Stimulated Proteoglycan Synthesis Research Articles

Pentosan Polysulfate Affords Pleotropic Protection to Multiple Cells and Tissues.

Pentosan polysulfate (PPS), a small semi-synthetic highly sulfated heparan sulfate (HS)-like molecule, shares many of the interactive properties of HS. The aim of this review was to outline the potential of PPS as an interventional therapeutic protective agent in physiological processes affecting pathological tissues. PPS is a multifunctional molecule with diverse therapeutic actions against many disease processes. PPS has been used for decades in the treatment of interstitial cystitis and painful bowel disease, it has tissue-protective properties as a protease inhibitor in cartilage, tendon and IVD, and it has been used as a cell-directive component in bioscaffolds in tissue engineering applications. PPS regulates complement activation, coagulation, fibrinolysis and thrombocytopenia, and it promotes the synthesis of hyaluronan. Nerve growth factor production in osteocytes is inhibited by PPS, reducing bone pain in osteoarthritis and rheumatoid arthritis (OA/RA). PPS also removes fatty compounds from lipid-engorged subchondral blood vessels in OA/RA cartilage, reducing joint pain. PPS regulates cytokine and inflammatory mediator production and is also an anti-tumor agent that promotes the proliferation and differentiation of mesenchymal stem cells and the development of progenitor cell lineages that have proven to be useful in strategies designed to effect repair of the degenerate intervertebral disc (IVD) and OA cartilage. PPS stimulates proteoglycan synthesis by chondrocytes in the presence or absence of interleukin (IL)-1, and stimulates hyaluronan production by synoviocytes. PPS is thus a multifunctional tissue-protective molecule of potential therapeutic application for a diverse range of disease processes.

Stability Indicating Method Development and Validation for the Simultaneous Estimation of Diacerin, Glucosamine sulphate and Methyl sulfonyl methane by RP-HPLC

Treatment for osteoarthritis includes a mixture of Diacerin (DIA), Glucosamine sulphate (GLU), and Methyl sulfonyl methane (MSM). Methyl sulfonyl methane functions as a dietary supplement, whereas Diacerin and Glucosamine sulphate stimulate proteoglycan synthesis. The simultaneous quantification of DIA, GLU, and MSM in pharmaceutical dosage forms has been developed using the RP-HPLC method. With a flow rate of 0.9 mL/min, chromatography was performed on an Agilent C18 150x 4.6, 5.0 mm column. pH 3.0 0.1 percent orthophosphoric acid buffer and 60:40 (percent v/v) acetonitrile made up the mobile phase. 2.233, 2.823, and 3.286 minutes were found to be the retention times for DIA, GLU, and MSM, respectively. In the concentration ranges of 2.5-15 g/mL (R2 = 0.999) for DIA, 37.5-225 g/mL (R2 = 0.999) for GLU, and 12.5-75 g/mL (R2 = 0.999) for MSM, the approach follows Beer's law. Diacerin's LOD and LOQ were discovered to be 0.05 g/mL and 0.14 g/mL, Glucosamine sulphate's LOD and LOQ were 0.90 g/mL and 2.73 g/mL, and Methyl sulfonyl methane's LOD and LOQ were 0.88 g/mL and 2.67 g/mL, respectively. Recovery studies in the dose form at three concentration levels were used to assess the accuracy of the established approach. The mean percent recovery for DIA, GLU, and MSM was determined to be 99.62 percent, 99.62 percent, and 99.94 percent, respectively. The approach was used to calculate the dosage of Orcerin GM tablets. As per ICH requirements, the devised approach has been statistically validated. As a result, the chromatographic method developed for the estimation has been described as rapid, simple, precise, accurate, robust, and reliable, and it can be used for routine analysis in research institutions, quality control departments in industries, approved testing laboratories, biopharmaceutics and bioequivalence studies, and clinical pharmacokinetic studies.

