Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes

The liver participates in inflammation via the elaboration of acute phase proteins from hepatocytes in response to IL-1, TNF-alpha, and IL-6/INF-beta 2/hepatocyte-stimulating factor. In addition, some inflammatory states of the liver are characterized by leukocyte infiltrates. Here we demonstrate that human hepatocyte lines are capable of expressing mRNA and biologic activity for a neutrophil chemotactic factor (NCF)/IL-8 in response to the inflammatory mediators IL-1 alpha, IL-1 beta, and TNF. Two human hepatoma cell lines (SK-Hep and Hep-G2) displayed a time- and dose-dependent increase in steady state levels of NCF/IL-8 mRNA and secretion of chemotactic activity in response to TNF and IL-1. Neutralizing antibody to NCF/IL-8 inhibited hepatocyte-derived chemotactic activity by 88%. In contrast to IL-1 and TNF, hepatocytes did not respond to LPS or IL-6 within the time and dose parameters used above. Although the expression of NCF/IL-8 mRNA (1.8 kb) was first detectable between 1 and 2 h poststimulation, significant chemotactic bioactivity was not observed until about 4 h. Heat-inactivated (100 degrees C, 30 min) cytokine failed to induced NCF/IL-8 mRNA synthesis, and cotreatment of cells with cytokine and cycloheximide super-induced NCF/IL-8 mRNA while inhibiting production of bioactivity. Thus, NCF/IL-8 expression is a primary induction phenomenon. Our data demonstrate the stimulus specific induction of NCF/IL-8 in hepatocytes and suggest that cytokine cell-to-cell communication circuits may be important in neutrophil-mediated inflammatory processes in the liver.     

Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.     

Heat shock of rabbit synovial fibroblasts increases expression of mRNAs for two metalloproteinases, collagenase and stromelysin

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.     

Interleukin-1 induced gene expression of neutrophil activating protein (interleukin-8) and monocyte chemotactic peptide in human synovial cells

We report here that human synovial cells stimulated by interleukin-1 alpha and interleukin-1 beta express mRNA for both IL-8 (neutrophil chemotactic peptide) and monocyte chemotactic protein. IL-1 stimulated synovial cells from both osteoarthritis and rheumatoid arthritis patients exhibited similar mRNA expression of interleukin-8 and monocyte chemotactic protein. A capacity to produce factors selectively chemotactic for neutrophils, lymphocytes and monocytes provides a mechanism whereby synovial cells can facilitate inflammatory arthritis.     

Inflammatory cytokines induce synthesis and secretion of gro protein and a neutrophil chemotactic factor but not beta 2-microglobulin in human synovial cells and fibroblasts.

Exposure of human synovial cells and fibroblasts in monolayer culture to interleukin 1 results in prominent secretion of proteins with Mr values of 6000 and 7000. By N-terminal sequence analysis, the Mr-6000 protein is identified as the protein encoded by a recently described gro mRNA. The Mr-7000 protein is identical to a neutrophil chemotactic factor released from monocytes. Stimulation of normal human fibroblasts with tumour necrosis factor alpha also results in expression and secretion of these two proteins. In addition to these cytokine-induced proteins, we have identified beta 2-microglobulin as an Mr-8000 protein constitutively secreted by synovial cells.     

Morphologic alterations in cultured human synovial fibroblasts induced by blood mononuclear cells

Effects of peripheral blood mononuclear cells on cultured synovial fibroblasts were studied. When mononuclear cells from normal or rheumatoid blood were incubated on synovial fibroblast cultures, a part of the cells adhered to the fibroblasts. They were mainly T lymphocytes but also some B lymphocytes and monocytes. After a 10-hour incubation, adhered mononuclear cells induced morphologic alterations to synovial fibroblasts: appearance of stellate cells and thinning and branching of fibroblasts. No changes were seen when the cells were incubated in the presence of indomethacin. Cytotoxicity of peripheral blood mononuclear cells from 8 rheumatoid patients was also tested against three rheumatoid and three normal synovial fibroblast strains. Only 2 out of 48 combinations were cytotoxicity. The potentially cytotoxic mononuclear cells were bound equally well to rheumatoid and control synovial fibroblast cultures.     

