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1.  CXCL16-Mediated Cell Recruitment to Rheumatoid Arthritis Synovial Tissue and Murine Lymph Nodes Is Dependent Upon the MAPK Pathway 
Arthritis and rheumatism  2006;54(3):765-778.
Objective
Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor α (TNFα), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process.
Methods
Using a human RA ST–SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo.
Results
CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFα. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFα in vitro, suggesting that CXCL16 protein may function in large part independently of TNFα.
Conclusion
Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.
doi:10.1002/art.21662
PMCID: PMC1472704  PMID: 16508941
2.  Effect of Oxidative Stress on Protein Tyrosine Phosphatase-1B in Scleroderma Dermal Fibroblasts 
Arthritis and Rheumatism  2011;64(6):1978-1989.
Objective
Platelet-derived growth factor (PDGF) and its receptor (PDGFR) promote fibrosis in scleroderma (SSc) dermal fibroblasts, which produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. In this study we determine whether the thiol-sensitive PTP1B is affected by ROS, thus enhancing PDGFR phosphorylation (p-PDGFR) and collagen I (Col I) synthesis. The effect of a thiol antioxidant, n-acetylcysteine (NAC), was also investigated.
Methods
Fibroblasts were isolated from skin. A phosphate release assay was used for PTP1B activity.
Results
ROS and Col I were significantly higher in SSc fibroblasts, accompanied by significantly lower amounts of free thiols compared to normal fibroblasts. After PDGF stimulation, not only were the PDGFR and ERK1/2 phosphorylated to a greater extent, but the ability to produce PTP1B was also hampered in SSc fibroblasts. PTP1B activity was significantly inactivated in SSc fibroblasts, which resulted from cysteine oxidation by higher levels of ROS, since oxidation of multiple PTPs, including PTP1B, was observed. Decreased PTP1B expression in normal fibroblasts led to increased Col I. NAC restored the low PTP1B activity, improved the profile of p-PDGFR, decreased the numbers of tyrosine-phosphorylated proteins and Col I, and scavenged ROS in SSc fibroblasts.
Conclusion
We introduce a new mechanism by which ROS promote a profibrotic phenotype in SSc fibroblasts through oxidative inactivation of PTP1B leading to pronounced PDGFR activation. Our study also provides a novel molecular mechanism by which NAC therapy may act on ROS and PTP1B to benefit SSc patients.
doi:10.1002/art.34336
PMCID: PMC3309078  PMID: 22161819
3.  Blocking ERK1/2 reduces TNF-α-induced-IL-18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of IL-18BPa Role of ERK1/2 in IL-18 bioactivity regulation 
Arthritis and rheumatism  2010;62(3):722-731.
Objective
To examine the mechanism of regulation of interleukin-18 (IL-18) bioactivity by IL-18 binding protein (IL-18BP) induction.
Methods
Levels of IL-18 and IL-18BPa expression were determined by enzyme-linked immunosorbent assays (ELISA) in osteoarthritis (OA) and rheumatoid arthritis (RA) synovial fluids, followed by free IL-18 calculation. IL-18 and IL-18BPa synthesis in RA synovial fibroblasts treated with pro- and anti-inflammatory cytokines were assessed by qRT-PCR and ELISA, respectively, followed by IL-18 bioactivity determination using KG-1 cells. Chemical signaling inhibitors and antisense oligonucleotides were used for validation of the signal transduction pathways involved in IL-18BPa/IL-18 regulation. TNF-α-induced caspase-1 activity was determined by a colorimetric assay.
Results
IL-18BPa was lower in RA synovial fluid than in OA synovial fluid (n=8; P < 0.05) and free IL-18 was higher in RA synovial fluid than in OA synovial fluid. TNF-α induced RA synovial fibroblast IL-18BPa and IL-18 in a time dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNF-α induced IL-18 production through extracellular signal-regulated kinases (ERK)1/2, protein kinase C (PKC)δ, and Src pathways, whereas IL-18BPa synthesis was mediated through nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), PKC, Src, and c-Jun N-terminal kinases (JNK) pathways. Furthermore, addition of exogenous IL-18BPa-Fc reduced the RA synovial fibroblast phosphorylation of ERK1/2 induced by TNF-α.