EGF Receptor Transactivation by Endothelin-1 Increased CHSY-1 Mediated by NADPH Oxidase and Phosphorylation of ERK1/2.

Objective Growth factors [transforming growth factor-β (TGF-β), epidermal growth factor (EGF), endothelin-1 (ET- 1)] stimulate proteoglycan synthesis resulting in retention and accumulation of low density lipoprotein (LDL) in vessel intima and leading to atherosclerosis development. This study investigated the role of ET-1 on the expression of CHSY1, proteoglycan synthesizing enzyme, through both EGF and TGF-β receptor transactivation in human vascular smooth muscle cells (VSMCs). Also, we explored the involvement of NADPH oxidase (NOX), an important intermediate of redox signaling, in ET-1 transactivated EGF receptor (EGFR) through endothelin receptors. Materials and Methods In this experimental study, phosphorylated ERK1/2 and CHSY1 protein levels in the human VSMCs were measured by Western blot analysis using anti phospho-ERK1/2 (Thr202/Tyr204) and anti CHSY1 antibodies. Results ET-1 (100 nM) and EGF (100 ng/ml) stimulated ERK1/2 phosphorylation and inhibited in the presence of bosentan (ET receptor inhibitor), AG1478 (EGFR inhibitor), and DPI (NOX antagonist). Also, ET-1 treatment increased CHSY1 enzyme level; this response was suppressed by bosentan, AG1478, DPI, and SB431542, TGF-β receptor antagonist. This study revealed that ET-1 increases expression of CHSY1 through transactivation of EGF and TGF-β receptors. ConclusionTransactivation through the EGF receptor mediated by phospho-ERK1/2 leads to expression of CHSY1 protein. EGF receptor transactivation by ET-1 is shown for the first time, to be dependent on NOX enzymes.

Chondrogenic Differentiation of Mesenchymal Stem Cells on Silk Fibroin:chitosan–Glucosamine Scaffold in Dynamic Culture

Cartilage damage is a common age-related problem that leads to progressive proteoglycan loss. Glucosamine stimulates proteoglycan synthesis and, therefore, its effect on the cartilage extracellular matrix synthesis over silk fibroin:chitosan (SF:CS) tissue-engineered scaffold was investigated for cartilage construct generation. Human mesenchymal stem cells (hMSCs) were cultured and differentiated over SF:CS-glucosamine porous scaffold, under dynamic culture condition in spinner flask bioreactor. hMSCs-seeded scaffold in dynamic culture exhibited homogenous cell distribution, proliferation and higher cell density at the core than static culture. Glucosamine in scaffold promoted proteoglycan and collagenous matrix synthesis as revealed by histological and immunofluorescence studies. Quantitative-PCR analysis showed upregulation of cartilage-specific genes, thereby confirming the chondrogenic differentiation. The chondrogenic differentiation of hMSCs was enhanced by the synergistic effect of glucosamine incorporated in SF:CS scaffold and influence of 3D dynamic culture environment, thereby resulting in chondrogenic phenotype of the cells that promoted cartilage regeneration.

Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues

ABSTRACTPurpose: Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues. Materials and Methods: Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents. Results: Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus. Conclusions: This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.

Evaluation of the potential synergism of imatinib-related poly kinase inhibitors using growth factor stimulated proteoglycan synthesis as a model response.

Tyrosine kinase inhibitors were the first class of smart drugs being specifically designed to inhibit a disease causing target. There is a very important but unresolved question as whether or not the overall therapeutic role of an individual tinib results from an action at its primary target, a single most likely, tyrosine kinase, or from the combined or aggregate action at the multiple targets which each tinib addresses. We selected a series of ten tinibs (gefitinib, sunitinib, lapatinib, erlotinib, imatinib, sorafenib, axitinib, vanitinib, bosutinib, dasatinib) with various known targets and investigated their activities in the inhibition of proteoglycan synthesis and GAG hyperelongation stimulated by a tyrosine kinase receptor agonist, platelet derived growth factor (PDGF) and for contrast, a serine/threonine kinase receptor agonist, TGF β and some downstream signalling pathways. The inhibitory activity varied from little to total inhibition. The actions of the tinibs were directed more towards inhibition of the tyrosine kinase, PDGF receptor signalling pathway compared to the TGF β. There was no suggestion of any synergistic effect arising from inhibition of multiple kinases as the most potent compound, dasatinib, is known to inhibit the broadest spectrum of kinases.

