Aquaporin expression in the human and canine intervertebral disc during maturation and degeneration.

The intervertebral disc (IVD) is a highly hydrated tissue, the rich proteoglycan matrix imbibes water, enabling the disc to withstand compressive loads. During aging and degeneration increased matrix degradation leads to dehydration and loss of function. Aquaporins (AQP) are a family of transmembrane channel proteins that selectively allow the passage of water in and out of cells and are responsible for maintaining water homeostasis in many tissues. Here, the expression of all 13 AQPs at gene and protein level was investigated in human and canine nondegenerate and degenerate IVDs to develop an understanding of the role of AQPs during degeneration. Furthermore, in order to explore the transition of notochordal cells (NCs) towards nucleus pulposus (NP) cells, AQP expression was investigated in canine IVDs enriched in NCs to understand the role of AQPs in IVD maturation. AQP0, 1, 2, 3, 4, 5, 6, 7, and 9 were expressed at gene and protein level in both nondegenerate and degenerate human NP tissue. AQP2 and 7 immunopositivity increased with degeneration in human NP tissue, whereas AQP4 expression decreased with degeneration in a similar way to AQP1 and 5 shown previously. All AQP proteins that were identified in human NP tissue were also expressed in canine NP tissue. AQP2, 5, 6, and 9 were found to localize to vacuole-like membranes and cell membranes in NC cells. In conclusion, AQPs were abundantly expressed in human and canine IVDs. The expression of many AQP isotypes potentially alludes to multifaceted functions related to adaption of NP cells to the conditions they encounter within their microenvironment in health and degeneration. The presence of AQPs within the IVD may suggest an adaptive role for these water channels during the development and maintenance of the healthy, mature IVD.

Expression of Cannabinoid Receptors in Human Osteoarthritic Cartilage: Implications for Future Therapies.

Introduction: Cannabinoids have shown to reduce joint damage in animal models of arthritis and reduce matrix metalloproteinase expression in primary human osteoarthritic (OA) chondrocytes. The actions of cannabinoids are mediated by a number of receptors, including cannabinoid receptors 1 and 2 (CB1 and CB2), G-protein-coupled receptors 55 and 18 (GPR55 and GPR18), transient receptor potential vanilloid-1 (TRPV1), and peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). However, to date very few studies have investigated the expression and localization of these receptors in human chondrocytes, and expression during degeneration, and thus their potential in clinical applications is unknown. Methods: Human articular cartilage from patients with symptomatic OA was graded histologically and the expression and localization of cannabinoid receptors within OA cartilage and underlying bone were determined immunohistochemically. Expression levels across regions of cartilage and changes with degeneration were investigated. Results: Expression of all the cannabinoid receptors investigated was observed with no change with grade of degeneration seen in the expression of CB1, CB2, GPR55, PPARα, and PPARγ. Conversely, the number of chondrocytes within the deep zone of cartilage displaying immunopositivity for GPR18 and TRPV1 was significantly decreased in degenerate cartilage. Receptor expression was higher in chondrocytes than in osteocytes in the underlying bone. Conclusions: Chondrocytes from OA joints were shown to express a wide range of cannabinoid receptors even in degenerate tissues, demonstrating that these cells could respond to cannabinoids. Cannabinoids designed to bind to receptors inhibiting the catabolic and pain pathways within the arthritic joint, while avoiding psychoactive effects, could provide potential arthritis therapies.

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc.

INTRODUCTION: The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration. METHODS: Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates ('infiltrated'). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples. RESULTS: LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes. CONCLUSIONS: Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the 'degenerate niche' prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.

Tumor necrosis factor α- and interleukin-1ß-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1.

OBJECTIVE: To investigate tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. METHODS: Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPß), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. RESULTS: An increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1ß treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPß on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKß significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1ß promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. CONCLUSION: Our findings indicate that TNFα and IL-1ß modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPß signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.

Expression of ADAMTS-1, ADAMTS-4, ADAMTS-5 and TIMP3 by hepatocellular carcinoma cell lines.

Little is known about the expression or role of ADAMTS-1, -4 and -5 and their endogenous inhibitor TIMP3 in the liver in physiological and pathological conditions. Their expression was, therefore, investigated in the hepatocellular carcinoma cell lines HepG2 and HuH-7 using qRT-PCR and western blotting, and their cellular localisation by immunocytochemistry. Cytokine treatments were used to assess mRNA and protein modulation. ADAMTS-1, -4, -5 and TIMP3 mRNA and protein were detected in both HepG2 and HuH-7 cells. IL-1ß and IL-6 treatments significantly modulated ADAMTS-1 mRNA expression and IL-1ß treatment ADAMTS-4 mRNA expression in HepG2 cells. Modulations of mRNA by ≥ 5-fold did not translate to increased protein expression. This study showed that ADAMTS-1, -4, -5 and TIMP3 were expressed at differential levels in hepatocellular carcinoma cell lines. The pro-inflammatory cytokines IL-1ß, TNF-α or IL-6 induced changes in mRNA expression, although these did not translate to the protein level.

siRNA knockdown of ADAM-10, but not ADAM-17, significantly reduces fractalkine shedding following pro-inflammatory cytokine treatment in a human adult brain endothelial cell line.

