Interleukin-1beta Stimulation Research Articles

TAF15 regulates the BRD4/GREM1 axis and activates the gremlin-1-NF-κB pathway to promote OA progression

BackgroundAnterior cruciate ligament (ACL) injury is recognized as a risk factor for osteoarthritis (OA) progression. Herein, the function of TAF15 in ACL injury-induced OA was investigated. MethodsOA cell model and OA mouse model were established by interleukin-1 beta (IL-1β) stimulation and ACL transection administration, respectively. The pathological changes were analyzed by histopathology. The mRNA and protein expressions were assessed using qRT-PCR, Western blot and IHC. Chondrocyte viability and apoptosis were examined by CCK8 assay and TUNEL staining, respectively. The interactions between TAF15, BRD4 and GREM1 were analyzed by RIP or ChIP assay. ResultsTAF15 expression was markedly elevated in OA, and its knockdown suppressed IL-1β-induced chondrocyte apoptosis and ECM degradation in vivo and cartilage pathological changes in vitro. TAF15 promoted BRD4 mRNA stability, and TAF15 silencing's repression on chondrocyte apoptosis and ECM degradation induced by IL-1β was abrogated following BRD4 overexpression. BRD4 promoted GREM1 expression by directly binding with GREM1. In addition, the GREM1/NF-κB pathway functioned as the downstream pathway of BRD4 in promoting OA progression. ConclusionTAF15 upregulation facilitated chondrocyte apoptosis and ECM degradation during OA development by acting on the BRD4/GREM1/NF-κB axis, which provided a theoretical basis for the development of novel therapies for OA.

Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-KB) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-KB inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-KB-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF-KB-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF-KB signaling. Administering direct pharmacological inhibitors of NF-KB did reduce NF-KB-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF-KB, signalling.

Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5

In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1β) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval:-51.7,-37.1], 51.5% [-80.5,-22.5], 62.3% [-96.6,-28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8,-8.6]) further with IL-1β (44.5% [-36.9,-23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8,-5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9,-0.8]), histological damage ([-5.5,-0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56d. This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

Diverse inflammatory threats modulate astrocytes Ca2+ signaling via connexin43 hemichannels in organotypic spinal slices

Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Protective and beneficial effects of temsirolimus on human chondrocyte under interleukin 1-beta stimulation

Purpose: Osteoarthritis (OA) is a cartilage degenerative disease associated with aging. The mammalian target of rapamycin (mTOR) is a signal transduction protein that plays a central role in protein synthesis and cell growth. Importantly, rapamycin which is an mTOR inhibitor has been reported to extend lifespan and suppress aging-related degeneration of various tissues. We previously reported protective effects of rapamycin on human chondrocytes and preventive roles in mouse OA models. Since mTOR inhibitors are immunosuppressive drugs, intra-articular administration would be desirable, considering the side effects by systemic administration. Temsirolimus, a converted form of rapamycin, is made by converting rapamycin into a water-soluble form. Currently, only temsirolimus is approved for intravenous administration in mTOR inhibitors and thus may be suitable for intra-articular administration. However, effects of temsirolimus against OA have not yet been examined. Therefore, the purpose of this study was to evaluate the effect of temsirolimus on interleukin (IL) 1beta-induced responses and changes in human chondrocytes. Methods: NHAC-Kn (Normal Human Articular, Chondrocyte-knee, Lonza) were treated by temsirolimus in 10% FBS-supplemented DMEM under 5% CO2. Cell viability was determined by Cell Counting Kit-8 (CCK8) assay. To explore effects on cell death, autophagy, inflammation, and matrix metabolism, cells were treated by temsirolimus in no serum-containing DMEM with 10-ng/ml interleukin-1β (IL-1β) to simulate inflammation. Relative mRNA expression levels of catabolic MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, and anabolic ACAN encoding aggrecan and COL2A1 encoding collagen type II alpha 1 chain, and inflammatory cytokines IL-1β and IL-6 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were assessed by real-time RT-PCR. To assess effects on autophagy, autophagy markers LC3-II/I ratio and beclin 1 were assessed by Western blotting. β-Actin was used as loading control. To assess effects on apoptosis, apoptotic cells were identified using a fluorescein-labeled TUNEL assay kit. Results: Temsirolimus showed dose-dependent decreases in CCK8 dehydrogenase activity (P<0.01 in >1 μM). Based on the CCK8 findings, 100 nM was selected as an effective but non-toxic concentration. Real-time RT-PCR demonstrated that IL-1β-induced mRNA upregulations of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 (P<0.02), IL-1β and IL-6 were suppressed by temsirolimus. Meanwhile, temsirolimus treatment resulted in a trend toward up-regulation of these anabolic genes, ACAN and COL2A1 (P<0.03). Gene expression of those anabolic genes was down-regulated by IL-1β but recovered by the treatment with temsirolimus, Western blotting showed that temsirolimus increased LC3-II/I ratio and expression of beclin 1 (P<0.05). Further, western blotting analysis showed that temsirolimus reduced the percentage of IL-1β-induced apoptotic TUNEL-positive cells (P<0.05). Conclusions: Temsirolimus attenuated IL-1β-induced upregulation of catabolic and inflammatory gene expressions and apoptosis while promoting anabolic gene expressions and autophagy in human chondrocyte. Considering systemic side effects of mTOR inhibitors, local delivery by intra-articular injection would be recommend for their clinical use. The Results of this study at least suggested that temsirolimus would be a promising drug for intra-articular injection in treatment of OA.

