Expression Of Catabolic Factors Research Articles

The matrix synthesis and anti-inflammatory effect of autologous leukocyte-poor platelet rich plasma in human cartilage explants.

To determine the effects of autologous leukocyte-poor platelet-rich plasma (LP-PRP) on the expression of markers involved in cartilage-extracellular matrix production and inflammation in cartilage explants bearing osteoarthritis. Cartilage explants and LP-PRP were obtained from 10 patients who underwent total knee arthroplasty. The explants were cultured in spinner flasks for 28 days in the presence of interleukin (IL)-1β and/or LP-PRP. The gene expression of catabolic (MMP13, ADAMTS5, and IL1β) and anabolic factors (COL2A1, ACAN, and SOX9) was quantified. A histological assessment was performed according to a modified Mankin score, and quantification of type II and I collagen deposition. The gene expression of catabolic factors and the Mankin score were lower in LP-PRP- and LP-PRP/IL-1β- than in IL-1β-treated explants, suggesting less matrix degradation in explants cultured in the presence of LP-PRP. Higher expression of genes involved in cartilage matrix restoration was observed in LP-PRP and LP-PRP/IL-1β- when compared to IL-1β-treated explants. The explants treated with LP-PRP and LP-PRP/IL-1β exhibited a higher deposition of type II collagen as well as a lower deposition of type I collagen and also better surface integrity and a significant increase in the number of chondrocytes. LP-PRP treatment favored restoration in early osteoarthritic cartilage and reduced the pro-inflammatory effect of IL-1β. LP-PRP is a promising therapy for early osteoarthritis, as it promotes extracellular matrix repair, reduces inflammation, and slows cartilage degeneration.

MicroRNA-365 regulates IL-1\u03b2-induced catabolic factor expression by targeting HIF-2\u03b1 in primary chondrocytes

Endothelial Per-Arnt-Sim domain protein-1/hypoxia-inducible factor-2α (EPAS-1/ HIF-2α) is a catabolic transcription factor that regulates osteoarthritis (OA)-related cartilage destruction. Here, we examined whether microRNA-365 (miR-365) affects interleukin (IL)-1β-induced expression of catabolic factors in chondrocytes via regulation of HIF-2α. MiR-365 levels were significantly decreased in human OA cartilage relative to normal cartilage. Overexpression of miR-365 significantly suppressed IL-1β-induced expression of HIF-2α in human articular chondrocytes. Pharmacological inhibition of various IL-1β-associated signaling pathways revealed mitogen-activated protein kinase and nuclear factor-κB as the primary pathways driving IL-1β-mediated decreases in miR-365 and subsequent increase in HIF-2α expression. Using a luciferase reporter assay encoding the 3′ untranslated region (UTR) of human HIF-2α mRNA, we showed that overexpression of miR-365 significantly suppressed IL-1β-induced up-regulation of HIF-2α. AGO2 RNA-immunoprecipitation (IP) assay demonstrated that miR-365 and HIF-2α mRNA were enriched in the AGO2-IP fraction in miR-365-transfected primary chondrocytes compared to miR-con-transfected cells, indicating that HIF-2α is a target of miR-365. Furthermore, miR-365 overexpression significantly suppressed IL-1β-induced expression of catabolic factors, including cyclooxygenase-2 and matrix metalloproteinase-1, -3 and -13, in chondrocytes. In pellet culture of primary chondrocytes miR-365 prevented IL-1β-stimulated extracellular matrix loss and matrix metalloproteinase-13 expression. MiR-365 regulates IL-1β-stimulated catabolic effects in human chondrocytes by modulating HIF-2α expression.

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of β-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released β-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.

Exposure to fermentation supernatant of Staphylococcus aureus accelerated dedifferentiation of chondrocytes and production of antimicrobial peptides.