Fractalkine shedding is believed to occur constitutively and following induction via the activity of two membrane-bound enzymes, ADAM-10 and ADAM-17. However, our previous work suggested that ADAM-17 is not involved in the proteolytic release of fractalkine under TNF treatment of a human adult brain endothelial cell line, hCMEC/D3. The pro-inflammatory cytokine, TNF, has previously been shown to be expressed in the perivascular cuffs in multiple sclerosis. Here we sought to identify, using siRNAs to silence the expression of ADAM-10 and ADAM-17, whether ADAM-10 is responsible for TNF-induced shedding of fractalkine from the cell membrane in hCMEC/D3. Our findings suggest that ADAM-10, and not ADAM-17, is the major protease involved in fractalkine release under pro-inflammatory conditions in this human adult brain endothelial cell model.

Cannabinoids: novel therapies for arthritis?

A key feature of osteoarthritis and rheumatoid arthritis is the loss of articular cartilage. Cartilage breakdown is mediated by complex interactions of proinflammatory cytokines, such as IL-1, inflammatory mediators, including nitric oxide and prostaglandin E(2), and proteases, including matrix metalloproteinases and aggrecanases, such as ADAMTS-4 and -5. Cannabinoids have been shown to reduce joint damage in animal models of arthritis. They have also been shown to prevent IL-1-induced matrix breakdown of collagen and proteoglycan, indicating that cannabinoids may mediate chondroprotective effects. Cannabinoids produce their effects via several cannabinoid receptors and it is important to identify the key cannabinoids and their receptors that are involved in chondroprotection. This review aims to outline the current and future prospects of cannabinoids as anti-arthritic therapeutics, in terms of their ability to prevent cartilage breakdown.

Effects of pro-inflammatory cytokines on the production of soluble fractalkine and ADAM17 by HepG2 cells.

BACKGROUND & AIMS: Soluble fractalkine is increased in the liver during times of injury; however the effect of pro-inflammatory cytokines in this process is currently unknown. The aim of this study was to determine whether pro-inflammatory cytokines elevated in patients with hepatocellular carcinoma influence fractalkine shedding from HepG2 cells and whether ADAM17 was involved in this process. METHODS: In vitro experiments were performed in the human hepatocellular carcinoma cell line HepG2. Soluble fractalkine was detected using an ELISA. ADAM17 expression was investigated using quantitative real time (reverse transcription)-polymerase chain reaction and flow cytometry. Short interfering RNA transfection was used to down-regulate ADAM17 expression. RESULTS: Soluble fractalkine was present in supernatants of HepG2 cells, and was significantly increased by interleukin-1ß (p ≤ 0.005) and tumour necrosis factor-α (p ≤ 0.043), but not by interleukin-6 (p ≥ 0.316). This corresponded to minor increases in ADAM17 protein, but not ADAM17 mRNA, following the same treatments. However, the down-regulation of ADAM17 protein did not affect fractalkine shedding. CONCLUSIONS: This study showed that soluble fractalkine is up-regulated under inflammatory conditions associated with hepatocellular carcinoma development, but ADAM17 does not appear to be responsible for regulating this process.

ADAMTS-9 expression is up-regulated following transient middle cerebral artery occlusion (tMCAo) in the rat.

The ADAMTS enzymes (a disintegrin and metalloproteinase with thrombospondin type 1-like motifs) have important roles in central nervous system (CNS) physiology and pathology. This current study aimed to analyse the expression of ADAMTS-9 following transient middle cerebral artery occlusion (tMCAo) in the rat, a model of focal cerebral ischaemia. Using real-time RT-PCR, ADAMTS-9 mRNA was demonstrated to be significantly up-regulated in tMCAo brain tissue compared to sham-operated at 24h post-ischaemia. The mature form of the ADAMTS-9 protein was only detected by Western blotting in brains subjected to tMCAo at 24h. In situ hybridisation demonstrated that ADAMTS-9 mRNA was expressed by neurones in tMCAo tissue. This study indicates that ADAMTS-9 expression is modulated in response to cerebral ischaemia in vivo and further research will resolve whether it plays a role in the subsequent degenerative or repair processes.

Expression of ADAM-17, TIMP-3 and fractalkine in the human adult brain endothelial cell line, hCMEC/D3, following pro-inflammatory cytokine treatment.