Fibrin(ogen)-induced expression of ICAM-1 and chemokines in human synovial fibroblasts

It has long been recognized that in most inflamed arthritic joints the coagulation system is activated, leading to the local generation of fibrin, and it has long been hypothesized that the local fibrin deposition promotes inflammation and tissue destruction. However, only recently has the direct effect of fibrin on the inflammatory process been seriously investigated, and specific roles assigned to fibrin or its products as mediators of the inflammatory process. Although fibrin and/or fibrinogen (fibrin(ogen)) is abundantly present in inflamed tissues and joints rich in fibroblastic cells, no significant data on fibrin(ogen)-induced gene expression by fibroblasts have been published. We now demonstrate that coculture of human synovial fibroblasts with fibrin(ogen) results in the up-regulation of ICAM-1 as well as increased production of the chemokines IL-8 and growth-related oncogene-alpha. Increased ICAM-1 expression was fibrin(ogen) dose-dependent and was demonstrated by ELISA, flow cytometry, and functional adhesion assays. Levels of ICAM-1 induced by fibrin(ogen) were comparable to those that could be induced by cytokine stimulation. Fibrin(ogen) stimulation of ICAM-1 could be suppressed by pyrrolidinedithiocarbamate, an inhibitor of NF-kappaB activation. Chemokine production was induced by fibrin(ogen) in cell culture supernatants >100-fold as compared with controls. Thus, through its activation of synovial fibroblasts, fibrin(ogen) deposition may promote the recruitment (via chemokines) and retention (via adhesion molecules) of lymphocytes within the arthritic joint.     

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Platelet-derived growth factor in chemotactic for fibroblasts

Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet- derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.     

Enhancement of placenta growth factor expression by oncostatin M in human rheumatoid arthritis synovial fibroblasts

Oncostatin M (OSM) belongs to IL‐6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of rheumatoid arthritis (RA) patients and it may arouse the inflammation responses in joints and eventually leads to bone erosion. Placenta growth factor (PLGF) is an angiogenic factor and highly homologous with vascular endothelial growth factor (VEGF). It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines including TNF‐α and IL‐6. Here, we demonstrated that OSM increased mRNA and protein levels of PLGF in a time‐ and concentration‐dependent manner in RA synovial fibroblasts. Inhibitors of JAK3 and PI3K antagonized OSM‐induced production of PLGF. OSM enhanced the phosphorylation of Tyr705‐STAT3, Ser727‐STAT3, Ser473‐Akt, and increased the nuclear translocation of phosphorylated STAT3 time‐dependently. Transfection of dominant negative Akt or application of PI3K inhibitorLY294002 significantly inhibited p‐Tyr705‐STAT3, p‐Ser727‐STAT3, and PLGF expression, indicating that Akt is involved in JAK3/STAT3/PLGF signaling cascade. To further examine whether STAT3 binds to the promoter region of PLGF, Chip assay was used and it was found that OSM could bind with PLGF promoter, which was inhibited by JAK3 and PI3K inhibitors. Accumulation of PLGF in the pannus may contribute to the inflammation, angiogenesis and joints destruction in RA patients. These findings demonstrated the important role of OSM in the pathology network of RA and provided novel therapeutic drug targets for RA treatment. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

Introduction

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs).

Methods

Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.

Conclusion

Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.     

Closely related alpha-tropomyosin mRNAs in quail fibroblasts and skeletal muscle cells

We describe the analysis of two quail cDNA clones representing distinct but closely related alpha-tropomyosin mRNAs. cDNA clone cC101 corresponds to a 1.2-kilobase RNA which accumulates to high levels during myoblast differentiation and which encodes the major isoform of skeletal muscle alpha-tropomyosin. cDNA clone cC102 corresponds to a 2-kilobase RNA which is abundant in cultured embryonic skin fibroblasts and which encodes one of two alpha-tropomyosin-related fibroblast tropomyosins of 35,000 and 34,000 daltons apparent molecular mass (class 1 tropomyosins). The cC102 protein is unique among reported nonstriated-muscle tropomyosins in being identical in amino acid sequence to the major isoform of skeletal muscle alpha-tropomyosin over an uninterrupted stretch of at least 183 amino acids (residues 75-257). The two protein sequences differ in the COOH-terminal region beginning with residue 258. Because the cC101 and cC102 RNAs share an extensive region (at least 373 nucleotides) of nucleotide sequence identity upstream of the codon for residue 258, they are likely derived from a single gene by alternative RNA splicing, as was recently proposed in the case of related beta-tropomyosin mRNAs in human fibroblasts and skeletal muscle (MacLeod, A. R., Houlker, C., Reinach, R. C., Smillie, L. B., Talbot, K., Modi, G., and Walsh, F. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7835-7837). No alpha-tropomyosin-related RNAs are abundant in undifferentiated myoblasts. This suggests the possibility of a fibroblast-specific function, as opposed to a general nonmuscle-cell function for class 1 tropomyosins and also has implications for the regulation of alpha-tropomyosin gene expression during embryonic development.     