Conclusion
These results suggest that IL-18BPa reduces IL-18 bioactivity induced by TNF-α, by regulating the ERK1/2 pathway in RA synovial fibroblasts. Targeting IL-18 bioactivity by induction or addition of IL-18BPa may provide another therapeutic option in the management of RA.
doi:10.1002/art.27269
PMCID: PMC2855552  PMID: 20131228
4.  Junctional Adhesion Molecule C Mediates Leukocyte Adhesion to Rheumatoid Arthritis Synovium 
Arthritis and rheumatism  2008;58(10):3020-3029.
Objective
Leukocyte infiltration into the rheumatoid arthritis (RA) synovium is a multistep process in which leukocytes leave the bloodstream and invade the synovial tissue (ST). Leukocyte transendothelial migration and adhesion to RA ST requires adhesion molecules on the surface of endothelial cells and RA ST fibroblasts. This study was undertaken to investigate the role of junctional adhesion molecule C (JAM-C) in mediating leukocyte recruitment and retention in the RA joint.
Methods
Immunohistologic analysis was performed on RA, osteoarthritis (OA), and normal ST samples to quantify JAM-C expression. Fibroblast JAM-C expression was also analyzed using Western blotting, cell surface enzyme-linked immunosorbent assay, and immunofluorescence. To determine the role of JAM-C in leukocyte retention in the RA synovium, in vitro and in situ adhesion assays and RA ST fibroblast transmigration assays were performed.
Results
JAM-C was highly expressed by RA ST lining cells, and its expression was increased in OA ST and RA ST endothelial cells compared with normal ST endothelial cells. JAM-C was also expressed on the surface of OA ST and RA ST fibroblasts. Furthermore, we demonstrated that myeloid U937 cell adhesion to both OA ST and RA ST fibroblasts and to RA ST was dependent on JAM-C. U937 cell migration through an RA ST fibroblast monolayer was enhanced in the presence of neutralizing antibodies against JAM-C.
Conclusion
Our results highlight the novel role of JAM-C in recruiting and retaining leukocytes in the RA synovium and suggest that targeting JAM-C may be important in combating inflammatory diseases such as RA.
doi:10.1002/art.23867
PMCID: PMC2911024  PMID: 18821692
5.  Junctional adhesion molecule-A is abnormally expressed in diffuse cutaneous systemic sclerosis skin and mediates myeloid cell adhesion 
Annals of the rheumatic diseases  2010;69(1):249-254.
Objective
To investigate the role of Junctional adhesion molecule A (JAM-A) in the pathogenesis of systemic sclerosis (SSc).
Methods
Biopsies from proximal and distal arm skin and serum were obtained from patients with SSc and normal (NL) volunteers. To determine the expression of JAM-A on SSc dermal fibroblasts and in SSc skin, cell surface ELISAs and immunohistology were performed. An ELISA was designed to determine the amount of soluble JAM-A (sJAM-A) in serum. Myeloid U937 cell-SSc dermal fibroblast and skin adhesion assays were performed to determine the role of JAM-A in myeloid cell adhesion.
Results
The stratum granulosum and dermal endothelial cells (ECs) from distal arm SSc skin exhibited significantly decreased expression of JAM-A compared to NL. However, sJAM-A was elevated in the serum of patients with SSc compared to NL. Conversely, JAM-A was increased on the surface of SSc compared to NL dermal fibroblasts. JAM-A accounted for a significant portion of U937 binding to SSc dermal fibroblasts. In addition, JAM-A contributed to U937 adhesion to both distal and proximal SSc skin.
