Microparticles stimulate angiogenesis by inducing ELR(+) CXC-chemokines in synovial fibroblasts

Abstract

Microparticles (MPs) are small membrane-vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro-inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co-cultured with increasing numbers of MPs. The effects of supernatants from co-cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans-well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro-angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co-cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio-chamber assay, supernatants from RASFs co-cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro-angiogenic ELR⁺ chemokines. These pro-angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.

Fig 1

MPs derived from leucocytes potently induce the expression of pro-angiogenic chemokines in SFs. Co-incubation of RASFs patients with pathophysiologically relevant numbers of MPs derived from U937 cell stimulated the expression of the ELR+ CXC chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in dose-dependent manner as analysed by real-time PCR (A). * indicates statistically significant differences compared to controls (P < 0.05). Comparable effects on the expression of ELR+ chemokines were also observed with MPs isolated from the synovial fluid of inflamed joints of three different RA patients (B).

Fig 1

MPs derived from leucocytes potently induce the expression of pro-angiogenic chemokines in SFs. Co-incubation of RASFs patients with pathophysiologically relevant numbers of MPs derived from U937 cell stimulated the expression of the ELR+ CXC chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in dose-dependent manner as analysed by real-time PCR (A). * indicates statistically significant differences compared to controls (P < 0.05). Comparable effects on the expression of ELR+ chemokines were also observed with MPs isolated from the synovial fluid of inflamed joints of three different RA patients (B).

Fig 2

Supernatants from RASFs incubated with MPs are chemotactic for microvascular ECs. Supernatants from RASFs co-cultured with MPs derived from leucocytes stimulated migration of ECs. The chemotactic effects can be partially inhibited by blocking antibodies against CXCL1, 2, 3, CXCL5 or CXCL6, demonstrating that the increased chemotactic activity is mediated by these ELR+ CXC chemokines. * indicates statistically significant differences compared to controls (P < 0.05). # indicates statistically significant compared to supernatants from RASFs co-incubated with MPs with irrelevant, non-blocking antibodies (P < 0.05).

Fig 3

Supernatants from RASFs co-incubated with microparticles do not alter the metabolic activity or affect the viability of ECs. Incubation of ECs with increasing concentrations of supernatants from RASFs cocultured with MPs did not alter the metabolic activity as analysed with the MTT assay (A). The number of annexin V positive, apoptotic ECs (B) or the number of PI positive necrotic ECs (C) also did not change.

Fig 3

Supernatants from RASFs co-incubated with microparticles do not alter the metabolic activity or affect the viability of ECs. Incubation of ECs with increasing concentrations of supernatants from RASFs cocultured with MPs did not alter the metabolic activity as analysed with the MTT assay (A). The number of annexin V positive, apoptotic ECs (B) or the number of PI positive necrotic ECs (C) also did not change.

Fig 4

Supernatants from murine SFs co-cultured with MPs stimulate angiogenesis in the matrigel plague assay in vivo. For this assay, chambers filled with supernatants from murine SFs co-incubated with MPs or control supernatants re-suspended in matrigel and chambers were implanted into subcutaneous pockets of C57Bl/6 mice. Blood vessels infiltrating into the matrigel chamber were quantified after 10 days. The number of blood vessels, defined by the presence of erythrocytes in the luminal space lined by CD34 positive endothelial cells, increased significantly higher in chambers filled with supernatants from SFs co-incubated with MPs compared to supernatants from SF cultured without MPs. * indicates statistically significant differences (P < 0.05).

Fig 5

Microparticles as key-players in the pathogenesis of RA. MPs are released from infiltrating leucocytes upon activation and accumulated in the synovial fluid of inflamed joints. MPs activate SFs and potently stimulate the release of pro-angiogenic ELR+ chemokines, pro-inflammatory CC chemokines and matrix degrading enzymes from SFs. Aberrant activation of SFs by MPs might therefore directly contribute to hypervascularization, inflammation and joint destruction in RA.