ADAM-17 expression is localised to endothelial cells in the human central nervous system (CNS) and is increased in multiple sclerosis (MS) white matter, suggesting a role in MS pathogenesis. Expression of ADAM-17, TIMP-3, and fractalkine were investigated in a human brain endothelial cell line (hCMEC/D3) after pro-inflammatory cytokine treatment. Tumour necrosis factor (TNF) significantly increased fractalkine mRNA (>100 fold) and protein expression, which was associated with increased shedding of fractalkine from the cell. Fractalkine shedding may regulate immune cell trafficking into the CNS, however, this does not appear to be directly controlled by ADAM-17 activity.

Arthritis and cannabinoids: HU-210 and Win-55,212-2 prevent IL-1alpha-induced matrix degradation in bovine articular chondrocytes in-vitro.

Cannabinoids have analgesic, immunomodulatory and anti-inflammatory properties and attenuate joint damage in animal models of arthritis. In this study the mechanisms of action of the synthetic cannabinoid agonists, HU-210 and Win-55,212-2, were studied to determine if they affected interleukin-1 alpha (IL-1alpha)-induced proteoglycan and collagen degradation in bovine nasal cartilage explant cultures and prostaglandin E2 (PGE2) production in primary cultures of bovine articular chondrocytes. The effects of the inactive enantiomer, Win-55,212-3, were compared with those of the active enantiomer, Win-55,212-2, to determine if the effects were cannabinoid (CB)-receptor mediated. The chondrocytes and explants were stimulated by IL-1alpha (100 U mL(-1) identical with 0.06 nM and 500 U mL(-1) identical with 0.3 nM, respectively). Proteoglycan breakdown was determined as sulfated glycosaminoglycan (sGAG) release using the dimethylmethylene blue assay. Collagen degradation was determined as hydroxyproline in the conditioned culture media and cartilage digests. PGE2 was determined by ELISA. Expression of cannabinoid receptors, CB1 and CB2; cyclooxygenase-1 and -2 (COX-1 and COX-2); inducible nitric oxide synthase (iNOS); as well as activation of nuclear factor-kappa B (NF-kappaB) in chondrocytes were studied using immunoblotting techniques and immunofluorescence. The results showed that HU-210 and Win-55,212-2 (5-15 microM) significantly inhibited IL-1-alpha stimulated proteoglycan (P < 0.001) and collagen degradation (P < 0.001). Win-55,212-2 (5-10 microM) also significantly inhibited PGE2 production (P < 0.01). At 5 microM, Win-55,212-2 inhibited the expression of iNOS and COX-2 and activation of NF-kappaB. Chondrocytes appeared to constitutively express cannabinoid receptors CB1 and CB2. It is concluded that biologically stable synthetic cannabinoids protect cartilage matrix from degradation induced by cytokines and this effect is possibly CB-receptor mediated and involves effects on prostaglandin and nitric oxide metabolism. Cannabinoids could also be producing these effects via inhibition of NF-kappaB activation.

ADAM-17 and TIMP3 protein and mRNA expression in spinal cord white matter of rats with acute experimental autoimmune encephalomyelitis.

Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the naïve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.

Effects of cannabinoids on nitric oxide production by chondrocytes and proteoglycan degradation in cartilage.

Cannabinoids have been reported to have anti-inflammatory effects and reduce joint damage in animal models of arthritis. This suggests a potential therapeutic role in arthritis of this group of compounds. Cannabinoids were studied to determine whether they have direct effects on chondrocyte metabolism resulting in cartilage protection. Synthetic cannabinoids, R-(+)-Win-55,212 (Win-2) and S-(-)-Win-55,212 (Win-3) and the endocannabinoid, anandamide, were investigated on unstimulated or IL-1-stimulated nitric oxide (NO) production in bovine articular chondrocytes as well as on cartilage proteoglycan breakdown in bovine nasal cartilage explants. Win-2 significantly inhibited (P < 0.05) NO production in chondrocytes at 1-10 microM concentrations. The combined CB(1) and CB(2) cannabinoid receptor antagonists, AM281 and AM630, respectively, at 100 microM did not block this effect, but instead they potentiated it. Anandamide and Win-2 (5-50 microM) also inhibited the release of sulphated glycosaminoglycans in bovine cartilage explants. The results suggest that some cannabinoids may prevent cartilage resorption, in part, by inhibiting cytokine-induced NO production by chondrocytes and also by inhibiting proteoglycan degradation.

Cannabinoids in pain and inflammation.

Cannabinoids exhibit medicinal properties including analgesic, anti-inflammatory and immunosuppressive properties. This paper reviews some of the recent findings in the study of cannabinoids in pain and inflammation. Some of the effects of cannabinoids are receptor mediated and others are receptor independent. Endocannabinoids naturally reduce pain and are cerebroprotective. Natural and synthetic cannabinoids have the potential to reduce nociception, reverse the development of allodynia and hyperalgesia, reduce inflammation and inflammatory pain and protect from secondary tissue damage in traumatic head injury.