Staphylococcus aureus (S. aureus) is the most popular pathogen found in septic arthritis. Despite bacteria was eradicated from joint cavity during acute infection, destruction of articular cartilage often continues for years, leading to permanent joint damage. The mechanism responsible for this consistent catabolic reaction in septic arthritis remains unclear. Here, we found that fermentation supernatant (FS) of S. aureus accelerated dedifferentiation of chondrocytes and induced expression of catabolic factors including A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs 5, NO synthase 2, matrix metalloproteinase-3, -13. In response to FS of S. aureus stimulation, expression of antimicrobial peptides (AMPs) including β-defensin-1, -2, -3, -4, cathelicidin antimicrobial peptide (CAMP) in dedifferentiated chondrocytes was significantly higher than that in chondrocytes which maintained their differentiated phenotype. Among AMPs detected, expression of CAMP in dedifferentiated chondrocytes was observed to increase 170 times higher than that in differentiated ones. When exposed to FS of S. aureus, expression of interleukin (IL)-1β, IL-17F, and IL-22 were remarkably increased in dedifferentiated chondrocytes. These results indicated that dedifferentiation of chondrocytes caused by exposure to S. aureus might be responsible for secondary osteoarthritis (OA) after acute S. aureus infection in joint. While, one potential benefit of dedifferentiation resulted from S. aureus exposure is that chondrocytes initiates a self-protective responsiveness by producing more AMPs against bacterial infection. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:443-451, 2018.

RANK/RANKL/OPG system in the intervertebral disc

BackgroundThe receptor activator of NF-κB ligand (RANKL), a member of the TNF ligand superfamily, is known to regulate bone metabolism. The expression of each component of the RANK/RANKL/osteoprotegerin (OPG) system in the intervertebral disc (IVD) has not been examined in detail. The purposes of this study were to examine the expression of the RANK/RANKL/OPG system and to evaluate the function of RANKL in the matrix metabolism of the rat IVD.MethodsSprague-Dawley, 12-week-old, male rats were used in this study. Anulus fibrosus (AF), nucleus pulposus (NP) and cartilaginous endplate (CEP) cells isolated from dissected thoracolumbar discs were monolayer-cultured. RANK/RANKL/OPG expression in rat IVDs was examined using real-time polymerase chain reaction (PCR) and immunohistochemical analysis (cultured cells and IVD tissues). To examine the effect of interleukin-1β (IL-1β) stimulation on the mRNA levels of RANK, RANKL and OPG, the cells were cultured with or without recombinant human IL-1β (rhIL-1β). To evaluate the effect of RANKL on the mRNA expression of catabolic factors (IL-1β, matrix metalloproteinase-3 (MMP-3) and MMP-13), the cells were cultured with RANKL in the presence or absence of rhIL-1β. The immunohistochemical expression of this system was also evaluated using human IVD tissues with different grades of degeneration.ResultsmRNA expression levels of RANK, RANKL, and OPG were clearly identified in AF, NP and CEP cells. Immunoreactivity to RANK, RANKL and OPG was distributed in the cell membranes and/or cytoplasm of the three types of cells. The mRNA level of RANKL was significantly upregulated by treatment with rhIL-1β of the three types of cells. Treatment with RANKL without rhIL-1β did not induce significant effects on the mRNA expression of catabolic factors by AF, NP and CEP cells. However, the expression was significantly upregulated by stimulation with RANKL in the presence of rhIL-1β. There was a general trend for more RANK/RANKL/OPG-positive cells in human IVD tissues in an advanced stage of degeneration compared to an early stage.ConclusionsOur study showed the possibility that the RANK/RANKL/OPG system may play a part in the process of intervertebral disc degeneration.

The nuclear phosphatase SCP4 regulates FoxO transcription factors during muscle wasting in chronic kidney disease

Chronic kidney disease (CKD) and related inflammatory responses stimulate protein-energy wasting, a complication causing loss of muscle mass. Primarily, muscle wasting results from accelerated protein degradation via autophagic/lysosomal and proteasomal pathways, but mechanisms regulating these proteolysis pathways remain unclear. Since dephosphorylation of FoxOs regulates ubiquitin/proteasome protein metabolism, we tested whether a novel nuclear phosphatase, the small C-terminal domain phosphatase (SCP) 4, regulates FoxOs signaling and, in turn, muscle wasting. In cultured mouse myoblast cells, SCP4 overexpression stimulated proteolysis, while knockdown of SCP4 prevented the proteolysis stimulated by inflammatory cytokines. SCP4 overexpression led to nuclear accumulation of FoxO1/3a followed by increased expression of catabolic factors including myostatin, Atrogin-1, and MuRF-1, and induction of lysosomal-mediated proteolysis. Treatment of C2C12 myotubes with proinflammatory cytokines stimulated SCP4 expression in an NF-κB-dependent manner. In skeletal muscle of mice with CKD, SCP4 expression was up-regulated. Similarly, in skeletal muscle of patients with CKD, SCP4 expression was significantly increased. Knockdown of SCP4 significantly suppressed FoxO1/3a-mediated expression of Atrogin-1 and MuRF-1 and prevented muscle wasting in mice with CKD. Thus, SCP4 is a novel regulator of FoxO transcription factors and promotes cellular proteolysis. Hence, targeting SCP4 may prevent muscle wasting in CKD and possibly other catabolic conditions.