VIP and CRF reduce ADAMTS expression and function in osteoarthritis synovial fibroblasts

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family is known to play an important role in the pathogenesis of osteoarthritis (OA), working on aggrecan degradation or altering the integrity of extracellular matrix (ECM). Thus, the main purpose of our study was to define the role of vasoactive intestinal peptide (VIP) and corticotrophin‐releasing factor (CRF), as immunoregulatory neuropeptides, on ADAMTS production in synovial fibroblasts (SF) from OA patients and healthy donors (HD). OA‐ and HD‐SF were stimulated with pro‐inflammatory mediators and treated with VIP or CRF. Both neuropeptides decreased ADAMTS‐4, ‐5, ‐7 and ‐12 expressions, aggrecanase activity, glycosaminoglycans (GAG), and cartilage oligomeric matrix protein (COMP) degradation after stimulation with fibronectin fragments (Fn‐fs) in OA‐SF. After stimulation with interleukin‐1β, VIP reduced ADAMTS‐4 and ‐5, and both neuropeptides decreased ADAMTS‐7 production and COMP degradation. Moreover, VIP and CRF reduced Runx2 and β‐catenin activation in OA‐SF. Our data suggest that the role of VIP and CRF on ADAMTS expression and cartilage degradation could be related to the OA pathology since scarce effects were produced in HD‐SF. In addition, their effects might be greater when a degradation loop has been established, given that they were higher after stimulation with Fn‐fs. Our results point to novel OA therapies based on the use of neuropeptides, since VIP and CRF are able to stop the first critical step, the loss of cartilage aggrecan and the ECM destabilization during joint degradation.     

Increase in the expression of human leukocyte antigen class I in human fibroblasts by soluble factors secreted from human cytomegalovirus-infected cells.

The human cytomegalovirus (HCMV) is known to downregulate the expression of the human leukocyte antigen (HLA) class I for escape from immune surveillance. In order to understand the HCMV immune evasion mechanism, expression of HLA class I on the surface of HCMV-infected cells was investigated. A decrease in the HLA class I expression was observed at higher MOI; whereas at a lower MOI a slight increase in the HLA class I expression was observed. When HCMV-infected and uninfected cells were separately prepared on coverslips and co-cultured, the increased HLA class I expression was observed in uninfected cells. Treatment of the uninfected cells with the culture supernatant from HCMV-infected cells resulted in an increase in the HLA class I expression. A biochemical analysis of the HCMV-infected cell culture supernatant revealed the presence of interferon (IFN) beta interleukin (IL)-1beta, and IL-6. The HLA class I-enhancing activity of the culture supernatant was mimicked by IFN beta, but not by IL1-beta or IL-6, and was partially reversed by pretreatment with an antibody to IFN beta. Therefore, it appears that the HCMV infection of human foreskin fibroblast cells induces interferon beta and other soluble factor(s) that are responsible for the up-regulation of the HLA class I expression.     

CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression

Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts.Osteoarthritis (OA) refers to clinical syndrome of joint pain accompanied by varying degrees of functional limitation and reduced quality of life.¹ Cause of the OA is unclear, although obesity, aging, sex, genetic factors, and injury have been associated with increased risk of OA.² Development and progression of OA are now believed to involve synovial inflammation even in early stages of the disease.³ Biochemical mediators like cytokines, chemokines, and growth factors were found in OA synovial fibroblasts (OASFs) that affect cellular functions of knee joints. These mediators promote inflammation, cartilage degradation, and neovascularization via activation of angiogenetic factors like vascular endothelial growth factor (VEGF),^4,5 reportedly secreted from mechanically overloaded chondrocytes⁶ and in OA joints in vivo.⁷ VEGF also affects chondrocytic metabolism, leading to release of matrix metalloproteinases that degrade cartilage matrix.⁸ Anti-VEGF polyclonal antibody markedly attenuated disease severity in arthritis,⁹ indicating anti-angiogenesis as novel OA treatment.Connective tissue growth factor (CTGF, a.k.a. CCN2) is a member of the CCN family, secreted multifunctional proteins that contain high levels of cysteine. It has been proven associated with several biological functions such as fibrosis, tissue remodeling, and tumorgenesis even to OA.¹⁰ The mRNA expression of CTGF has been upregulated adjacent to areas of cartilage surface damage, and present in chondro-osteophytes.¹¹ In animal model, CTGF overexpression in synovial lining of mouse knee joints results in reversible synovial fibrosis and cartilage damage.¹² Both plasma and synovial fluid CTGF concentration in OA patients were correlated with radiographic severity and could be useful for monitoring progression of OA.¹³ We previously indicated CTGF enhancing IL-6 and MCP-1 expression and promoting inflammation in OASFs,^14,15 meaning CTGF contributes to pathogenesis of OA.The small, noncoding microRNAs (miRNAs) transcribed from DNA are 18–24 nucleotides in length, modulating targeted gene expression via either translational repression or mRNA cleavage.¹⁶ It is recently reported that miRNA expression was associated with well-defined clinical pathological features and disease outcomes;^17,18 miRNAs also have been linked with OA pathogenesis, especially for expression of genes encoding catabolic factors like matrix metalloproteinases and ADAMTS.¹⁹ Many evidences indicated that miR-210 as angiogenic miRNA.^{20, 21, 22} In addition, overexpression of miR-210 can stimulate formation of capillary-like structures in vitro when cells are cultured in Matrigel.²³ However, the exact etiological mechanism of miR-210 in angiogenesis and OA pathogenesis is largely unknown.Angiogenesis is essential for the development, growth, and progression of OA.²⁴ VEGF, a potent angiogenic factor, is pivotal in OA pathogenesis.⁷ CTGF is cited as promoting inflammatory cytokine release during OA;¹² its role in angiogenesis is implied in many cell types,^25,26 but its signal pathway in VEGF production and angiogenesis in synovial fibroblasts has not been extensively studied. We explored intracellular signal pathway in CTGF-induced VEGF production in OASFs and found CTGF activating PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathway to upregulate miR-210 expression and contributing to inhibit GPD1L expression and prolyl hydroxylases 2 (PHD2) activity as well as trigger HIF-1α-dependent VEGF expression and angiogenesis in human OASFs.     

