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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.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
14.  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
15.  Chemokines and angiogenesis in rheumatoid arthritis 
In rheumatoid arthritis, chemokines mediate the migration of inflammatory leukocytes into the synovium. Among the four known chemokine families, CXC, CC chemokines and fractalkine seem to be of outstanding importance in this process. Angiogenesis, the formation of new vessels, is also important during the perpetuation of inflammation underlying rheumatoid arthritis. In this review, authors discuss the role of the most important chemokines and chemokine repetors in arthritis-associated neovascularization. The process and regulation of angiogenesis are described in this context as well. Apart from discussing the pathogenic role of chemokines and chemokine receptors in arthritic vessel formation, authors also review the important relevance of chemokines and angiogenesis for therapeutic intervention.
PMCID: PMC2884394  PMID: 19482623
Angiogenesis; Chemokines; Chemokine Receptors; Rheumatoid Arthritis; Angiogenic Mediators; Review
16.  CXCR6 Induces Prostate Cancer Progression by the AKT/Mammalian Target of Rapamycin Signaling Pathway 
Cancer research  2008;68(24):10367-10376.
Previous studies show that the chemokine CXCL16 and its receptor CXCR6 are likely to contribute to prostate cancer (PCa). In this investigation, the role of the CXCR6 receptor in PCa was further explored. CXCR6 protein expression was examined using high-density tissue microarrays and immunohistochemistry. Expression of CXCR6 showed strong epithelial staining that correlated with Gleason score. In vitro and in vivo studies in PCa cell lines suggested that alterations in CXCR6 expression were associated with invasive activities and tumor growth. In addition, CXCR6 expression was able to regulate expression of the proangiogenic factors interleukin (IL)-8 or vascular endothelial growth factor (VEGF), which are likely to participate in the regulation of tumor angiogenesis. Finally, we found that CXCL16 signaling induced the activation of Akt, p70S6K, and eukaryotic initiation factor 4E binding protein 1 included in mammalian target of rapamycin (mTOR) pathways, which are located downstream of Akt. Furthermore, rapamycin not only drastically inhibited CXCL16-induced PCa cell invasion and growth but reduced secretion of IL-8 or VEGF levels and inhibited expression of other CXCR6 targets including CD44 and matrix metalloproteinase 3 in PCa cells. Together, our data shows for the first time that the CXCR6/AKT/mTOR pathway plays a central role in the development of PCa. Blocking the CXCR6/AKT/mTOR signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for PCa.
doi:10.1158/0008-5472.CAN-08-2780
PMCID: PMC2884407  PMID: 19074906
17.  Bim-BH3 mimetic therapy is effective at suppressing inflammatory arthritis through the activation of myeloid cell apoptosis 
Arthritis and rheumatism  2010;62(2):441-451.
Objective
Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies which activate the apoptotic cascade may have potential as a future therapy for RA, however few therapeutics fit this category. Recently, therapies that mimic the action of Bcl-2 homology 3 (BH3) domain-only proteins such as Bim have shown success in preclinical studies of cancer but their potential in autoimmune disease is unknown.
Methods
Synovial tissue from RA and osteoarthritis (OA) patients were analyzed for expression of Bim and CD68 using immunohistochemistry. Macrophages from mice lacking (Bim−/−) were examined for response to lipopolysaccharide (LPS) using flow cytometry, real time PCR, ELISA, and immunoblot analysis. Bim−/− mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum-transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT-BH3) was administered as a prophylactic and as a therapeutic. Edema of the ankles and histopathogical analysis of ankle sections were used to determine severity of arthritis, cellular composition, and apoptosis.
Results
The expression of Bim was reduced in RA synovial tissue as compared to controls, particularly in macrophages. Bim−/− macrophages displayed elevated expression of markers of inflammation and secreted more IL-1β following stimulation with LPS or thioglycollate. TAT-BH3 ameliorated arthritis development, reduced the number of myeloid cells in the joint, and enhanced apoptosis without inducing cytotoxicity.
Conclusion
These data demonstrate that BH3 mimetic therapy may have significant potential for RA treatment.
doi:10.1002/art.27198
PMCID: PMC2848986  PMID: 20112357
Bim; arthritis; macrophages; apoptosis
18.  Vasculogenesis in rheumatoid arthritis 
Decreased number and impaired functions of endothelial progenitor cells (EPCs) leading to impaired vasculogenesis have been associated with rheumatoid arthritis (RA). Defective vasculogenesis has also been implicated in premature atherosclerosis in RA. Recently, early-outgrowth monocytic and late-outgrowth hemangioblastic EPC subsets have been characterized. Hemangioblastic EPCs may exert increased numbers in active RA and may play a role in vascular repair underlying RA.
doi:10.1186/ar2943
PMCID: PMC2888181  PMID: 20346090
19.  Monocyte chemoattractant protein-1 (MCP) and macrophage inflammatory protein-1α (MIP) as possible biomarkers for the chronic pelvic pain syndrome 
The Journal of urology  2008;179(5):1857-1862.
