M. Asif Amin

Fractalkine: A Novel Angiogenic Chemokine in Rheumatoid Arthritis

Angiogenesis is an important aspect of the vasculoproliferation found in the rheumatoid arthritic (RA) pannus. We have previously implicated members of the CXC chemokine family as potent angiogenic mediators in RA. We investigated the possibility that the sole member of the CX(3)C chemokine family, fractalkine (fkn), induces angiogenesis and that fkn might mediate angiogenesis in RA. Recombinant human fkn significantly induced migration of human dermal microvascular endothelial cells (HMVECs), a facet of the angiogenic response, in the pmol/L range in a concentration-dependent manner (P < 0.05). Fkn also induced the formation of significantly more endothelial tubes on Matrigel than did a negative control (P < 0.05). Fkn significantly induced 2.3-fold more blood vessel growth than control in the in vivo Matrigel plug assays (P < 0.05). We identified HMVEC expression of the fkn receptor, CX(3)CR1. Next, we determined if RA synovial fluid (SF)-induced angiogenesis was fkn-dependent. SFs from six RA patients immunodepleted of soluble fkn induced 56% less migration of HMVECs than did sham-depleted RA SFs (P < 0.05). In vivo, immunodepletion of fkn from six RA SFs significantly inhibited their angiogenic activity in Matrigel plug assays (P < 0.05). Immunodepletion of fkn from five RA synovial tissue homogenates inhibited their ability to induce angiogenesis in in vivo Matrigel plug assays (P < 0.05). These results establish a new function for fkn as an angiogenic mediator and suggest that it may mediate angiogenesis in RA.

Migration Inhibitory Factor Mediates Angiogenesis via Mitogen-Activated Protein Kinase and Phosphatidylinositol Kinase

Abstract— In this study, we investigated the effects of migration inhibitory factor (rhMIF) on angiogenesis-related signaling cascades and apoptosis in human endothelial cells (ECs). We show that in vitro rhMIF induces migration and tube formation in Matrigel of human dermal microvascular endothelial cells (HMVECs), with potency comparable to that of basic fibroblast growth factor. In vivo, rhMIF induces angiogenesis in Matrigel plugs and in the corneal bioassay. Using panels of relatively specific kinase inhibitors, antisense oligonucleotides, and dominant-negative mutants, we show that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) are critical for MIF-dependent HMVEC migration, whereas Src and p38 kinases are nonessential. Moreover, we demonstrate that rhMIF induces time-dependent increases in phosphorylation levels of MEK1/2, Erk1/2, and Elk-1, as well as PI3K, and its effector kinase, Akt, in HMVECs. Studies with dominant-negative mutants and antisense oligonucleotides corroborate these effects in HMVECs. Furthermore, we demonstrate that rhMIF-induced angiogenesis in the rat cornea in vivo and in the ex vivo endothelial cell morphogenesis assay is also MAPK- and PI3K-dependent. Our findings support a role for MIF as an angiogenic factor and provide a rationale for the use of MIF as a therapeutic inducer of neovascularization in the development of collateral circulation in coronary artery disease.

IL-4 Adenoviral Gene Therapy Reduces Inflammation, Proinflammatory Cytokines, Vascularization, and Bony Destruction in Rat Adjuvant-Induced Arthritis

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-α, IL-1β, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.

Interleukin-13 Gene Therapy Reduces Inflammation, Vascularization, and Bony Destruction in Rat Adjuvant-Induced Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial pannus formation, leukocyte infiltration, and angiogenesis. Adenoviral production of interleukin-13 (IL-13) reduces levels of proinflammatory mediators in an explant model of RA synovial tissue in vitro. To assess this approach in an animal model of arthritis, we compared intra-articular injections of an adenovirus producing rat IL-13 (AxCArIL-13), a control virus, and rat ankles receiving phosphate-buffered saline (PBS) in rat adjuvant-induced arthritis (AIA). We demonstrate that IL-13 levels are normally low in ankles throughout the course of rat AIA. We show that administration of AxCArIL-13 before arthritis onset significantly reduces ankle circumference, paw volume, bony destruction, the number of polymorphonuclear cells (PMNs), the quantity of blood vessels, and levels of monocyte chemoattractant protein (MCP)-1 in ankles. When administered as a treatment to inflamed ankles, AxCArIL-13 decreases articular index scores, paw volumes, bony destruction, vascularization, tumor necrosis factor-alpha (TNF-alpha) levels, and the quantity of monocytes, lymphocytes, and PMNs. Thus, increasing IL-13 levels significantly ameliorates the course of rat AIA, suggesting that similar strategies for the treatment of human RA are worthy of further study.

Treatment with sulfasalazine or sulfapyridine, but not 5‐aminosalicylic acid, inhibits basic fibroblast growth factor–induced endothelial cell chemotaxis

OBJECTIVE Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression. METHODS SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites. RESULTS HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78. CONCLUSION These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.