Silymarin modulates catabolic cytokine expression through Sirt1 and SOX9 in human articular chondrocytes

BackgroundSilymarin (SMN), a polyphenolic flavonoid, is involved in multiple bioactive functions including anti-inflammation. Pretreatment with SMN demonstrated chondroprotection against tumour necrosis factor-alpha (TNF-α) stimulation in a chondrocyte cell line. However, pre- and posttreatment with phytochemicals have varying effects on osteoarthritis (OA) chondrocytes, and the therapeutic potential of SMN after catabolic cytokine stimulation is not fully elucidated.MethodsThe cytotoxicity of SMN (12.5, 25, 50 and 100 μM) was evaluated in human primary chondrocytes. The chondrocytes were supplemented with SMN (25 and 50 μM) after interleukin-1beta (IL-1β) stimulation. The mRNA expression and protein production of catabolic/anabolic cytokines as well as extracellular matrix (ECM) components were evaluated.ResultsHigh-dose SMN (100 μM) impaired the mitochondrial activity in chondrocytes, and 50 μM SMN further caused cell death in IL-1β-stimulated cells. The addition of 25 μM SMN ameliorated cell senescence; downregulated the catabolic genes of inducible nitric oxide synthase, IL-1β, TNF-α, matrix metalloproteinase-3 (MMP-3), MMP-9 and MMP-13; upregulated the anabolic genes of tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen type II alpha 1; and restored the expression of chondrogenic phenotype genes SOX9 and sirtuin-1 (Sirt1). In addition, the production of IL-1β, MMP-3 and MMP-9 decreased with an increase in TIMP-1 secretion. However, the mRNA levels of IL-6, IL-8 and IL-10 and protein production remained high. The addition of nicotinamide, a Sirt1 inhibitor, downregulated SOX9 and attenuated the therapeutic effects of SMN on IL-1β-stimulated chondrocytes.ConclusionSMN regulates the chondrocyte phenotype through Sirt1 and SOX9 to improve ECM homeostasis and may serve as a complementary therapy for early-stage knee OA.

