Kelly McKelvey

Endogenous MMP-9 and not MMP-2 promotes rheumatoid synovial fibroblast survival, inflammation and cartilage degradation

OBJECTIVE The aim of this study was to investigate the effect of endogenous matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) on the invasive characteristics of RA synovial fibroblasts. METHODS Synovial fibroblasts isolated from patients with RA or OA were treated with MMP small interfering RNA (siRNA), inhibitors and recombinant proteins or TNF-α, with or without cartilage explants. Cell viability and proliferation were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and 5-bromo-2-deoxyuridine (BrdU) proliferation assays, respectively; apoptosis by an in situ cell death detection kit; migration and invasion by CytoSelect invasion assay, scratch migration and collagen gel assays; cartilage degradation by 1,9-dimethylmethylene blue assay; and inflammatory mediators and MMPs by ELISA, western blot and zymography. RESULTS MMP-2 was expressed by both OA and RA synovial fibroblasts, whereas only RA synovial fibroblasts expressed MMP-9. Suppressing MMP-2 or MMP-9 reduced RA synovial fibroblast proliferation equally. However, MMP-9 siRNA had greater effects compared with MMP-2 siRNA on promoting apoptosis and suppressing RA synovial fibroblast viability, migration and invasion. Suppression/inhibition of MMP-9 also decreased the production of IL-1β, IL-6, IL-8 and TNF-α, inactivated nuclear factor κB (NF-κB), extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and suppressed RA synovial fibroblast-mediated cartilage degradation. In contrast, suppression/inhibition of MMP-2 stimulated TNF-α and IL-17 secretion and activated NF-κB, while recombinant MMP-2 (rMMP-2) inactivated NF-κB and suppressed RA synovial fibroblast-mediated cartilage degradation. Results using specific inhibitors and rMMPs provided supportive evidence for the siRNA results. CONCLUSION Endogenous MMP-2 or MMP-9 contribute to RA synovial fibroblast survival, proliferation, migration and invasion, with MMP-9 having more potent effects. Additionally, MMP-9 stimulates RA synovial fibroblast-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters. Overall, our data indicate that MMP-9 derived from RA synovial fibroblasts may directly contribute to joint destruction in RA.

Exosomes: Mechanisms of Uptake

Exosomes are 30–100 nm microvesicles which contain complex cellular signals of RNA, protein and lipids. Because of this, exosomes are implicated as having limitless therapeutic potential for the treatment of cancer, pregnancy complications, infections, and autoimmune diseases. To date we know a considerable amount about exosome biogenesis and secretion, but there is a paucity of data regarding the uptake of exosomes by immune and non-immune cell types (e.g., cancer cells) and the internal signalling pathways by which these exosomes elicit a cellular response. Answering these questions is of paramount importance.

Cell-specific expression of TLR9 isoforms in inflammation

Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

Activated protein C: A regulator of human skin epidermal keratinocyte function

Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APCs function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

Treatment of chronic diabetic lower leg ulcers with activated protein C: a randomised placebo-controlled, double-blind pilot clinical trial

Lower leg ulcers are a serious and long‐term complication in patients with diabetes and pose a major health concern because of the increasing number of patients diagnosed with diabetes each year. This study sought to evaluate the clinical benefit of topical activated protein C (APC) on chronic lower leg ulcers in patients with diabetes. Twelve patients were randomly assigned to receive either APC (N = 6) or physiological saline (placebo; N = 6) in a randomised, placebo‐controlled, double‐blind pilot clinical trial. Treatment was administered topically, twice weekly for 6 weeks with final follow‐up at 20 weeks. Wound area was significantly reduced to 34·8 ± 16·4% of week 0 levels at 20 weeks in APC‐treated wounds (p = 0·01). At 20 weeks, three APC‐treated wounds had completely healed, compared to one saline‐treated wound. Full‐thickness wound edge skin biopsies showed reduced inflammatory cell infiltration and increased vascular proliferation following APC treatment. Patient stress scores were also significantly reduced following APC treatment (p < 0·05), demonstrating improved patient quality of life as assessed by the Cardiff Wound Impact Questionnaire. This pilot trial suggests that APC is a safe topical agent for healing chronic lower leg ulcers in patients with diabetes and provides supporting evidence for a larger clinical trial.

Low Circulating Protein C Levels Are Associated with Lower Leg Ulcers in Patients with Diabetes

Activated protein C (APC) promotes angiogenesis and reepithelialisation and accelerates healing of diabetic ulcers. The aim of this study was to determine the relationship between the incidence of lower leg ulcers and plasma levels of APCs precursor, protein C (PC), in diabetic patients. Patients with diabetes who had a lower leg ulcer(s) for >6 months (n = 36) were compared with age-, type of diabetes-, and sex-matched subjects with diabetes but without an ulcer (n = 36, controls). Total PC was assessed using a routine PC colorimetric assay. There was a significantly (P < 0.001) lower level of plasma PC in patients with ulcers (103.3 ± 22.7, mean ± SD) compared with control (127.1 ± 34.0) subjects, when corrected for age and matched for gender and type of diabetes. Ulcer type (neuropathic, ischaemic, or mixed) was not a significant covariate for plasma PC levels (P = 0.35). There was no correlation between PC levels and gender, type of diabetes, HbA1c, or C-reactive protein in either group. In summary, decreased circulating PC levels are associated with, and may predispose to, lower leg ulceration in patients with diabetes.

Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2.

IntroductionRheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA2V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.MethodsHuman synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA2V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA2V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.ResultsEPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA2V co-localized with EPCR on RASFs. Suppression of sPLA2V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA2V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.ConclusionsOur results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA2V.

Embryonic/fetal mortality and intrauterine growth restriction is not exclusive to the CBA/J sub-strain in the CBA × DBA model

Inbred strains of mice are powerful models for understanding human pregnancy complications. For example, the exclusive mating of CBA/J females to DBA/2J males increases fetal resorption to 20–35% with an associated decline in placentation and maintenance of maternal Th1 immunity. More recently other complications of pregnancy, IUGR and preeclampsia, have been reported in this model. The aim of this study was to qualify whether the CBA/CaH substrain female can substitute for CBA/J to evoke a phenotype of embryonic/fetal mortality and IUGR. (CBA/CaH × DBA/2J) F1 had significantly higher embryonic/fetal mortality mortality (p = 0.0063), smaller fetuses (p < 0.0001), and greater prevalence of IUGR (<10th percentile; 47% vs 10%) than (CBA/CaH × Balb/c) F1. Placentae from IUGR fetuses from all mating groups were significantly smaller (p < 0.0001) with evidence of thrombosis and fibrosis when compared to normal-weight fetuses ( > 10th percentile). In addition, placentae of “normal-weight” (CBA/CaH × DBA/2J) F1 were significantly smaller (p < 0.0006) with a greater proportion of labyrinth (p = 0.0128) and an 11-fold increase in F4/80 + macrophage infiltration (p < 0.0001) when compared to placentae of (CBA/CaH × Balb/c) F1. In conclusion, the embryonic/fetal mortality and IUGR phenotype is not exclusive to CBA/J female mouse, and CBA/CaH females can be substituted to provide a model for the assessment of novel therapeutics.

Aberrant levels of natural IgM antibodies in osteoarthritis and rheumatoid arthritis patients in comparison to healthy controls.

Natural IgM antibodies (nIgM) are polyreactive autoantibodies that have diverse roles in regulating autoimmunity, systemic inflammation and removal of oxidized low-density lipoproteins (oxLDL). We hypothesized that aberrant states of nIgM may exist in persons with osteoarthritis (OA) and rheumatoid arthritis (RA). Herein, we characterized and compared the levels of nIgM specific for phosphorylcholine (anti-PC), double-stranded DNA (anti-dsDNA), and galactosyl (anti-Gal) in persons with OA, RA and healthy controls (HC). Levels of anti-PC nIgM in OA patients were significantly lower than both HC and RA patients in an age-adjusted analysis (P<0.05). In contrast, anti-Gal nIgM levels were significantly higher in RA patients than OA patients (P<0.05) and markedly increased in comparison to HC. Anti-PC nIgM significantly correlated with anti-dsDNA and anti-Gal nIgM levels in HC and RA (P<0.05) but not in OA patients. Elevated CRP levels were associated with RA conditions and old ages in general. There was no significant correlation between anti-PC nIgM and CRP or oxLDL levels. Our study highlights for the first time the evidence of aberrant state of nIgM in human OA compared to healthy individuals that implicates a deficiency in immune responses to oxLDL which may contribute to the metabolic syndromes in the development of OA.

Activated protein C inhibits proliferation and tumor necrosis factor α-stimulated activation of p38, c-Jun NH2-terminal kinase (JNK) and Akt in rheumatoid synovial fibroblasts.

Synovial fibroblast proliferation Is a hallmark of the Invasive pannus in the rheumatoid joint. Activated protein C (APC) is a natural anticoagulant that exerts antiinflammatory and cyto-protective effects in various diseases via endothelial protein C receptor (EPCR) and proteinase-activated receptor (PAR)-mediated pathways. In this study, we investigated the effect and the underlying cellular signaling mechanisms of APC on proliferation of human rheumatoid synovial fibroblasts (RSFs). We found that APC stimulated proliferation of mouse dermal fibroblasts (MDFs) and normal human dermal fibroblasts (HDFs) by up to 60%, but robustly downregulated proliferation of RSFs. APC induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) and enhanced expression of p21 and p27 in a dose-dependent manner in RSFs. The latter effect was inhibited by pretreatment with the ERK inhibitors PD98059 and U0126 but not by p38 inhibitor SB203580. In addition, APC significantly downregulated tumor necrosis factor (TNF)α-stimulated cell proliferation and activation of p38, c-Jun NH2-terminal kinase (JNK) and Akt in RSFs. These results provide the first evidence that APC selectively inhibits proliferation and the inflammatory signaling pathways of RSFs. Thus, APC may reduce synovial hyperplasia and pannus invasion in rheumatoid arthritis.