Conclusions
JAM-A expression is dysregulated in SSc skin. Decreased expression of JAM-A on SSc ECs may result in a reduced response to proangiogenic basic fibroblast growth factor. While increased JAM-A expression on SSc fibroblasts may serve to retain myeloid cells, which in turn secrete angiogenic factors.
doi:10.1136/ard.2008.102624
PMCID: PMC2795028  PMID: 19153103
Systemic sclerosis; Scleroderma; JAM-A; Cell adhesion
6.  Amelioration of Rat Adjuvant-Induced Arthritis by Met-RANTES 
Arthritis and rheumatism  2005;52(6):1907-1919.
Objective
CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal–modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1α (MIP-1α; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat.
Methods
Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA.
Results
Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1α and MIP-1β at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor α, interleukin-1β, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint.
Conclusion
The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.
doi:10.1002/art.21033
PMCID: PMC1282452  PMID: 15934086
8.  Evidence for CXCL16 as a potent angiogenic mediator and endothelial progenitor cell chemotactic factor 
Arthritis and rheumatism  2013;65(7):10.1002/art.37981.
Background
We examined the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium.
Methods
We utilized the RA synovial tissue (ST) severe combined immunodeficient (SCID) mouse chimera system to examine human dermal microvascular endothelial cell (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium injected intragraft with RA synovial fluid (SF) immunodepleted of CXCL16. CXCR6 deficient (CXCR6−/−) and wild-type (Wt) C57BL/6 mice were primed to develop K/BxN serum induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression by immunofluorescence and signaling activity for CXCL16.
Results
We found that CXCR6 is prominently expressed on human EPCs and HMVECs and can be upregulated by interleukin-1β (IL-1β). SCID mice injected intragraft with RA SF immunodepleted of CXCL16 showed a significant reduction in EPC recruitment. Using the K/BxN serum induced inflammatory arthritis model, CXCR6−/− mice showed profound reductions in hemoglobin (Hb) levels that correlated with reductions in monocyte and T-cell recruitment to arthritic joint tissue in CXCR6−/− compared to wildtype (Wt) mice. We also found that HMVECs and EPCs respond to CXCL16 stimulation but have unique signal transduction pathways and homing properties.
Conclusion
These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand-receptor pair that can be highly correlated with EPC recruitment and blood vessel formation in the RA joint.
doi:10.1002/art.37981
PMCID: PMC3701743  PMID: 23633118
CXCL16; CXCR6; endothelial progenitor cells; vasculogenesis; angiogenesis; rheumatoid arthritis; chemotaxis
9.  Blocking the janus-activated kinase pathway reduces tumor necrosis factor alpha-induced interleukin-18 bioactivity by caspase-1 inhibition 
Arthritis Research & Therapy  2014;16(2):R102.
Introduction
Our objective was to examine the role of the janus-activated kinase (JAK) pathway in the modulation of tumor necrosis factor-α (TNF)-induced-IL-18 bioactivity by reduction of caspase-1 function.
Methods
Caspase-1 expression in rheumatoid arthritis (RA) synovial fibroblasts treated with TNF was assessed by qRT-PCR and Western blot. Interleukin (IL)-18 was assessed by enzyme-linked immunosorbent assay (ELISA) in cell lysates and conditioned media and detected by immunofluorescence (IF) staining in RA synovial fibroblasts. The critical pathways for TNF-induced caspase-1 expression were determined by using chemical inhibitors of signaling followed by TNF stimulation. IL-18 bioactivity was assessed using human myelomonocytic KG-1 cells.
Results
TNF induced RA synovial fibroblast caspase-1 expression at the protein level in a time-dependant manner (P < 0.05). Blocking the JAK pathway reduced TNF-induced-caspase-1 expression at the transcriptional and protein levels by approximately 60% and 40%, respectively (P < 0.05). Blocking the JAK pathway reduced TNF-induced-caspase-1 expression at the transcriptional, protein, and activity levels by approximately 60%, 40%, and 53%, respectively (P < 0.05). We then confirmed by IF that TNF-induced IL-18 and investigated roles of the ERK1/2 and JAK pathways. Blocking the JAK pathway, TNF induced intracytoplasmic granular IL-18 expression suggesting a defect of caspase-1. Finally, blocking the JAK pathway, we observed a reduction of IL-18 bioactivity by 52% in RA synovial fibroblasts (P < 0.05).