The Role of Bone Morphogenetic Proteins 2, 7, and 14 in Approaches for Intervertebral Disk Restoration

Bone morphogenetic proteins (BMPs) are a group of cytokines in the transforming growth factor beta superfamily. Transforming growth factor beta originally was described as a group of substances that have the ability to produce exostosis formation, but it was later discovered to have a wide range of chondroinductive and osteoinductive actions during embryogenesis and later in life. Recombinant BMPs were successfully introduced into clinical practice for augmentation of bone fusion in spinal and orthopedic surgeries; however, BMPs are now gaining attention from researchers for their role in intervertebral disk (IVD) pathology. We summarize the current understanding and future directions for the role of BMPs in IVD degradation and restoration. Recombinant human BMP-2, a component of INFUSE bone graft (Medtronic, Minneapolis, Minnesota, USA), was the first clinically available BMP drug. INFUSE bone grafts are used to enhance bone formation in lumbar fusion procedures, decreasing the rate of nonunions compared with autologous iliac crest bone grafts. It is also approved by the U.S. Food and Drug Administration for several other bone fusion procedures. Initial studies for BMP-2 in the IVD showed regenerative potential in the nucleus pulposus, where BMP2 resulted in a mitogenic effect and stimulated proteoglycan synthesis without ossification; additionally, studies in explanted rabbit IVDs showed that the presence of BMP-2 favorably increased expression of collagen II and aggrecans, while decreasing expression of catabolic matrix metalloproteinases in the anulus fibrosus. However, BMP-2 also decreased glycosaminoglycan content and increased type I collagen production in the nucleus pulposus as well as initiating ossification of the anulus fibrosus. Furthermore, exogenous application of BMP-2 can accelerate osteophyte formation, typically seen clinically in late-stage degenerative disk disease as a compensatory mechanism for spinal instability. Endogenous BMP-2 in the IVD increases with age and in mechanically induced models of disk degeneration. Recombinant human BMP-7, also known as osteogenic protein1, is currently marketed as OP-1 (Stryker Biotech, Hopkinton, Massachusetts, USA), an aid for posterior spinal fusion and long

Link N and Mesenchymal Stem Cells Can Induce Regeneration of the Early Degenerate Intervertebral Disc

Link N is a naturally occurring peptide that can stimulate proteoglycan synthesis in intervertebral disc (IVD) cells. IVD repair can also potentially be enhanced by mesenchymal stem cell (MSC) supplementation to maximize extracellular matrix (ECM) production. In a previous study, we have shown that Link N can inhibit osteogenesis and increase the chondrogenesis of MSCs in vitro. The aim of the present study was to determine the potential of MSCs and Link N alone or in combination with regard to tissue repair in the degenerate disc. Bovine IVDs with trypsin-induced degeneration were treated with MSCs, Link N, or a combination of MSCs and Link N. Trypsin-treated discs were also injected with phosphate-buffered saline to serve as a degeneration control. The ECM proteins and proteoglycans were extracted from the inner nucleus pulposus (NP) of the discs, and sulfated glycosaminoglycans (GAGs) were analyzed by the dimethyl methylene blue dye-binding assay. The expression of type II collagen was analyzed by western blot. To track the MSCs after injection, MSCs were labeled with PKH67 and observed under confocal microscopy after the 2 week culture period. The GAG content significantly increased compared with the degeneration control when degenerate discs were treated with MSCs, Link N, or a combination of both Link N and MSCs. Histological analysis revealed that the newly synthesized proteoglycan was able to diffuse throughout the ECM and restore tissue content even in areas remote from the cells. The quantity of extractable type II collagen was also increased when the degenerate discs were treated with MSCs and Link N, either alone or together. MSCs survived, integrated in the tissue, and were found distributed throughout the NP after the 2 week culture period. MSCs and Link N can restore GAG content in degenerate discs, when administered separately or together. Treatment with MSCs and Link N can also increase the expression of type II collagen. The results support the concept that biological repair of disc degeneration is feasible, and that the administration of either MSCs or Link N has therapeutic potential in early stages of the disease.

