Microparticles stimulate the synthesis of prostaglandin E₂ via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1

Abstract

Objective. Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype.Methods. Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real-time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF-B and activator protein 1 (AP-1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and IB expression vectors.Results. Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase 1 (mPGES-1), and prostaglandin E 2 (PGE 2 ). In contrast, no up-regulation of COX-1, mPGES-2, cytosolic PGES, or phospholipase A 2 was observed. The induction of PGE 2 was blocked by selective inhibition of COX-2. Microparticles activated NF-B, AP-1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF-B, AP-1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE 2 by synovial fibroblasts.Conclusion. These results demonstrate that microparticles up-regulate the production of PGE 2 in synovial fibroblasts by inducing COX-2 and mPGES-1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA.

Induction of apoptosis in circulating angiogenic cells by microparticles

“…To analyze whether microparticles derived from apoptotic ECs affect the number of CACs, CACs from patients with SSc and healthy volunteers were coincubated with microparticles for 4 days. The number of microparticles used for these experiments was based on the numbers of microparticles in the blood of patients with SSc and has also been used for other studies (22,23,29,30).…”

“…Apoptosis in primary human microvascular ECs (HMVECs; Invitrogen), immortalized HMVECs (27), and immortalized murine glomerular ECs (28) was induced by incubation with staurosporine (Sigma-Aldrich) at a concentration of 10 M. After 12 hours, supernatants were collected, and microparticles were isolated by differential centrifugation as previously described (23,29,30). For coculture experiments, CACs were incubated for 4 days with freshly isolated microparticles (1.0 ϫ 10 4 to 1.0 ϫ 10 5 ) derived from apoptotic ECs.…”

“…CACs were stimulated with arachidonic acid (Sigma-Aldrich) in concentrations from 25 nM to 250 nM for 24 hours, 48 hours, or 4 days. The concentrations used in our study represent pathophysiologically relevant concentrations, because they cover the mean levels of arachidonic acid stored within microparticles in the blood of patients with SSc and healthy volunteers (22,30).…”

Histone deacetylase 7, a potential target for the antifibrotic treatment of systemic sclerosis

“…Quantification of specific mRNA was performed by single-reporter and SYBR Green real-time PCR using an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as described previously (14). Predeveloped primer probes were used for HDACs 1, 2, and 8, platelet-derived growth factor B (PDGF-B), and PDGF receptor ␤ (PDGFR␤) (all from Applied Biosystems).…”

“…Fibroblasts from patients with SSc were transfected using an Amaxa Basic Nucleofector kit (Amaxa, Gaithersburg, MD) as described previously (14). For silencing of HDAC-7, we used HDAC-7 siRNA (Santa Cruz Biotechnology/Qiagen) and scrambled siRNA representing an irrelevant coding siRNA (Applied Biosystems) as a control, which does not target any gene product.…”