Conclusions
These results provide a new way to regulate TNF-induced-IL-18 bioactivity by blocking capase-1. These data present a novel role for JAK inhibition in RA patients and emphasize JAK inhibition use as a new therapeutic option in RA management.
doi:10.1186/ar4551
PMCID: PMC4060193  PMID: 24762050
10.  Inhibitor of DNA binding 1 as a secreted angiogenic transcription factor in rheumatoid arthritis 
Introduction
Rheumatoid arthritis (RA) is characterized by enhanced blood vessel development in joint synovium. This involves the recruitment of endothelial progenitor cells (EPCs), allowing for de novo vessel formation and pro-inflammatory cell infiltration. Inhibitor of DNA Binding 1 (Id1) is a transcription factor characteristic of EPCs that influences cell maturation.
Method
Enzyme-linked immunosorbant assay (ELISA) and polymerase chain reaction (PCR) were used to examine Id1 levels in synovial fluid (SF) and endothelial cells (ECs), respectively. Immunohistology was used to determine the expression of Id1 in synovial tissue (ST). Human dermal microvascular EC (HMVEC) migration and tube forming assays were used to determine if recombinant human Id1 (rhuId1) and/or RA SF immunodepleted Id1 showed angiogenic activity. We also utilized the RA ST severe combined immunodeficient (SCID) mouse chimera to examine if Id1 recruits EPCs to RA synovium.
Results
ST samples immunostained for Id1 showed heightened expression in RA compared to osteoarthritis (OA) and normal (NL) ST. By immunofluorescence staining, we found significantly more Id1 in RA compared to OA and NL vasculature, showing that Id1 expressing cells, and therefore EPCs, are most active in vascular remodeling in the RA synovium. We also detected significantly more Id1 in RA compared to OA and other arthritis SFs by ELISA, which correlates highly with Chemokine (C-X-C motif) ligand 16 (CXCL16) levels. In vitro chemotaxis assays showed that Id1 is highly chemotactic for HMVECs and can be attenuated by inhibition of Nuclear Factor κB and phosphoinositide 3-kinase. Using in vitro Matrigel assays, we found that HMVECs form tubes in response to rhuId1 and that Id1 immunodepleted from RA SF profoundly decreases tube formation in Matrigel in vitro. PCR showed that Id1 mRNA could be up-regulated in EPCs compared to HMVECs in response to CXCL16. Finally, using the K/BxN serum induced arthritis model, we found that EC CXCR6 correlated with Id1 expression by immunohistochemistry.
Conclusions
We conclude that Id1 correlates highly with CXCL16 expression, EPC recruitment, and blood vessel formation in the RA joint, and that Id1 is potently angiogenic and can be up-regulated in EPCs by CXCL16.
doi:10.1186/ar4507
PMCID: PMC4060463  PMID: 24620998
11.  Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation 
Introduction
We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.
Methods
Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.
Results
Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.
Conclusions
These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.
doi:10.1186/ar4456
PMCID: PMC3978694  PMID: 24467809
12.  NK4 therapy: a new approach to target angiogenesis and inflammation in rheumatoid arthritis 
Rheumatoid arthritis (RA) is a progressive autoimmune disease characterized by synovial membrane hyperplasia, inflammation, and angiogenesis. Hepatocyte growth factor (HGF) and its receptor, c-Met, are both overexpressed in the RA synovium. NK4 is an antagonist of HGF which has been shown to inhibit tumor growth, metastasis, and angiogenesis. In an experimental model of RA, NK4 gene therapy inhibited joint damage and inflammation in both preventative and therapeutic models. NK4 treatment therefore represents a possible therapeutic option in combating RA.
doi:10.1186/ar4320
PMCID: PMC3979028  PMID: 24074223
13.  Type I Interferons Modulate Vascular Function, Repair, Thrombosis and Plaque Progression in Murine Models of Lupus and Atherosclerosis 
Arthritis and rheumatism  2012;64(9):2975-2985.