Regulation of a disintegrins and metalloproteinase with thrombospondin motifs 7 during inflammation in nucleus pulposus (NP) cells: role of AP-1, Sp1 and NF-κB signaling.

The objective of this study is to explore the effect of inflammatory cytokines on a disintegrins and metalloproteinase with thrombospondin motifs 7 (ADAMTS7) and to demonstrate the role of Sp1, AP-1 and NF-κB signaling on the ADAMTS7 regulation during inflammation in NP cells. Real-time PCR was to detect the effect of ADAMTS7 knockdown on the expression of catabolic enzymes during inflammatory condition in NP cells. Real-time PCR, western blot, immunofluorescence and transfection experiments were used to observe the effect of tumor necrosis factor-α (TNF-α) or interleukin-1β on the expression and the activity of ADAMTS7, and demonstrated the role to Sp1, AP-1 and NF-κB in the regulation of ADAMTS7 during inflammation. As other cells, ADAMTS7 knockdown suppressed the mRNA expression of catabolic factors during inflammation in human NP cells. However, the expression of ADAMTS7 mRNA and protein and the activity of ADAMTS7 promoter were refractory to inflammatory cytokines. In addition, Sp1, AP-1, not NF-κB signaling sustained the expression of ADAMTS7 mRNA, protein, as well as promoter activity during inflammation in NP cells. ADAMTS7 played a crucial role in the expression of catabolic genes in the presence of TNF-α and AP-1, Sp1, not NF-κB signaling were critical for the maintenance of ADAMTS7 expression during inflammation in NP cells.

Upregulation of Atrogin-1/FBXO32 is not necessary for cartilage destruction in mouse models of osteoarthritis

In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2α and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the invivo functions of Atrogin-1 in mouse models of OA. The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32-knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin-1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1β, HIF-2α, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice.

Advanced oxidation protein products induce catabolic effect through oxidant-dependent activation of NF-κ B pathway in human chondrocyte

Advanced oxidation protein products (AOPPs) have been shown to participate in the progression of rheumatoid arthritis (RA). However, the effect of AOPPs accumulation on catabolic effect in human chondrocyte and the underlying mechanism(s) remain unclear. The present study demonstrated that AOPPs inhibited cell viability and glycosaminoglycan (GAG) production in human chondrocyte. Exposure of chondrocyte to AOPPs significantly increased the production of catabolic factors, such as cyclooxygenase-2 (COX-2), matrix metalloproteinase 3 (MMPs)-3 and MMP-13. AOPPs stimulation induced ROS generation and NF-κ B p65 phosphorylation, which could be inhibited by soluble receptor for advanced glycan end products (sRAGE), NADPH oxidase inhibitor (apocynin), ROS scavenger (N-acetyl-cysteine, NAC). Furthermore, NF-κ B inhibitor Bay11-7082 significantly reversed the AOPPs-induced expression of catabolic factors and phosphorylation of NF-κ B p65. Targeting AOPPs-triggered catabolic effect might be as a promising option for patients with RA.

Context-specific protection of TGFα null mice from osteoarthritis.

Transforming growth factor alpha (TGFα) is a growth factor involved in osteoarthritis (OA). TGFα induces an OA-like phenotype in articular chondrocytes, by inhibiting matrix synthesis and promoting catabolic factor expression. To better understand TGFα’s potential as a therapeutic target, we employed two in vivo OA models: (1) post-traumatic and (2) aging related OA. Ten-week old and six-month old male Tgfa null mice and their heterozygous (control) littermates underwent destabilization of the medial meniscus (DMM) surgery. Disease progression was assessed histologically using the Osteoarthritis Research Society International (OARSI) scoring system. As well, spontaneous disease progression was analyzed in eighteen-month-old Tgfa null and heterozygous mice. Ten-week old Tgfa null mice were protected from OA progression at both seven and fourteen weeks post-surgery. No protection was seen however in six-month old null mice after DMM surgery, and no differences were observed between genotypes in the aging model. Thus, young Tgfa null mice are protected from OA progression in the DMM model, while older mice are not. In addition, Tgfa null mice are equally susceptible to spontaneous OA development during aging. Thus, TGFα might be a valuable therapeutic target in some post-traumatic forms of OA, however its role in idiopathic disease is less clear.