Endotoxins of enteric pathogens are chemotactic factors for human neutrophils

Early activation of human peripheral blood polymorphonuclear neutrophils is characterized by their morphological changes from spherical to polarized shapes. The endotoxins from enteric pathogens (S. dysenteriae type 1, V. cholerae Inaba 569B, S. typhimurium, and K. pneumoniae) were assessed by their ability to induce morphological polarization of the neutrophils as measures of early activation. Phagocytic activity, adhesion, chemokinetic locomotion, and nitroblue tetrazolium (NBT) dye-reduction ability measured the later activation of the cells. Neutrophils showed distinct morphological polarization in suspension over a wide range of concentrations of these endotoxins when were compared with those that were induced by the standard chemotactic factor, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). It was discovered that all of the endotoxins induced locomotor responses in neutrophils in suspension that were dose- and time-dependent. The optimum concentration for the endotoxins of S. dysenteriae, V. cholerae, and K. pneumoniae was 1 mg/ml in which 71, 69, and 66% of the neutrophils were polarized. However, the S. typhimurium dose was 2 mg/ml in which 50% of the cells responded. Neutrophils that were stimulated with endotoxins also showed increased random locomotion (p<0.005) through cellulose nitrate filters, but an enhanced adhesion of the cells to glass surfaces (p<0.03). These are important functions of these cells to reach and phagocytose damaged cells, as well as invading microorganisms. Interestingly, the endotoxins had a highly-significant inhibitory effect upon the proportions of neutrophils phagocytosing opsonized yeast (p<0.01) with a small number of yeast that were engulfed by the cells (p<0.02). Further, endotoxin-treated cells showed an enhanced ability to reduce NBT dye (p<0.03). Therefore, we concluded that endotoxins of enteric pathogens are neutrophil chemotactic factors.     

Ion channel expression and function in normal and osteoarthritic human synovial fluid progenitor cells

Osteoarthritis (OA) is a chronic disease affecting the cartilage of over 15% of Canadians. Synovial fluid mesenchymal progenitor cells (sfMPCs) are present in joints and are thought to contribute to healing. OA sfMPCs have a greater proliferative ability but decreased chondrogenic potential. However, little is known about the factors influencing/regulating the differences between normal and OA sfMPCs. Recently, our lab has shown that sfMPC chondrogenic differentiation in vitro is favorably biased toward a similar osmotic environment as they experience in vivo. The current study now examines the expression and functionality of a variety of ion channels in sfMPCs derived from normal individuals and early OA patients. Results indicated that there is differential ion channel regulation at the functional level and expression level in early OA sfMPCs. All ion channels were upregulated in early OA compared to normal sfMPCs with the exception of KCNMA1 at the mRNA level. At the protein level, TRPV4 was over expressed in early OA sfMPCs, while KCNJ12 and KCNMA1 were unchanged between normal and early OA sfMPCs. At the functional level, the inward rectifying potassium channel was under expressed in early OA sfMPCs, however the membrane potential was unchanged between normal and early OA sfMPCs. In the synovial environment itself, a number of differences in ion concentration between normal and early OA synovial fluid were observed. These findings suggest that normal and OA progenitor cells demonstrate functional differences in how they interact with the synovial ion environment.