Objectives
Patients with systemic lupus erythematosus (SLE) have a striking increase in atherothrombotic cardiovascular disease (CVD), not explained by the Framingham risk equation. In vitro studies indicate that type-I Interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regards to the development of CVD, has not been characterized. We examined the role of type-I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis.
Methods
Lupus-prone New Zealand Mixed-2328 mice (NZM) and atherosclerosis-prone Apolipoprotein-E-knockout mice (ApoE−/−) were compared to mice lacking type-I IFN-receptor (INZM and ApoEIFNR−/−, respectively) in their endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development and occlusive thrombosis. Similar experiments were performed when NZM and ApoE−/− received an IFN-α-containing or an empty adenovirus.
Results
Loss of type IIFN-receptor signaling improves endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function and neoangiogenesis in lupus-prone mice, independent of disease activity or gender. Further, acute exposure to IFN-α impairs endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. ApoEIFNR−/− mice have decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis, compared to ApoE−/− mice, while NZM and ApoE−/− mice exposed to IFN-α develop accelerated thrombosis and platelet activation.
Conclusions
These results support the hypothesis that type I-IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.
doi:10.1002/art.34504
PMCID: PMC3411886  PMID: 22549550
Angiogenesis; atherosclerosis; systemic lupus erythematosus
14.  125I-Labeled Gold Nanorods for Targeted Imaging of Inflammation 
ACS nano  2011;5(11):8967-8973.
For better examination of inflammation, we designed inflammation-targeted nuclear and optical dual-modality contrast agents prepared by I-125 radiolabeling of gold nanorods (GdNRs) conjugated with anti-intercellular adhesion molecule 1 (ICAM-1) antibody. The bioactivity and specific binding of the PEGylated 125I-ICAM-GdNRs conjugates to the ICAM-1 was validated through ELISA testing. Inflammation-targeted imaging was then conducted on an adjuvant-induced arthritic rat model which demonstrated an elevation of ICAM-1 level in the affected ankle joints. Facilitated by the I-125 radioisotope and the whole-body imaging via the Gamma camera, the time-dependent distribution of the systemically injected agent as well as the uptake of the agent in the inflammatory articular tissues could be examined conveniently and quantitatively. The success in targeted delivery of gold nanoparticles to inflammatory tissue enables both nuclear and optical imaging of inflammation at molecular or cellular level. Other than diagnosis, radiolabeled gold nanoparticles also hold promise for targeted therapy of a variety of disorders.
doi:10.1021/nn203138t
PMCID: PMC3222780  PMID: 22003968
Gold nanorods; Iodine-125; γ-imaging; Radiolabeled nanoparticles; inflammation
15.  Suppression of endothelial cell activity by inhibition of TNFα 
Introduction
TNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis.
Methods
Human dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation.
Results
Certolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05).
Conclusion
Our data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.
doi:10.1186/ar3812
PMCID: PMC3446462  PMID: 22534470
17.  Macrophage Migration Inhibitory Factor Promotes Tumor Growth in the Context of Lung Injury and Repair 
Rationale: Tissue injury and repair involve highly conserved processes governed by mechanisms that can be co-opted in tumors. We hypothesized that soluble factors released during the repair response to lung injury would promote orthotopic tumor growth.
Objectives: To determine whether lung injury promoted growth of orthotopic lung tumors and to study the molecular mechanisms.
Methods: We initiated lung injury in C57Bl6 mice using different stimuli, then injected Lewis lung carcinoma cells during the repair phase. We assessed tumor growth 14 days later. We measured tumor angiogenesis, cytokine expression, proliferation, and apoptosis.
