Targeting CD13/aminopeptidase N as a novel therapeutic approach for scleroderma fibrosis.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune multisystem disease with poorly understood pathogenesis and ineffective treatment options. Soluble CD13 (sCD13), generated by cleavage of cell surface CD13 via matrix metalloproteinase 14 (MMP14), signals through the bradykinin receptor B1 (B1R) to elicit pro-inflammatory, pro-arthritic, and pro-angiogenic responses. In this study we explored the anti-fibrotic potential of targeting the sCD13-B1R axis in SSc. METHODS: The expression of CD13, B1R and MMP14 was examined in SSc skin and explanted dermal fibroblasts. The efficacy of B1R antagonists in the inhibition on fibrosis was determined in vitro and in vivo. RESULTS: Expression of the genes for CD13, B1R and MMP14 was elevated in skin biopsies from patients with diffuse cutaneous (dc)SSc. Notably, single cell analysis of SSc skin biopsies revealed the highest BDKRB1 expression in COL8A1-positive myofibroblasts, a population exclusively seen in SSc. TGF-ß induced the expression of BDKRB1 and production of sCD13 by dcSSc skin fibroblasts. Treatment of dcSSc fibroblasts with sCD13 promoted fibrotic gene expression, signaling, cell proliferation, migration, and gel contraction. The profibrotic sCD13 or TGFß responses were prevented by a B1R antagonist. Mice lacking Cd13 or Bdkrb1 were resistant to bleomycin-induced skin fibrosis and inflammation. Pharmacological B1R inhibition had a comparable antifibrotic effect. CONCLUSION: These results are the first to demonstrate a key role for sCD13 in SSc skin fibrosis, and suggest that targeting the sCD13-B1R signaling axis is a promising novel therapeutic approach for SSc.

Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Activation of Cytotoxic Lymphocytes Through CD6 Enhances Killing of Cancer Cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8+ T cells. Tumor-infiltrating cytotoxic lymphocytes were found in higher proportions and were activated in UMCD6-treated mice compared to controls. Similar changes in gene expression were observed by RNA-seq analysis of NK cells treated with UMCD6. Particularly, UMCD6 up-regulated the NKG2D-DAP10 complex and activated PI3K. Thus, the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

Soluble CD13 induces inflammatory arthritis by activating the bradykinin receptor B1.

CD13, an ectoenzyme on myeloid and stromal cells, also circulates as a shed, soluble protein (sCD13) with powerful chemoattractant, angiogenic, and arthritogenic properties, which require engagement of a G protein-coupled receptor (GPCR). Here we identify the GPCR that mediates sCD13 arthritogenic actions as the bradykinin receptor B1 (B1R). Immunofluorescence and immunoblotting verified high expression of B1R in rheumatoid arthritis (RA) synovial tissue and fibroblast-like synoviocytes (FLSs), and demonstrated binding of sCD13 to B1R. Chemotaxis, and phosphorylation of Erk1/2, induced by sCD13, were inhibited by B1R antagonists. In ex vivo RA synovial tissue organ cultures, a B1R antagonist reduced secretion of inflammatory cytokines. Several mouse arthritis models, including serum transfer, antigen-induced, and local innate immune stimulation arthritis models, were attenuated in Cd13-/- and B1R-/- mice and were alleviated by B1R antagonism. These results establish a CD13/B1R axis in the pathogenesis of inflammatory arthritis and identify B1R as a compelling therapeutic target in RA and potentially other inflammatory diseases.

Inhibition of bromodomain extraterminal histone readers alleviates skin fibrosis in experimental models of scleroderma.