PURPOSE
The chronic pelvic pain syndrome (CPPS) is characterized by pelvic pain, voiding symptoms and varying degrees of inflammation within expressed prostatic secretions (EPS). We evaluated the chemokines MCP-1 (CCL2) and MIP-1α (CCL3) in EPS to identify marker elevations associated with both inflammatory (IIIA) and non-inflammatory (IIIB) CPPS. In addition, chemokine levels were correlated with clinical pain as determined by the NIH chronic prostatitis symptom index (CPSI).
MATERIALS AND METHODS
EPS were collected by digital rectal examination and evaluated by ELISA for MCP-1 and MIP-1α in 154 patients; controls (n = 13), BPH (n = 54), CPPS IIIA (n = 37), CPPS IIIB (n = 50). MCP-1 and MIP-1α levels were compared between IIIA, IIIB, and the control subgroups and correlated against the CPSI and pain sub-score using a Spearman test.
RESULTS
Mean levels of MCP-1 in the control, inflammatory BPH, non-inflammatory BPH, inflammatory CPPS, and non-inflammatory CPPS were 599.4, 886.0, 1636.5, 3261.2, and 2272.7 pg/ml, respectively. Mean levels of MIP-1α in the control, inflammatory BPH, non-inflammatory BPH, IIIA CPPS, and IIIB CPPS were 140.1, 299.4, 238.7, 1057.8, and 978.4 pg/ml, respectively. For each cytokine, both CPPS subtypes had statistically higher levels than the control group and BPH patients (p=0.0002). Receiver operating curves utilizing MCP-1 levels greater than 704 pg/ml and MIP-1α greater than 146 pg/ml identified patients with CPPS with an accuracy of 90% from control patients. MIP-1α levels (p=0.0007) correlated with the pain sub-score of the CPSI while MCP-1 (p=0.71) did not.
CONCLUSIONS
MCP-1 and MIP-1α within the prostatic fluid in both CPPS subtypes provide candidate future biomarkers for CPPS. In addition, MIP-1α elevation in EPS provides a new marker for clinical pain in CPPS patients. Given these findings, prostatic dysfunction likely plays a role in the pathophysiology of some patients with this syndrome. These chemokines may serve as effective diagnostic markers and modulators against the chemokines could provide an attractive treatment strategy in individuals with CPPS.
doi:10.1016/j.juro.2008.01.028
PMCID: PMC2786778  PMID: 18353390
chronic pelvic pain syndrome; prostatitis; cytokines; monocyte chemoattractant protein; macrophage inflammatory protein
20.  Vascular involvement in rheumatic diseases: 'vascular rheumatology' 
The vasculature plays a crucial role in inflammation, angiogenesis, and atherosclerosis associated with the pathogenesis of inflammatory rheumatic diseases, hence the term 'vascular rheumatology'. The endothelium lining the blood vessels becomes activated during the inflammatory process, resulting in the production of several mediators, the expression of endothelial adhesion molecules, and increased vascular permeability (leakage). All of this enables the extravasation of inflammatory cells into the interstitial matrix. The endothelial adhesion and transendothelial migration of leukocytes is a well-regulated sequence of events that involves many adhesion molecules and chemokines. Primarily selectins, integrins, and members of the immunoglobulin family of adhesion receptors are involved in leukocyte 'tethering', 'rolling', activation, and transmigration. There is a perpetuation of angiogenesis, the formation of new capillaries from pre-existing vessels, as well as that of vasculogenesis, the generation of new blood vessels in arthritis and connective tissue diseases. Several soluble and cell-bound angiogenic mediators produced mainly by monocytes/macrophages and endothelial cells stimulate neovascularization. On the other hand, endogenous angiogenesis inhibitors and exogenously administered angiostatic compounds may downregulate the process of capillary formation. Rheumatoid arthritis as well as systemic lupus erythematosus, scleroderma, the antiphospholipid syndrome, and systemic vasculitides have been associated with accelerated atherosclerosis and high cardiovascular risk leading to increased mortality. Apart from traditional risk factors such as smoking, obesity, hypertension, dyslipidemia, and diabetes, inflammatory risk factors, including C-reactive protein, homocysteine, folate deficiency, lipoprotein (a), anti-phospholipid antibodies, antibodies to oxidized low-density lipoprotein, and heat shock proteins, are all involved in atherosclerosis underlying inflammatory rheumatic diseases. Targeting of adhesion molecules, chemokines, and angiogenesis by administering nonspecific immunosuppressive drugs as well as monoclonal antibodies or small molecular compounds inhibiting the action of a single mediator may control inflammation and prevent tissue destruction. Vasoprotective agents may help to prevent premature atherosclerosis and cardiovascular disease.