Measurements and Main Results: Regardless of the mechanism, injured lungs contained more numerous and larger tumors than sham-injured lungs. Tumors from injured lungs were no more vascular, but had higher levels of proliferation and reduced rates of apoptosis. The cytokine macrophage migration inhibitory factor (MIF) was highly expressed in both models of tissue injury. We observed no increase in tumor growth after lung injury in MIF knockout mice. We induced lung-specific overexpression of MIF in a double-transgenic mouse, and observed that MIF overexpression by itself was sufficient to accelerate the growth of orthotopic Lewis lung carcinoma tumors.
Conclusions: Lung injury leads to increased expression of the cytokine MIF, which results in protection from apoptosis and increased proliferation in orthotopic tumors injected after the acute phase of injury.
doi:10.1164/rccm.201001-0120OC
PMCID: PMC2970845  PMID: 20581170
cytokines; angiogenesis; stroma
18.  Expression and Function of CXCL16 in a Novel Model of Gout 
Arthritis and rheumatism  2010;62(8):2536-2544.
Objective
To better define the activity of soluble CXCL16 to recruit polymorphonuclear cells (PMNs) in vivo, we developed a novel animal model of gout pathology. We tested CXCL16 to recruit PMNs in a human normal synovial tissue (NL ST) severe combined immunodeficient (SCID) mouse chimera injected intragraft with gouty SF.
Methods
For in vitro studies, we utilized the modified Boyden chemotaxis system to identify CXCL16 as an active recruitment factor for PMNs. Cell migration could be reduced by neutralizing gouty SF CXCL16 activity. For in vivo analysis, fluorescently dye-tagged PMNs were injected i.v., while a simultaneous injection of diluted gouty SF containing CXCL16 was administered intragraft. We similarly inhibited the receptor for CXCL16, CXCR6, by incubating PMNs with neutralizing CXCR6 antibodies and examined PMN recruitment to gouty tissues in the SCID mouse chimera system.
Results
CXCL16 is highly elevated in gouty SF and PMNs migrate to CXCL16 in vitro. NL ST SCID mouse chimeras injected intragraft with gouty SF depleted of CXCL16 during PMN transfer showed a significant 50% reduction of PMN recruitment to engrafted tissue compared to grafts administered sham depleted gouty SF. Similar findings were achieved when incubating PMNs with neutralizing anti-CXCR6 antibody before injection into chimeras administered gouty SF.
Conclusion
Overall, this study outlines the effectiveness of the human SCID mouse chimera system as a viable animal model for gout, identifying a primary function of CXCL16 as a significant mediator of in vivo PMN recruitment to gouty SF.
doi:10.1002/art.27518
PMCID: PMC2921006  PMID: 20506383
Inflammation; chemokines; SCID; gout; CXCL16
19.  Junctional adhesion molecule-C is a soluble mediator of angiogenesis 
Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed by endothelial cells that plays a role in tight junction formation, leukocyte adhesion, and trans-endothelial migration. In the present study, we investigated whether JAM-C is found in soluble form and if soluble JAM-C (sJAM-C) mediates angiogenesis. We found that JAM-C is present in soluble form in normal serum and elevated in rheumatoid arthritis (RA) serum. The concentration of sJAM-C is also elevated locally in RA synovial fluid compared to RA serum or osteoarthritis synovial fluid. sJAM-C was also present in the culture supernatant of human microvascular endothelial cells (HMVECs) and immortalized human dermal microvascular endothelial cells (HMEC-1s), and its concentration was increased following cytokine stimulation. In addition, sJAM-C cleavage from the cell surface was mediated in part by a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17. In functional assays, sJAM-C was both chemotatic and chemokinetic for HMVECs, and induced HMVEC tube formation on Matrigel in vitro. Neutralizing anti-JAM-C antibodies inhibited RA synovial fluid induced HMVEC chemotaxis and sJAM-C induced HMVEC tube formation on Matrigel. sJAM-C also induced angiogenesis in vivo in the Matrigel plug and sponge granuloma models. Moreover, sJAM-C mediated HMVEC chemotaxis was dependent on Src, p38, and PI3K. Our results show that JAM-C exists in soluble form, and suggest that modulation of sJAM-C may provide a novel route for controling pathological angiogenesis.
doi:10.4049/jimmunol.1000556
PMCID: PMC3003428  PMID: 20592283
20.  Green tea extract inhibits chemokine production, but up-regulates chemokine receptor expression, in rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis 
Rheumatology (Oxford, England)  2009;49(3):467-479.