Expression of the RANK/RANKL/OPG system in the human intervertebral disc: implication for the pathogenesis of intervertebral disc degeneration

BackgroundThe expression of the receptor activator of nuclear factor kappa B (RANK) /RANK ligand (RANKL) /osteoprotegerin (OPG) system and its association with the progression of intervertebral disc (IVD) degeneration has recently been reported in a human IVD. However, the effect of the RANK/RANKL/OPG system on the matrix metabolism of human IVD cells, especially on the expression of catabolic factors relevant to IVD degeneration, remains unknown. The purpose of this study was to examine the expression of the RANK/RANKL/OPG system, and then to evaluate the effect of this system on the expression of catabolic factors by human IVD cells.MethodsAnnulus fibrosus (AF) and nucleus pulposus (NP) cells isolated by sequential enzyme digestion from human IVD tissues obtained during spine surgeries were monolayer cultured. The expression of the RANK/RANKL/OPG system was determined using immunohistochemical methods and real-time polymerase chain reaction (PCR). To evaluate the influence of interleukin-1 beta (IL-1β) stimulation on the mRNA expression of RANK, RANKL, and OPG, recombinant human IL-1β (rhIL-1β) was administered in the culture media of IVD cells. To examine the influence of RANKL signaling on the expression of matrix metalloprotease-3 (MMP-3), MMP-13, and IL-1β, the cells were cultured with exogenous recombinant human RANKL (rhRANKL), recombinant human OPG (rhOPG) or anti-human RANKL mouse monoclonal antibody (ahRANKL-mAB) with or without rhIL-1β.ResultsImmunoreactivity to RANK/RANKL/OPG and the mRNA expression of the three genes were obviously identified in both AF and NP cells. rhIL-1β stimulation significantly upregulated the mRNA expression level of RANK/RANKL/OPG. The mRNA expression of catabolic factors was significantly upregulated by stimulation of rhRANKL in the presence of rhIL-1β. On the other hand, the administration of either rhOPG or ahRANKL-mAB significantly suppressed the mRNA expression of catabolic factors that had been upregulated by rhIL-1β stimulation. The suppressive effect of ahRANKL-mAB against rhIL-1β stimulation was also confirmed by the protein expression of MMP-3.ConclusionsThe present study showed that the RANK/RANKL/OPG system may be involved in the progression of IVD degeneration. This study also suggested the potential use of anti-RANKL monoclonal antibody and OPG as therapeutic agents to suppress the progression of IVD degeneration.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1β) stimulation. Western blotting showed significant decreases in specific proteins by each RNAi (all P<0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1β-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1β-induced senescence-associated beta-galactosidase (SA-β-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1β-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

Delivery of anti-inflammatory peptides from hollow PEGylated poly(NIPAM) nanoparticles reduces inflammation in an ex vivo osteoarthritis model

Targeted delivery of anti-inflammatory osteoarthritis treatments have the potential to significantly decrease undesirable systemic side effects and reduce required therapeutic dosage. Here we present a targeted, non-invasive drug delivery system to decrease inflammation in an osteoarthritis model. Hollow thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) nanoparticles have been synthesized via degradation of a N,N′-bis(acryloyl)cystamine (BAC) cross-linked core out of a non-degradable pNIPAM shell. Sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was copolymerized in the shell to increase passive loading of an anti-inflammatory mitogen-activated protein kinase-activated protein kinase 2 (MK2)-inhibiting cell-penetrating peptide (KAFAK). The drug-loaded hollow nanoparticles were effective at delivering a therapeutically active dose of KAFAK to bovine cartilage explants, suppressing pro-inflammatory interleukin-6 (IL-6) expression after interleukin-1 beta (IL-1β) stimulation. This thermosensitive hollow nanoparticle system provides an excellent platform for the delivery of peptide therapeutics into highly proteolytic environments such as osteoarthritis.

Elevated Expression of Long Non-coding RNA MALAT1 Associates with Increased Autophagy in Interleukin-1 Beta Stimulated ATDC5 Cells

Elevated Expression of Long Non-coding RNA MALAT1 Associates with Increased Autophagy in Interleukin-1 Beta Stimulated ATDC5 Cells

L-Glutathione enhances antioxidant capacity of hyaluronic acid and modulates expression of pro-inflammatory cytokines in human fibroblast-like synoviocytes.

