EPC Angiogenesis we examined whether S1P promotes osteoblastic VEGF expression

Summary

Introduction

Angiogenesis is a critical driver of rheumatoid arthritis (RA) development [1], as are endothelial progenitor cells (EPCs) [2,3], which contain the cell surface markers CD34, CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), which stimulate postnatal vasculogenesis [4] and angiogenic function [5]. VEGF induces EPC proliferation and migration, and facilitates angiogenesis [5], enabling the development of RA [6,7]. We have previously demonstrated that the proinflammatory cytokine cysteine-rich 61 (CCN1) stimulates VEGF expression in osteoblasts and upregulates EPC angiogenesis in RA [7]. Proangiogenic factors can stimulate the sphingosine kinase 1 (SphK1)/S1P/S1P1 pathway to upregulate fibroblast-like synoviocyte (FLS) proliferation and migration and facilitate angiogenesis in a rat model of RA [14]. We have previously shown that S1P upregulates the expression of IL-1β and IL-6 in human osteoblasts [15,16], which suggests that S1P may serve as an inflammatory mediator during arthritis progression

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