Bone marrow-derived cells and biophysical stimulation for talar osteochondral lesions: a randomized controlled study.

Osteochondral lesions of the talus (OLT) frequently occur after ankle sprains in young patients participating in sports activities. These injuries may lead to chronic pain, joint swelling, and finally osteoarthritis, therefore, surgical repair is frequently needed. A collagen scaffold seeded with bone marrow-derived cells (BMDCs) harvested from patient's iliac crest and implanted into the OLT through a single arthroscopic procedure has been recently proposed as an effective treatment option. Nevertheless, BMDCs, embedded in an inflammatory environment, tend to differentiate toward a fibroblast phenotype with a consequential loss of mechanical characteristics. Biophysical stimulation with pulsed electromagnetic fields (PEMFs) has been shown to promote anabolic chondrocyte activity, stimulate proteoglycan synthesis, and reduce the release of the most relevant pro-inflammatory cytokines. The aim of this randomized controlled trial was to evaluate the effects of PEMFs on clinical outcome in patients who underwent BMDCs transplantation for OLT. Thirty patients affected by grade III and IV Outerbridge OLT underwent BMDCs transplantation. After surgery, patients were randomly assigned to either experimental group (PEMFs 4 hours per day for 60 days starting within 3 days after operation) or control group. Clinical outcome was evaluated with (American Orthopaedic Foot and Ankle Society) AOFAS score, Visual Analog Scale (VAS), and Short Form-36 (SF-36). Significantly higher AOFAS score was recorded in the experimental group both at 6 or 12 months follow-up. At 60 days and 6 and 12 months follow-up, significant lower pain was observed in the experimental group. No significant difference was found in SF-36 between groups. A superior clinical outcome was found in the experimental group with more than 10 points higher AOFAS score at final follow-up. Biophysical stimulation started soon after surgery aided patient recovery leading to pain control and a better clinical outcome with these improvements lasting more than 1 year after surgery. Level II, prospective comparative study.

Link-N Exerts Its Growth Factor-Like Effects through Activation of Smad 1/5 Signaling in Human IVD Cells