Intra-articular injection of hyaluronic acid (HA) has been widely accepted for the treatment of osteoarthritis (OA) in early stage. l-Glutathione (GSH), an antioxidant, has an anti-inflammatory effect on protecting cells from reactive oxygen species and reactive nitrogen species (ROS/RNS). In this study, the therapeutic effects of HA (0.1%) supplemented with GSH (0, 5, 10, and 20% in weight ratios to HA) on human fibroblast-like synoviocytes (FLSs) were evaluated. The results showed that cell morphology and glycosaminoglycan production of FLSs were not changed under treatments. However, the addition of HA + 20% GSH significantly decreased cell survival (p < 0.001) relative to other groups. Relative to un-stimulated FLSs, interleukin-1 beta (IL-1β) stimulation significantly decreased the total antioxidant capacity (p < 0.001) of cells. The antioxidant capacity was restored and the intracellular ROS/RNS was decreased in HA or HA + GSH-treated FLSs. Real-time PCR analysis revealed the mRNA levels of IL-1β, tumor necrosis factor-alpha, and matrix metalloproteinase-3 were down-regulated significantly (all p < 0.05) when FLSs cultured in HA or HA + GSH. IL-6 mRNA expressions were down-regulated significantly in HA and HA + 5% GSH groups (both p < 0.05) but up-regulated when HA supplemented with 10% and 20% GSH (both p < 0.01). In addition, the protein levels of IL-1β were further decreased with significant differences (both p < 0.05) in the HA + 10% GSH and HA + 20% GSH groups when compared to FLSs cultured in normal medium. In conclusion, HA supplemented with GSH improves antioxidant capacity and modulates pro-inflammatory cytokines expressions in FLSs. GSH has the potential to augment the effect of viscosupplementation using HA on OA patients. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2071-2079, 2016.

Expression of lumican and fibromodulin following interleukin-1 beta stimulation of disc cells of the human temporomandibular joint

Small leucine-rich repeat proteoglycans (SLRP) are present in the extracellular matrix of the temporomandibular joint (TMJ) disc. Lumican and fibromodulin, classified as class 2 SLRPs, play important roles in TMJ assembly, proliferation and inflammation. Degenerative change in the TMJ disc gives rise to the process of internal derangement (ID). In this study, we immunohistochemically examined the expression of lumican and fibromodulin in nine human TMJ specimens and examined the gene expression of both proteoglycans in cultured human TMJ disc cells under interleukin-1 beta (IL-1 β)-stimulated conditions. An articular disc cell line was established by collagenase treatment of a TMJ disc. The subcultured cells were then incubated for 1, 3, 6, 12, 24 or 48 h under both normal and IL-1 β (1 ng/mL) conditions. The gene expression of lumican and fibromodulin was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in the deformed disc and that IL-1 β induces a significant increase in lumican mRNA, but not in fibromodulin mRNA, after 24∼48 h culture compared to cells cultured in the absence of IL-1 β (P<0.05). These results indicate that lumican and fibromodulin display different behaviors and that lumican may promote regeneration of the TMJ after degeneration and deformation induced by IL-1 β.

The P38 Mitogen-Activated Protein Kinase Mediates Osteogenic Transformation Of Human Aortic Valve Interstitial Cells By Interleukin-1 Beta: Implications For Aortic Stenosis