IL-6/STAT3 signalling blockade protects against experimental osteoarthritis in mice

IL-6/STAT3 signalling blockade protects against experimental osteoarthritis in mice

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes

Background: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. Methods: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. Results: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. Conclusion: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis.

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.

Chronic exposure to cigarette smoke causes extrapulmonary abnormalities in rats

Pathophysiological features of chronic obstructive pulmonary disease (COPD) include systemic abnormalities, such as weight loss and skeletal muscle wasting. Although cigarette smoke (CS) is a major risk factor in COPD, the systemic effects of CS exposure remain to be elucidated. In this study, rats were exposed to CS or smoke-free air for 12 weeks. CS-exposed rats developed emphysema and had significantly lower body weight and food intake than control rats. The plasma ghrelin levels significantly increased with an upregulation of gastric ghrelin mRNA expression induced by CS exposure. Further, we observed low plasma insulin-like growth factor-1 levels and high tumor necrosis factor-α levels. A significant reduction of skeletal muscle strength and an increase in the mRNA expression of catabolic factors was observed in CS-exposed rats. These results indicated that chronic CS exposure induced not only pulmonary emphysema but also systemic abnormalities related to muscle catabolism associated with inflammatory responses.

Role of HIF-1α and HIF-2α in osteoarthritis

The hallmark of OA is cartilage destruction, several factors such as catabolic enzymes and chondrocyte death include apoptosis and/or autophagy are considered for the pathogenesis. Articular cartilage is maintained in a low oxygen environment throughout life. Chondrocytes are therefore adapted to these hypoxic conditions. The increased HIF-1α and HIF-2α mediate the response of chondrocytes to hypoxia. HIF-1α regulates chondrogenesis by regulating SOX9 expression in the genetic level, HIF-1 also serves to regulate both autophagy and apoptosis. Therefore, HIF-1α may protect articular cartilage by promoting the chondrocyte phenotype, maintaining chondrocyte viability, and supporting metabolic adaptation to a hypoxic environment. In contrast with HIF-1α, HIF-2α is a catabolic factor in the osteoarthritic process. Although HIF-2α is essential for hypoxic induction of the human articular chondrocyte phenotype, HIF-2α directly induces the expression of catabolic factors in chondrocytes, and HIF-2α enhances Fas expression to mediate chondrocyte apoptosis and regulates autophagy in maturing chondrocytes. Taken together, manipulation of HIF-1α and HIF-2α could represent a promising approach to the treatment of OA. Further study should elucidate the exact machnism of HIF-1α and HIF-2α in cartilage and determine which is predominant in osteoarthritic process.

Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis.

IntroductionAngiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection.MethodsFirst, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group).ResultsHistologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group.ConclusionsConsidering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0427-y) contains supplementary material, which is available to authorized users.

Recombinant IL-1Ra Stimulation of Native NP Cells Inhibits a Multitude of Pathological Features

Introduction Interleukin-1 (IL-1) has been implicated in the pathogenesis of disc degeneration, with increased levels seen during degeneration without a concordant increase in its natural inhibitor: IL-1 receptor antagonist (IL-1Ra). IL-1 has been shown to stimulate a pleathora of catabolic actions including matrix degrading enzymes, cytokines, chemokines, and factors which promote nerve and blood vessel ingrowth. Here, we investigated the effect of the natural inhibitor of IL-1, to determine its ability to inhibit catabolic events regulated by natively produced IL-1 in degenerate human nucleus pulposus (NP) cells. Materials and Methods Human NP cells were isolated via collagenase digestion from four human intervertebral disc (IVD) obtained from degenerate disc samples from patients undergoing discetomy. Following expansion in monolayer culture, cells were transferred to alginate bead cultures at passage two and maintained for 2 weeks before stimulation with IL-1Ra to enable redifferentiation to take place. Cells were treated with IL-1Ra at 1, 10, 100 pg/mL and 1, 10, 100 ng/mL for 48 hours. Following stimulation, cells were released from alginate beads and RNA isolated using Trizol reagent. cDNA synthesized and real-time PCR used to determine the effects on a wide range of gene targets including matrix metalloproteases (MMPs), cytokines, chemokines, and factors involved in nerve ingrowth (NGF), pain pathways (substance P), and angiogenesis (vascular endothelial growth factor, VEGF). Results Baseline levels of all factors investigated varied between patients as did the overall responsiveness to IL-1Ra stimulation. IL-1Ra stimulation resulted in decreased gene expression for several MMPs including MMPs 3 and 13; cytokines including IL-1 and IL-6; chemokines including IL-8, CCL2, and CXCL3. NGF, substance P, and VEGF were also all decreased by IL-1Ra stimulation. Effective doses were target and patient-specific and while the highest dose of IL-1Ra often resulted in decreased expression of catabolic factors in all patients, lowest doses (1.10 pg/mL IL-1Ra) resulted in stimulation of some catabolic responses in some patients, for example, IL-6 and IL-8 were increased in two out of four patient samples investigated at low doses of IL-1Ra. Conclusion IL-1 has been implicated as a key factor in the pathogenesis of disc degeneration, however till date it is not fully understood whether inhibition of this cytokine in native disc cells can inhibit the plethora of pathogenic factors linked to IL-1. This study has demonstrated that treatment of NP cells with IL-1Ra results in decreased expression of MMPs, cytokines, chemokines, nerve, and blood vessel growth factors and neurotrophic factors, suggesting IL-1Ra could be a useful agent in the prevention of further degeneration to create the correct tissue niche to support tissue regeneration. However, patient responses were seen to be variable, with some patients displaying increased expression of some catabolic factors at low doses of IL-1Ra, thus selection of patients and carefully controlled drug release would be essential to enable such a therapy to be deployed successfully. Disclosure of Interest None declared