Interleukin 18 induces angiogenesis in vitro and in vivo via Src and Jnk kinases

Background Interleukin 18 (IL-18) is a novel mediator of angiogenesis in rheumatoid arthritis (RA). Objective To examine the role of IL-18 in RA angiogenesis and the signalling mechanisms involved. Methods Human dermal microvascular endothelial cell (HMVEC) chemotaxis, capillary morphogenesis assays and Matrigel plug angiogenesis assays were performed in vivo using IL-18 with or without signalling inhibitors. A novel model of angiogenesis was devised using dye-tagged HMVECs to study their homing into RA and normal (NL) synovial tissues (STs) engrafted in severe combined immunodeficient (SCID) mice. Results IL-18-mediated angiogenesis depended on Src and Jnk, as the inhibitors of Src and Jnk blocked IL-18-induced HMVEC chemotaxis, tube formation and angiogenesis in Matrigel plugs. However, inhibitors of Janus kinase 2, p38, MEK, phosphatidylinositol-3-kinase and neutralising antibodies to vascular endothelial growth factor or stromal derived factor-1α did not alter IL-18-induced HMVEC migration. These results were confirmed with Jnk or Src sense or antisense oligodeoxynucleotides. Moreover, IL-18 induced phosphorylation of Src and Jnk in HMVECs. As proof of principle, IL-18 null mice had a significantly decreased angiogenesis compared with wild-type mice in Matrigel plug angiogenesis assays in vivo. IL-18 markedly enhanced mature HMVEC homing to human RA ST compared with NL ST in SCID mice, confirming the role of IL-18-induced angiogenesis in RA ST in vivo. Conclusion Targeting IL-18 or its signalling intermediates may prove to be a potentially novel therapeutic strategy for angiogenesis-dependent diseases, such as RA.

Evidence that CXCL16 is a potent mediator of angiogenesis and is involved in endothelial progenitor cell chemotaxis: Studies in mice with K/BxN serum-induced arthritis

OBJECTIVE To examine the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium. METHODS We utilized the RA synovial tissue SCID mouse chimera system to examine human microvascular EC (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium that was injected intragraft with CXCL16-immunodepleted RA synovial fluid (SF). CXCR6-deficient 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 assessment of CXCL16 signaling activity. RESULTS CXCR6 was prominently expressed on human EPCs and HMVECs, and its expression on HMVECs could be up-regulated by interleukin-1β. SCID mice injected with CXCL16-depleted RA SF exhibited a significant reduction in EPC recruitment. In experiments using the K/BxN serum-induced inflammatory arthritis model, CXCR6(-/-) mice showed profound reductions in hemoglobin levels, which correlated with reductions in monocyte and T cell recruitment to arthritic joint tissue compared to that observed in WT mice. Additionally, HMVECs and EPCs responded to CXCL16 stimulation, but exhibited 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 is closely associated with EPC recruitment and blood vessel formation in the RA joint.

Citrullination of epithelial neutrophil-activating peptide 78/CXCL5 results in conversion from a non-monocyte-recruiting chemokine to a monocyte-recruiting chemokine.

To examine whether the citrullinated chemokines epithelial neutrophil–activating peptide 78 (ENA‐78)/CXCL5, macrophage inflammatory protein 1α/CCL3, and monocyte chemotactic protein 1/CCL2 are detected in the biologic fluid of patients with rheumatoid arthritis (RA), and if so, to determine the biologic activities of these chemokines.

Interleukin-18 as an in vivo mediator of monocyte recruitment in rodent models of rheumatoid arthritis

IntroductionThe function of interleukin-18 (IL-18) was investigated in pertinent animal models of rodent rheumatoid arthritis (RA) to determine its proinflammatory and monocyte recruitment properties.MethodsWe used a modified Boyden chemotaxis system to examine monocyte recruitment to recombinant human (rhu) IL-18 in vitro. Monocyte recruitment to rhuIL-18 was then tested in vivo by using an RA synovial tissue (ST) severe combined immunodeficient (SCID) mouse chimera. We defined monocyte-specific signal-transduction pathways induced by rhuIL-18 with Western blotting analysis and linked this to in vitro monocyte chemotactic activity. Finally, the ability of IL-18 to induce a cytokine cascade during acute joint inflammatory responses was examined by inducing wild-type (Wt) and IL-18 gene-knockout mice with zymosan-induced arthritis (ZIA).ResultsWe found that intragraft injected rhuIL-18 was a robust monocyte recruitment factor to both human ST and regional (inguinal) murine lymph node (LN) tissue. IL-18 gene-knockout mice also showed pronounced reductions in joint inflammation during ZIA compared with Wt mice. Many proinflammatory cytokines were reduced in IL-18 gene-knockout mouse joint homogenates during ZIA, including macrophage inflammatory protein-3α (MIP-3α/CCL20), vascular endothelial cell growth factor (VEGF), and IL-17. Signal-transduction experiments revealed that IL-18 signals through p38 and ERK½ in monocytes, and that IL-18-mediated in vitro monocyte chemotaxis can be significantly inhibited by disruption of this pathway.ConclusionsOur data suggest that IL-18 may be produced in acute inflammatory responses and support the notion that IL-18 may serve a hierarchic position for initiating joint inflammatory responses.

Accelerated development of arthritis in mice lacking endothelial selectins

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.