Introduction: Mechanisms of inflammation have been implicated in the pathogenesis of calcific aortic stenosis. In particular, the pro-inflammatory cytokine, interleukin-1 beta (IL-1B), has been shown to stimulate aortic valve interstitial cells (AVICs) to assume an osteogenic phenotype, characterized by the production of bone-forming proteins such as bone morphogenetic protein-2 (BMP-2). The p38 mitogen-activated protein kinase (MAPK) mediates mechanisms of inflammation in many cell types. Therefore, we hypothesized that the phenotypic change in AVICs is mediated by the p38 MAPK pathway. The purpose of this study was to examine the role of p38 MAPK in IL-1B-induced BMP-2 production in human AVICs. Methods: AVICs were isolated and grown in culture from normal aortic valves from the explanted hearts of patients undergoing cardiac transplantation (n=3). BMP-2 production (Western Blot) was determined following IL-1B stimulation (10 ng/mL) for 24 hours. P38 MAPK inhibition was accomplished by the p38 MAPK inhibitor, SB 203580 (SB, 1.0 μM, DMSO is the vehicle). Statistical analysis was by ANOVA. Results: BMP-2 production in response to IL-1B is mediated by the p38 MAPK signaling pathway. IL-1B stimulation resulted in a nearly four-fold increase in BMP-2 production (p<0.05). This BMP-2 production was prevented by p38 MAPK inhibition. p38 MAPK inhibition decreased BMP-2 levels by 76.3% versus IL-1B alone, and was 10.6% lower than BMP-2 expression in unstimulated controls (p<0.05). [See figure] Conclusions: IL-1B-stimulated changes in the phenotype of human AVICs are mediated by the p38 MAPK signaling pathway. These data provide mechanistic insight into the pathogenesis of calcific aortic stenosis.

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.

Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

IntroductionThe etiology of degenerative disc disease is unknown. Several investigators have reported the presence of proteolytic enzymes, such as the matrix metalloproteinase (MMP) and ADAMTS (a disintegrin and metalloprotease with thrombospondin-like repeats) families, in degenerated human discs. Glasson and colleagues recently reported that a significant reduction occurs in the severity of cartilage destruction in ADAMTS5 knockout mice compared with wild-type mice. The purpose of this study was to evaluate the suppressive effects of injections of ADAMTS5 small interference RNA (siRNA) oligonucleotide on intervertebral disc degeneration in the rabbit anular needle-puncture model.MethodsRabbit nucleus pulposus (NP) cells were transfected with siRNA oligonucleotides specific for ADAMTS5 or the control. The suppression of the ADAMTS5 gene by siRNA transfection was assessed by using real-time polymerase chain reaction (PCR), both in monolayer and alginate bead cultures with or without interleukin-1β (IL-1β) stimulation. The effect of siRNA was determined in vivo by using the rabbit anular needle-puncture model (control group: n = 8; ADAMTS5 group: n = 8). One week after the initial anular puncture, the animals received an injection of the control or anti-ADAMTS5 oligonucleotide (100 μg each at the L2/3 and L4/5 level; 16 discs/group). Disc height, magnetic resonance imaging (MRI) (Thompson classification and signal intensity), and safranin-O staining (histologic grade) were assessed.ResultsIL-1β treatment significantly increased the ADAMTS5 mRNA level in NP cells (P < 0.01). ADAMTS5 gene suppression was 70% compared with the control oligonucleotide in both monolayer and alginate bead culture with or without stimulation with IL-1β. The injection of anti-ADAMTS5 oligonucleotide in vivo resulted in improved MRI scores with increased signal intensity and improved histologic grade scores with statistical significance (P < 0.05). No significant change in disc height was observed.ConclusionsA single injection of ADAMTS5 siRNA induced the suppression of degradation in NP tissues, as shown by significantly improved MRI and histologic grades. The mechanism of response to siRNA may be worthy of exploration for possible therapeutic purposes.