Objective. Evaluation of the efficacy of green tea extract (GTE) in regulating chemokine production and chemokine receptor expression in human RA synovial fibroblasts and rat adjuvant-induced arthritis (AIA).
Methods. Fibroblasts isolated from human RA synovium were used in the study. Regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, growth-regulated oncogene (GRO)α/CXCL1 and IL-8/CXCL8 production was measured by ELISA. Western blotting was used to study the phosphorylation of protein kinase C (PKC)δ and c-Jun N-terminal kinases (JNK). Chemokine and chemokine receptor expression was determined by quantitative RT–PCR. The benefit of GTE administration in rat AIA was determined.
Results. GTE (2.5–40 μg/ml) inhibited IL-1β-induced MCP-1/CCL2 (10 ng/ml), RANTES/CCL5, GROα/CXCL1 and IL-8/CXCL8 production in human RA synovial fibroblasts (P < 0.05). However, GTE inhibited MCP-1/CCL2 and GROα/CXCL1 mRNA synthesis in RA synovial fibroblasts. Furthermore, GTE also inhibited IL-1β-induced phosphorylation of PKCδ, the signalling pathway mediating IL-1β-induced chemokine production. Interestingly, GTE preincubation enhanced constitutive and IL-1β-induced CCR1, CCR2b, CCR5, CXCR1 and CXCR2 receptor expression. GTE administration (200 mg/kg/day p.o.) modestly ameliorated rat AIA, which was accompanied by a decrease in MCP-1/CCL2 and GROα/CXCL1 levels and enhanced CCR-1, -2, -5 and CXCR1 receptor expression in the joints of GTE administered rats.
Conclusions. Chemokine receptor overexpression with reduced chemokine production by GTE may be one potential mechanism to limit the overall inflammation and joint destruction in RA.
doi:10.1093/rheumatology/kep397
PMCID: PMC2820264  PMID: 20032224
Green tea; Chemokines; Chemokine receptors; Rheumatoid arthritis; Synovial fibroblasts; Complementary and alternative medicine; Adjuvant-induced arthritis
21.  Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis 
Introduction
Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.
Methods
Immunohistology and enzyme-linked immunosorbent assays (ELISAs) were used to determine chemokine and chemokine receptor expression in the skin and serum, respectively, of SSc and normal patients. Endothelial cells (ECs) were isolated from SSc skin biopsies and chemokine and chemokine receptor expression was determined by quantitative PCR and immunofluorescence staining.
Results
Antiangiogenic IP-10/CXCL10 and MIG/CXCL9 were elevated in SSc serum and highly expressed in SSc skin. However, CXCR3, the receptor for these chemokines, was decreased on ECs in SSc vs. normal skin. CXCL16 was elevated in SSc serum and increased in SSc patients with early disease, pulmonary arterial hypertension, and those that died during the 36 months of the study. In addition, its receptor CXCR6 was overexpressed on ECs in SSc skin. At the mRNA and protein levels, CXCR3 was decreased while CXCR6 was increased on SSc ECs vs. human microvascular endothelial cells (HMVECs).