The Extracellular Calcium-Sensing Receptor as a Contributor in Human Cartilaginous Endplate Degeneration

Introduction Intervertebral discs (IVDs) are virtually avascular tissue relying on the diffusion of nutrients and metabolites from blood vessels in the outer annulus fibrosus, and in the vertebral bodies that partially penetrate the subchondral plate, and terminate at the cartilaginous endplate (CEP). Degeneration of CEP is viewed as a catalyst to IVD degeneration, as it impedes nutrient diffusion to the disc. Similar to articular cartilage, degeneration of CEP involves decreased proteoglycan and type II collagen (Col II) content, thinning, increased type X collagen (Col X), and advanced calcification. Although biological repair strategies of the degenerative disc involving supplementation of growth factors are being pursued, disc nutrition is an important factor in considering their success in disc repair. The extracellular calcium-sensing receptor (CaSR) is a G protein-coupled receptor, and currently, is the only known mechanism in sensing both local and systemic calcium fluctuations. CaSR is the principle regulator of parathyroid hormone synthesis and secretion from the parathyroid gland, and in addition to its expression in other calcium-handling tissues (intestine, kidney), evidence suggests a direct involvement in bone and cartilage development. Recently, CaSR has been shown to modulate vascular calcification; however, its function in the calcification of other tissues remains unknown. Objectives Calcification of CEP is suggested to be a catalyst in the degeneration of IVDs. However, the mechanism(s) of CEP degeneration are currently unknown. We propose that CaSR activation modulates the degeneration of CEP, by increasing the expression of catabolic factors in CEP chondrocytes, resulting from the accumulation of ionic calcium in the milieu. Materials and Methods Human CEPs were obtained from donor IVDs of Thompson grades 2, 3, and 4 through organ donations within 24 hours of death. CEP chondrocytes were isolated from tissue by sequential digestion with pronase followed by collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on CEP tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting, and MAPK signaling were performed on cultured CEP chondrocytes of various grades to demonstrate expression of CaSR and degenerative markers, including the function of the receptor. CEP cells of various grades were cultured in medium containing either high calcium (5 mM Ca2+), CaSR allosteric agonist (cinacalcet, 1 µM), or regular growth medium (control, 1 mM Ca2+) for 7 days, and monitored for changes in markers of degeneration and CaSR expression. Results Ionic calcium content in CEP tissue increased significantly with IVD degeneration (Fig. A). Interestingly, immunohistochemistry on CEP tissue also revealed an increase in the expression of CaSR that paralleled the degree of IVD degeneration. Immunocytochemistry and Western blotting on isolated CEP cells demonstrated an upregulation of CaSR (Fig. B) and type X collagen that varied with Thompson grades. The expression of matrix metalloproteinase (MMP)-13 was also increased with degeneration; however, the expression of Col II and the proteoglycan aggrecan (Agg) were inversely related to Thompson grade. The magnitude of CaSR signaling in CEP cells when stimulated with 5 mM Ca2+, as determined by ERK phosphorylation, was significantly enhanced in grade 4 versus grade 2 CEP chondrocytes. To determine a role of CaSR in the degeneration of CEP, we cultured Thompson grade 2 CEP chondrocytes in 1 mM Ca2+ (control medium), 5 mM Ca2+ or cinacalcet (allosteric CaSR agonist, 1 µM) for 7 days. Western blot analysis on lysate from G2 CEP cells treated with 5 mM Ca2+ or cinacalcet demonstrated significant increases in the expression of Col X and MMP-13 (Figs. D and E), however, decreased expression of Col II and Agg was observed (Fig. C). Conclusion CaSR has been described as a mediator in ectopic calcification, and its expression is upregulated in CEP of degenerative discs. We provide evidence that CaSR upregulation may be a hallmark of CEP calcification. Activation of CaSR either by its agonist, calcium or cinacalcet, lead to increases in the synthesis of degenerative factors (Col X and MMP-13), and decreases in the production of matrix proteins (Col II and Agg). An understanding on the mechanism(s) of CEP degeneration is key when considering growth factor substitution in IVD repair, as disc nutrition may be an important factor in determining their success. Disclosure of Interest None declared References Lotz JC, Fields AJ, Liebenberg EC. The role of the vertebral end plate in low back pain. Global Spine J 2013;3(3):153–164 Brown EM. Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best Pract Res Clin Endocrinol Metab 2013;27(3):333–343