Small molecule inhibitors of Hsp90 potently affect inflammatory disease pathways and exhibit activity in models of rheumatoid arthritis

To evaluate the ability of SNX-7081, a novel small molecule inhibitor of Hsp90, to block components of inflammation, including cytokine production, protein kinase activity, and angiogenic signaling. A close analog was evaluated in preclinical in vivo models of rheumatoid arthritis (RA). SNX-7081 binding to Hsp90 was characterized in Jurkat cells and RA synovial fibroblasts (RASFs). Inhibition of NF-kappaB nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor alpha, or interleukin-1beta stimulation. Suppression of cytokine production in THP-1 cells, human umbilical vein endothelial cells, and RASFs was studied. Disruption of MAPK signaling cascades by SNX-7081 following growth factor stimulation was assessed. SNX-7081 was tested in 2 relevant angiogenesis assays: platelet-derived growth factor activation of fibroblasts and LPS-induced nitric oxide (NO) release in J774 macrophages. A close analog, SNX-4414, was evaluated in rat collagen-induced arthritis and adjuvant-induced arthritis, following oral treatment. SNX-7081 showed strong binding affinity to Hsp90 and expected induction of Hsp70. NF-kappaB nuclear translocation was blocked by SNX-7081 at nanomolar concentrations, and cytokine production was potently inhibited. Growth factor activation of ERK and JNK signaling was significantly reduced by SNX-7081. NO production was also sharply inhibited. In animal models, SNX-4414 fully inhibited paw swelling and improved body weight. Scores for inflammation, pannus formation, cartilage damage, and bone resorption returned to normal. The present results demonstrate that a small molecule Hsp90 inhibitor can impact inflammatory disease processes. The strong in vivo efficacy observed with SNX-4414 provides preclinical validation for consideration of Hsp90 inhibitors in the treatment of RA.

Neutrophil-related immunoinflammatory disturbance in steroid-overdosed ulcerative colitis patients

There is some evidence that large preoperative doses of steroids are a causative factor for postoperative higher morbidity in ulcerative colitis (UC) patients. This study aimed to assess steroid-related changes in functional profiles of neutrophils in UC patients to estimate the immunological changes under surgical stress. Neutrophils were extracted from peripheral blood of 30 UC patients and 30 healthy controls. UC patients whose neutrophils were isolated were divided into two subgroups according to their total preoperative dosage of prednisolone: group H, > or =10,000 mg; group L, <10,000 mg. Expression of neutrophil surface antigens was analyzed and neutrophil phagocytosis was evaluated. Patterns of cell death of neutrophils were evaluated by co-culturing with Escherichia coli. Production of inflammatory mediators in cultured neutrophils was assessed. There were no significant differences in the expression rates of TLR4, CD11b, and CD16b on neutrophils (CD15(+) cells) between the two patient groups and controls. There was also no significant difference in neutrophil phagocytosis between the two patient groups and controls. The neutrophil necrosis rate in group H was higher than that in group L and the controls 3 h after exposure to E. coli. Neutrophils from group H released the highest levels of proinflammatory cytokines following interleukin-1beta or lipopolysaccharide stimulation. Neutrophils from group H also released the highest levels of proteolytic enzymes. Steroid-overdosed UC patients may have a functional deficit in neutrophils, which may cause a postsurgical systemic "storm" of inflammatory mediators.

The antioxidant resveratrol protects against chondrocyte apoptosis via effects on mitochondrial polarization and ATP production

To determine the effects of the antioxidant resveratrol on the functions of human chondrocytes in osteoarthritis (OA). Chondrocytes and cartilage explants were isolated from OA patients undergoing knee replacement surgery. Effects of resveratrol in the presence or absence of interleukin-1beta (IL-1beta) stimulation were assessed by measurement of prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) synthesis, cyclooxygenase (COX) activity, matrix metalloproteinase (MMP) expression, and proteoglycan production. To explore the mechanisms of action of resveratrol, its effects on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, cytochrome c release, and annexin V staining. Resveratrol inhibited both spontaneous and IL-1beta-induced PGE(2) production by >20% (P < 0.05) and by 80% (P < 0.001), respectively; similarly, LTB(4) production was reduced by >50% (P < 0.05). The production of PGE(2) was inhibited via a 70-90% suppression of COX-2 expression and enzyme activity (P < 0.05). Resveratrol also promoted anabolic effects in OA explant cultures, by elevating proteoglycan synthesis and decreasing production of MMPs 1, 3, and 13. Pretreatment of OA chondrocytes with resveratrol blocked mitochondrial membrane depolarization, loss of mitochondrial biomass, and IL-1beta-induced ATP depletion. Similarly, IL-1beta-mediated induction of the apoptotic markers cytochrome c and annexin V was also inhibited by resveratrol. Exogenous addition of PGE(2) abolished the protective effects of resveratrol on mitochondrial membrane integrity, ATP levels, expression of apoptotic markers, and DNA fragmentation. Resveratrol protects against IL-1beta-induced catabolic effects and prevents chondrocyte apoptosis via its inhibition of mitochondrial membrane depolarization and ATP depletion. These beneficial effects of resveratrol are due, in part, to its capacity to inhibit COX-2-derived PGE(2) synthesis. Resveratrol may therefore protect against oxidant injury and apoptosis, which are main features of progressive OA.