Conclusions
These results show that while the expression of MIG/CXCL9 and IP-10/CXCL10 are elevated in SSc serum, the expression of CXCR3 is downregulated on SSc dermal ECs. In contrast, CXCL16 and CXCR6 are elevated in SSc serum and on SSc dermal ECs, respectively. In all, these findings suggest angiogenic chemokine receptor expression is likely regulated in an effort to promote angiogenesis in SSc skin.
doi:10.1186/ar3242
PMCID: PMC3241362  PMID: 21303517
22.  Pathological Role of Fractalkine/CX3CL1 in Rheumatic Diseases: A Unique Chemokine with Multiple Functions 
Understanding rheumatic diseases from the perspective of chemokine biology has shaped and will continue to shape our approach for targeted drug design. Among different kinds of chemokines, fractalkine/CX3CL1 has been found to play an important role in inflammation, portraying unique functional, and structural characteristics. This review summarizes the emerging role of fractalkine/CX3CL1 from a functional and clinical perspective and provides evidence to validate it as a potential therapeutic target in rheumatic diseases such as rheumatoid arthritis, Sjögren’s syndrome, systemic lupus erythematosus, scleroderma, as well as diseases related to vascular inflammation. From this, recent studies investigating potential therapeutic agents against fractalkine/CX3CL1’s role in pathology have shown promise.
doi:10.3389/fimmu.2011.00082
PMCID: PMC3341950  PMID: 22566871
fractalkine/CX3CL1; rheumatic disorders; proteolytic shedding; ADAM17/TACE; ADAM10
23.  Synovial Inflammation in Patients with Osteonecrosis of the Femoral Head 
Much of the work aimed at elucidating the pathogenesis of osteonecrosis (ON) of the femoral head has focused on bone blood supply, with little attention to the surrounding synovial tissue (ST). We hypothesized that patients with ON exhibit synovial inflammation. Using immunohistological techniques, we found that a large population of patients with ON had synovial inflammation. Moreover, a population of ON patients had inflamed ST without having an inflammatory disease co-morbidity. The inflammatory infiltrate in these patients comprised primarily CD4+ T cells and CD68+ macrophages, the latter of which expressed increased levels of cellular adhesion molecules. Our results suggest the presence of a previously unrecognized population of ON patients without a diagnosed inflammatory co-morbidity and a highly inflammed synovium consisting primarily of a macrophage and CD4+ T-cell infiltrate.
doi:10.1111/j.1752-8062.2009.00133.x
PMCID: PMC2925228  PMID: 20443906
osteonecrosis; synovium; inflammation; macrophage; adhesion molecules
24.  Endothelial progenitor cells in arthritis-associated vasculogenesis and atherosclerosis 
Vasculogenesis is the generation of vessels from endothelial progenitor cells (EPCs). Attenuated numbers and function of EPCs associated with defective vasculogenesis are present in rheumatoid arthritis (RA), scleroderma and other autoimmune-inflammatory diseases, which have significant relevance for increased cardio- and cerebrovascular morbidity and mortality in arthritis [1–5]. Stimulation of EPCs and vasculogenesis may be beneficial to prevent and manage atherosclerosis related to arthritis. [1–5].
doi:10.1016/j.jbspin.2009.04.007
PMCID: PMC2919750  PMID: 19945323
Vasculogenesis; Atherosclerosis; Rheumatoid arthritis; Endothelial progenitor cells
25.  Targeting Angiogenesis in Rheumatoid Arthritis 
Current rheumatology reviews  2008;4(4):298-303.
Angiogenesis, the development of new capillaries, is a crucial process in health and disease. The perpetuation of neovascularization in rheumatoid arthritis is highly involved in leukocyte extravasation into the synovium and pannus formation. Numerous soluble and cell surface-bound angiogenic mediators, including growth factors, cytokines, proteases, matrix macromolecules, cell adhesion receptors, chemokines and chemokine receptors, have been implicated in the process of neovascularization. Endogenous angiostatic factors, primarily angiostatin, endostatin, IL-4, IL-13, some angiostatic chemokines may be used to downregulate neovascularization. In addition, angiogenesis might be targeted by several specific approaches against VEGF, angiopoietin, αvβ3 integrin or by exogenously administered compounds including DMARDs, anti-TNF agents, fumagillin analogues or thalidomide. Potentially all anti-angiogenic could be tried in order to control synovitis.
PMCID: PMC2919854  PMID: 20706557
Angiogenesis; rheumatoid arthritis; targeting; angiogenic mediators; angiostasis

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