Regulation of the Catabolic Cascade in Osteoarthritis by the Zinc-ZIP8-MTF1 Axis

Osteoarthritis (OA), primarily characterized by cartilage degeneration, is caused by an imbalance between anabolic and catabolic factors. Here, we investigated the role of zinc (Zn2+) homeostasis, Zn2+ transporters, and Zn(2+)-dependent transcription factors in OA pathogenesis. Among Zn2+ transporters, the Zn2+ importer ZIP8 was specifically upregulated in OA cartilage of humans and mice, resulting in increased levels of intracellular Zn2+ in chondrocytes. ZIP8-mediated Zn2+ influx upregulated the expression of matrix-degrading enzymes (MMP3, MMP9, MMP12, MMP13, and ADAMTS5) in chondrocytes. Ectopic expression of ZIP8 in mouse cartilage tissue caused OA cartilage destruction, whereas Zip8 knockout suppressed surgically induced OA pathogenesis, with concomitant modulation of Zn2+ influx and matrix-degrading enzymes. Furthermore, MTF1 was identified as an essential transcription factor in mediating Zn2+/ZIP8-induced catabolic factor expression, and genetic modulation of Mtf1 in mice altered OA pathogenesis. We propose that the zinc-ZIP8-MTF1 axis is an essential catabolic regulator of OA pathogenesis.

Modulation of endochondral ossification by MEK inhibitors PD0325901 and AZD6244 (Selumetinib)

MEK inhibitors (MEKi) PD0325901 and AZD6244 (Selumetinib) are drugs currently under clinical investigation for cancer treatment, however the Ras–MAPK pathway is also an important mediator of normal bone cell differentiation and function. In this study we examined the effects of these compounds on endochondral processes using both in vitro and in vivo models. Treatment with PD0325901 or AZD6244 significantly increased Runx2 and Alkaline phosphate gene expression in calvarial osteoblasts and decreased TRAP+ cells in induced osteoclast cultures. To test the effects of these drugs on bone healing, C57/Bl6 mice underwent a closed tibial fracture and were treated with PD0325901 or AZD6244 at 10mg/kg/day. Animals were culled at day 10 and at day 21 post-fracture for analysis of the fracture callus and the femoral growth plate in the contralateral leg. MEKi treatment markedly increased cartilage volume in the soft callus at day 10 post-fracture (+60% PD0325901, +20% AZD6244) and continued treatment led to a delay in cartilage remodeling. At the growth plate, we observed an increase in the height of the hypertrophic zone relative to the proliferative zone of +78% in PD0325901 treated mice. Osteoclast surface was significantly decreased both at the terminal end of the growth plate and within the fracture calluses of MEKi treated animals. The mechanistic effects of MEKi on genes encoding cartilage matrix proteins and catabolic enzymes were examined in articular chondrocyte cultures. PD0325901 or AZD6244 led to increased matrix protein expression (Col2a1 and Acan) and decreased expression of catabolic factors (Mmp13 and Adamts-5). Taken together, these data support the hypothesis that MEKi treatment can impact chondrocyte hypertrophy, matrix resorption, and fracture healing. These compounds can also affect bone architecture by expanding the hypertrophic zone of the growth plate and reducing osteoclast surface systemically.