Binding of the bromodomain and extraterminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription. We sought to determine the antifibrotic efficacy and potential mechanisms of BET inhibition in systemic sclerosis (SSc). Blockade of BETs was done using a pan-BET inhibitor, JQ1; BRD2 inhibitor, BIC1; or BRD4 inhibitors AZD5153 or ARV825. BET inhibition, specifically BRD4 blockade, showed antifibrotic effects in an animal model of SSc and in patient-derived diffuse cutaneous SSc (dcSSc) fibroblasts. Transcriptome analysis of JQ1-treated dcSSc fibroblasts revealed differentially expressed genes related to extracellular matrix, cell cycle, and calcium (Ca2+) signaling. The antifibrotic effect of BRD4 inhibition was mediated at least in part by downregulation of Ca2+/calmodulin-dependent protein kinase II α and reduction of intracellular Ca2+ concentrations. On the basis of these results, we propose targeting Ca2+ pathways or BRD4 as potentially novel therapeutic approaches for progressive tissue fibrosis.

CD6 is a target for cancer immunotherapy.

Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with the aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung, and prostate cancer cells through direct effects on both CD8+ T cells and NK cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xenotransplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and downregulated expression of the inhibitory receptor NKG2A on both NK cells and CD8+ T cells, with concurrent increases in perforin and granzyme B production. The combined capability of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4+ lymphocyte differentiation while enhancing killing of cancer cells through distinct effects on CD8+ and NK cells opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.

Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system.

Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.

Attenuation of Murine Collagen-Induced Arthritis by Targeting CD6.

OBJECTIVE: CD6 is an important regulator of T cell function that interacts with the ligands CD166 and CD318. To further clarify the significance of CD6 in rheumatoid arthritis (RA), we examined the effects of targeting CD6 in the mouse model of collagen-induced arthritis (CIA), using CD6-knockout (CD6-KO) mice and CD6-humanized mice that express human CD6 in lieu of mouse CD6 on their T cells. METHODS: We immunized wild-type (WT) and CD6 gene-KO mice with a collagen emulsion to induce CIA. For treatment studies using CD6-humanized mice, mice were immunized similarly and a mouse anti-human CD6 IgG (UMCD6) or control IgG was injected on days 7, 14, and 21. Joint tissues were evaluated for tissue damage, leukocyte infiltration, and local inflammatory cytokine production. Collagen-specific Th1, Th9, and Th17 responses and serum levels of collagen-specific IgG subclasses were also evaluated in WT and CD6-KO mice with CIA. RESULTS: The absence of CD6 reduced 1) collagen-specific Th9 and Th17, but not Th1 responses, 2) the levels of many proinflammatory joint cytokines, and 3) serum levels of collagen-reactive total IgG and IgG1, but not IgG2a and IgG3. Joint homogenate hemoglobin content was significantly reduced in CD6-KO mice with CIA compared to WT mice with CIA (P < 0.05) (reduced angiogenesis). Moreover, treating CD6-humanized mice with mouse anti-human CD6 monoclonal antibody was similarly effective in reducing joint inflammation in CIA. CONCLUSION: Taken together, these data suggest that interaction of CD6 with its ligands is important for the perpetuation of CIA and other inflammatory arthritides that are T cell driven.

Angiogenic and Arthritogenic Properties of the Soluble Form of CD13.

Aminopeptidase N/CD13 is expressed by fibroblast-like synoviocytes (FLS) and monocytes (MNs) in inflamed human synovial tissue (ST). This study examined the role of soluble CD13 (sCD13) in angiogenesis, MN migration, phosphorylation of signaling molecules, and induction of arthritis. The contribution of sCD13 was examined in angiogenesis and MN migration using sCD13 and CD13-depleted rheumatoid arthritis (RA) synovial fluids (SFs). An enzymatically inactive mutant CD13 and intact wild-type (WT) CD13 were used to determine whether its enzymatic activity contributes to the arthritis-related functions. CD13-induced phosphorylation of signaling molecules was determined by Western blotting. The effect of sCD13 on cytokine secretion from RA ST and RA FLS was evaluated. sCD13 was injected into C57BL/6 mouse knees to assess its arthritogenicity. sCD13 induced angiogenesis and was a potent chemoattractant for MNs and U937 cells. Inhibitors of Erk1/2, Src, NF-κB, Jnk, and pertussis toxin, a G protein-coupled receptor inhibitor, decreased sCD13-stimulated chemotaxis. CD13-depleted RA SF induced significantly less MN migration than sham-depleted SF, and addition of mutant or WT CD13 to CD13-depleted RA SF equally restored MN migration. sCD13 and recombinant WT or mutant CD13 had similar effects on signaling molecule phosphorylation, indicating that the enzymatic activity of CD13 had no role in these functions. CD13 increased the expression of proinflammatory cytokines by RA FLS, and a CD13 neutralizing Ab inhibited cytokine secretion from RA ST organ culture. Mouse knee joints injected with CD13 exhibited increased circumference and proinflammatory mediator expression. These data support the concept that sCD13 plays a pivotal role in RA and acute inflammatory arthritis.