Gene Expression of Endothelial Cells due to Interleukin-1 Beta Stimulation and Neutrophil Transmigration

During the inflammatory response, endothelial cell (EC) functions and mechanics change dramatically. To understand these responses, the authors analyzed changes in EC gene expression in an in vitro model of inflammation using cDNA microarrays. After interleukin-1 beta (IL1beta) stimulation, over 2500 genes were differentially expressed, of which approximately 2000 had not been previously identified by microarray studies of IL1beta stimulation in human umbilical vein endothelial cells (HUVECs). Functional grouping of these genes according to gene ontologies revealed genes associated with apoptosis, cell cycle, nuclear factor (NF)-kappa B cascade, chemotaxis, and immune response. Interestingly, claudin-1, known to exist in endothelial cell-cell junctions was up-regulated, but claudin-5 and occludin, which also exist in EC junctions, were down-regulated. Pre-b-cell colony enhancing factor (PBEF), a cytokine which may play a role in regulating endothelial permeability, was also up-regulated following IL1beta stimulation. Neutrophil transmigration across IL1beta-stimulated ECs did not induce changes in EC gene expression as strongly as IL1beta stimulation alone. Nineteen genes after 1 h and 22 genes after 3 h of neutrophil application were differentially expressed. These results indicate that, in terms of transcriptional effects on ECs, neutrophil transmigration is a relatively small perturbation in comparison to the background of large scale changes induced in ECs by cytokine stimulation. Supplementary materials are available for this article. Go to the publisher's online edition of Endothelium for the following free supplementary resources: supplementary figures and tables.

Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation

Oxidative stress and inflammation are fundamental for the onset of aging and appear to be causatively linked. Previously, we reported that hepatocytes from aged rats, compared with young rats, are hyperresponsive to interleukin-1beta (IL-1beta) stimulation and exhibit more potent c-Jun N-terminal kinase (JNK) activation and attenuated interleukin-1 receptor-associated kinase-1 (IRAK-1) degradation. An age-related increase in the activity of neutral sphingomyelinase-2 (NSMase-2), a plasma membrane enzyme, was found to be responsible for the IL-1beta hyperresponsiveness. The results reported here show that increased NSMase activity during aging is caused by a 60-70% decrease in hepatocyte GSH levels. GSH, at concentrations typically found in hepatocytes from young animals, inhibits NSMase activity in a biphasic dose-dependent manner. Inhibition of GSH synthesis in young hepatocytes activates NSMase, causing increased JNK activation and IRAK-1 stabilization in response to IL-1beta, mimicking the hyperresponsiveness typical for aged hepatocytes. Vice versa, increased GSH content in hepatocytes from aged animals by treatment with N-acetylcysteine inhibits NSMase activity and restores normal IL-1beta response. Importantly, the GSH decline, NSMase activation, and IL-1beta hyperresponsiveness are not observed in aged, calorie-restricted rats. In summary, this report demonstrates that depletion of cellular GSH during aging plays an important role in regulating the hepatic response to IL-1beta by inducing NSMase-2 activity.