doi:10.1186/ar2515
PMCID: PMC2592799  PMID: 18947376
21.  Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues 
The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin αvβ3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCγ in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MΦs) treated with tumor necrosis factor-α (TNFα) or interleukin-1β (IL1β). RA and OA STs showed a greater percentage of pFAK on lining cells and MΦs compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFα or IL1β stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MΦs. pPyk2 was expressed on RA ST fibrobasts but not in MΦs at baseline, however it was upregulated upon TNFα or IL1β activation in both cell types. pSrc was expressed in RA ST fibroblasts and MΦs at baseline and was further increased by TNFα or IL1β stimulation. pPaxillin and pPLCγ were upregulated in RA versus OA and ND lining cells and sublining MΦs. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.
doi:10.1186/ar2318
PMCID: PMC2212559  PMID: 17963503
22.  Vasculopathy and disordered angiogenesis in selected rheumatic diseases: rheumatoid arthritis and systemic sclerosis 
Arthritis Research & Therapy  2007;9(Suppl 2):S3.
Angiogenesis is important in the pathogenesis of systemic inflammatory rheumatic diseases, a family of related disorders that includes rheumatoid arthritis and systemic sclerosis. Rheumatoid arthritis is the rheumatic disease in which the role of angiogenesis has been studied most extensively. However, whereas rheumatoid arthritis is characterized by excessive angiogenesis, the situation is not as clear cut in other rheumatic diseases. For example, systemic sclerosis is characterized by reduced capillary density with insufficient angiogenic responses. Results with angiogenesis inhibitors are controversial, and there is – in parallel – a wide range of upregulated angiogenic factors such as vascular endothelial growth factor. Dysregulation of angiogenesis in systemic sclerosis is accompanied by other pathogenic processes, including fibrosis, autoimmunity and vasculopathy. Animal models with at least partial features of the vasculopathy observed in systemic sclerosis include wound healing models, graft versus host disease models and, in particular, the University of California at Davis line 200 chicken model of systemic sclerosis.
doi:10.1186/ar2187
PMCID: PMC2072889  PMID: 17767741
23.  Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis 
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.
In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCδ inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCδ, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.
doi:10.1186/ar2021
PMCID: PMC1779381  PMID: 16872482
24.  Accelerated development of arthritis in mice lacking endothelial selectins 
Arthritis Research & Therapy  2005;7(5):R959-R970.
The selectins, along with very late antigen-4 and CD44, have been implicated in mediating leukocyte rolling interactions that lead to joint recruitment and inflammation during the pathogenesis of rheumatoid arthritis. Previously, we showed that P-selectin deficiency in mice resulted in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. Here, we report that mice deficient either in E-selectin or in E-selectin and P-selectin (E/P-selectin mutant) also exhibit accelerated development of arthritis compared with wild type mice in the CIA model, suggesting that these adhesion molecules perform overlapping functions in regulating joint disease. Analyses of cytokine and chemokine expression in joint tissue from E/P-selectin mutant mice before the onset of joint swelling revealed significantly higher joint levels of macrophage inflammatory protein-1α and IL-1β compared to wild-type mice. IL-1β remained significantly increased in E/P-selectin mutant joint tissue during the early and chronic phases of arthritis. Overall, these data illustrate the novel finding that E-selectin and P-selectin expression can significantly influence cytokine and chemokine production in joint tissue, and suggest that these adhesion molecules play important regulatory roles in the development of arthritis in E/P-selectin mutant mice.
doi:10.1186/ar1770
PMCID: PMC1257424  PMID: 16207337
25.  Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation 
Arthritis Research & Therapy  2005;7(2):R392-R401.
During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.
doi:10.1186/ar1502
PMCID: PMC1065337  PMID: 15743487
cartilage destruction; experimental arthritis; interleukin-13; Fcγ receptors; MMPs

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