5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent New Class of Inhibitors of Rho/Myocardin-Related Transcription Factor (MRTF)/Serum Response Factor (SRF)-Mediated Gene Transcription as Potential Antifibrotic Agents for Scleroderma.

Through a phenotypic high-throughput screen using a serum response element luciferase promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated gene transcription with good potency (IC50 = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism. The series nevertheless has no observable cytotoxicity up to 100 µM. Ensuing pharmacokinetic optimization resulted in the development of two potent and orally bioavailable anti-fibrotic agents that were capable of dose-dependently reducing connective tissue growth factor gene expression in vitro as well as significantly reducing the development of bleomycin-induced dermal fibrosis in mice in vivo.

Citrullinated Inhibitor of DNA Binding 1 Is a Novel Autoantigen in Rheumatoid Arthritis.

OBJECTIVE: To explore the intrinsic role of inhibitor of DNA binding 1 (ID-1) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and to investigate whether ID-1 is citrullinated and autoantigenic in RA. METHODS: RA patient serum ID-1 levels were measured before and after infliximab treatment. RA FLS were transfected with a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 construct targeting ID-1 to examine the effects of ID-1 deletion. RA synovial fluid (SF) and homogenized synovial tissue (ST) were immunoprecipitated for ID-1 and measured for citrullinated residues using an enzyme-linked immunosorbent assay and Western blotting. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on in vitro-citrullinated recombinant human ID-1 (cit-ID-1) to localize the sites of citrullination. Normal and RA sera and SF were analyzed by immunodot blotting for anti-citrullinated protein antibodies (ACPAs) to cit-ID-1. RESULTS: RA patient serum ID-1 levels positively correlated with several disease parameters and were reduced after infliximab treatment. RA FLS displayed reduced growth and a robust increase in interleukin-6 (IL-6) and IL-8 production upon deletion of ID-1. ID-1 immunodepletion significantly reduced the levels of citrullinated residues in RA SF, and citrullinated ID-1 was detected in homogenized RA ST (n = 5 samples; P < 0.05). Immunodot blot analyses revealed ACPAs to cit-ID-1 but not to native ID-1, in RA peripheral blood (PB) sera (n = 30 samples; P < 0.001) and SF (n = 18 samples; P < 0.05) but not in normal PB sera. Following analyses of LC-MS/MS results for citrullination sites and corresponding reactivity in immunodot assays, we determined the critical arginines in ID-1 for autoantigenicity: R33, R52, and R121. CONCLUSION: Novel roles of ID-1 in RA include regulation of FLS proliferation and cytokine secretion as well as autoantigenicity following citrullination.

Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds.

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF-dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including significant reduction of melanoma metastasis and bleomycin- induced fibrosis. Although these compounds are efficacious in these disease models, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent cotranscription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. We also show with genetic approaches that pirin modulates MRTF- dependent luciferase reporter activity. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-ß- induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which co crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

A unique role for galectin-9 in angiogenesis and inflammatory arthritis.

BACKGROUND: Galectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis. METHODS: To determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees. RESULTS: Gal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9. CONCLUSIONS: Gal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.

Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease.

Tumor necrosis factor alpha (TNF-α) has both positive and negative roles in human disease. In certain cancers, TNF-α is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF-α antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNF-α-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNF-α stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated and non-phosphorylated TAK1 that binds within the ATP-binding pocket and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNF-α-induced cell death, with general implications for cancer and autoimmune disease treatment.

Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the Rho/MRTF/SRF transcriptional pathway as potential antifibrotic therapeutics for systemic scleroderma.

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.

Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis.

BACKGROUND: Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. METHODS: We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition. RESULTS: By PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-ß). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo. CONCLUSIONS: Id1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Activation of the Thromboxane A2 Receptor by 8-Isoprostane Inhibits the Pro-Angiogenic Effect of Vascular Endothelial Growth Factor in Scleroderma.

The pathogenesis of scleroderma (SSc) includes components of autoimmunity, vascular dysfunction, and accumulation of extracellular matrix. 8-isoprostane, an oxidized lipid created by oxidative stress, activates the thromboxane A2 receptor (TXAR) and the Rho-associated kinase (ROCK) pathway. In this study, we determined whether the TXAR was activated by 8-isoprostane in SSc endothelial cells (ECs) and whether this pathway inhibited VEGF-induced angiogenesis. Elevated 8-isoprostane was observed in plasma and conditioned media from SSc patients. SSc-conditioned media inhibited EC tube formation, whereas addition of vitamin E, by reducing 8-isoprostane, increased tube formation. VEGF did not induce angiogenesis in SSc ECs, but vitamin E or TXAR inhibition restored its effect. The expression of TXAR, RhoA, and ROCK1/2 was elevated in SSc ECs. ROCK activity and 8-isoprostane-induced ROCK activation were significantly higher in SSc ECs, whereas VEGF had no effect. The hyper-activation of the TXAR leads to inhibition of VEGF-induced angiogenesis, as inhibition of the TXAR pathway results in a blockade of 8-isoprostane-induced ROCK activation and restoration of VEGF activity. These results suggest that the TXAR pathway has a crucial role in angiogenesis and that 8-isoprostane is not just a by-product of oxidative stress but also has a significant role in the impaired angiogenesis that characterizes SSc.

Dynamic interactions between plasma IL-1 family cytokines and central endogenous opioid neurotransmitter function in humans.

Evidence in animal models suggests IL-1 family cytokines interact with central endogenous opioid neurotransmitter systems, inducing or perpetuating pathological states such as persistent pain syndromes, depression, substance use disorders, and their comorbidity. Understanding these interactions in humans is particularly relevant to understanding pathological states wherein this neurotransmitter system is implicated (ie, persistent pain, mood disorders, substance use disorders, etc). Here, we examined relationships between IL-1ß, IL-1ra, and functional measures of the endogenous opioid system in 34 healthy volunteers, in the absence and presence of a standardized sustained muscular pain challenge, a psychophysical challenge with emotionally and physically stressful components. Mu-opioid receptor availability in vivo was examined with [(11)C]carfentanil positron emission tomography (PET) scanning. Sex and neuroticism impacted IL-1 family cytokines; higher baseline IL-1ß and IL-1ra was identified in females with lower neuroticism. Higher baseline IL-1ß was also associated with reduced µ-opioid receptor availability (amygdala) and greater pain sensitivity. The pain challenge increased IL-1ß in females with high neuroticism. Strong associations between IL-1ra (an anti-nociceptive cytokine) and µ-opioid receptor activation (VP/NAcc) were identified during the pain challenge and the resulting analgesic effect of µ-opioid receptor activation was moderated by changes in IL-1ß whereby volunteers with greater pain induced increase in IL-1ß experienced less endogenous opioid analgesia. This study demonstrates the presence of relationships between inflammatory factors and a specific central neurotransmitter system and circuitry, of relevance to understanding interindividual variations in regulation of responses to pain and other physical and emotional stressors.