Introduction Back pain is a fairly common problem, which affects a large portion of the population and has an impact on quality of life. Intervertebral disc (IVD) degeneration is one of the most common causes of back pain. Link-N peptide represents the 16 amino acid sequence (DHLSDNYTLDHDRAIH) from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and IVDs. We have previously shown that Link-N can stimulate collagen and proteoglycan synthesis in IVD cells in vitro1 and in intact human IVDs ex vivo,2 as well as increase disc height in a rabbit model of disc degeneration.3 Recently, BMPII receptor has been shown to interact with Link-N in rabbit IVD cells.4 The purpose of this study was to determine the mechanism by which Link-N exerts its beneficial effects by promoting matrix production in human IVD cells. Materials and Methods Human lumbar spines were retrieved through the Transplant Quebec organ donation program. IVD cells were isolated from nucleus pulposus (NP) and annulus fibrosus (AF) regions. The cells were cultured to 90% confluence in NP Cell Medium (NPCM, Cat. No.4801, ScienCell). Then the cells were incubated overnight in serum-free DMEM followed by treatment with Link-N (1 μg/mL), TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) for 10 minutes to 4 hours. TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) was used as positive controls. Cell extracts were prepared using NP40 lysis buffer. Total protein in the cell extracts was determined by the bicinchoninic acid assay (BCA). Protein expression was analyzed by immunoblotting using specific antibodies. Western blot images were quantified using ImageJ (NIH) software. For gene expression studies, cells were incubated for 24 hours with Link-N (1 μg/mL) in the presence or absence of human recombinant noggin (100 ng/mL) and total RNA was isolated using Trizol reagent and relative gene expression was determined by RT PCR. For receptor-binding studies, AF and NP cells were treated with biotinylated Link-N (1 μg/mL) with or without untagged Link-N (10 μg/mL) for competitive displacement. Scrambled biotinylated Link-N (1 μg/mL) was used as a specificity control. After treatments, cells were fixed in 2% paraformaldehyde. Fixed cells were treated with fluorescent-dye conjugated streptavidin (Alexa Fluor 700 conjugate, streptavidin). We assessed whether noggin directly binds with biotinylated Link-N by dot-blot technique. Results Link-N addition to human IVD cells did not promote the phosphorylation of Smads 2 and 3. This suggested that Link-N signaling does not function in a fashion similar to TGF-β, which stimulates the phosphorylation/activation of Smads 2 and 3. Further, similar to BMP-2, Link-N promoted the phosphorylation of Smad ? (Fig. 1). These results suggest that Link-N exerts its growth factor-like effects via a BMP-signaling mechanism rather than TGF-β. Noggin is a known physiological negative modulator of BMP signaling and noggin levels are elevated in disease conditions. As we observed that Link-N signaling is similar to BMP, it is essential to assess if noggin can interfere with the function of Link-N. Dot-blot results showed that noggin does not directly bind with Link-N. Therefore, noggin inhibition of Link-N signaling is unlikely because of noggin binding with Link-N. This suggests that Link-N may have an added advantage as a therapeutic agent over BMPs. In addition, Link-N increased BMP 4 expression significantly in both AF and NP cells, with the increase in AF cells being significantly higher than that in NP cells. Specific binding (also internalization) of biotinylated Link-N was found both in AF and NP cells, as this binding was competitively displaced by nonbiotinylated Link-N. No binding was seen with scrambled biotinylated Link-N. Conclusion Link-N represents a potential economic growth factor with beneficial effects on disc repair and the identification of the intracellular signaling pathways that it transduces is a step further toward clinical trials on NP repair in the degenerate human IVD. Disclosure of Interest None declared References Mwale F, Demers CN, Petit A, et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem 2003;88(6):1202–1213 Gawri R, Antoniou J, Ouellet J, et al. Best paper NASS 2013: link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs. Eur Cell Mater 2013;26:107-119, discussion 119 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120 Wang Z, Weitzmann MN, Sangadala S, Hutton WC, Yoon ST. Link protein N-terminal peptide binds to bone morphogenetic protein (BMP) type II receptor and drives matrix protein expression in rabbit intervertebral disc cells. J Biol Chem 2013;288(39):28243–28253

Mechanism of Proteoglycan Synthesis by Bovine and Human Link N

IntroductionLink N peptide is the N-terminal region of link protein which stabilizes the interaction between aggrecan and hyaluronan in proteoglycan aggregates. In vivo, during tissue turnover, lin...

Atherogenic, fibrotic and glucose utilising actions of glucokinase activators on vascular endothelium and smooth muscle

BackgroundPharmaceutical interventions for diabetes aim to control glycaemia and to prevent the development of complications, such as cardiovascular diseases. Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role. Glucokinase activity in the pancreas is critical in enhancing insulin release in response to hyperglycaemia. Glucokinase activators (GKAs) are novel agents for diabetes which act by enhancing the formation of glucose-6-phosphate leading to increased insulin production and subsequent suppression of blood glucose. Little, however, is known about the direct effects of GKAs on cardiovascular cells.MethodsThe effect of the GKAs RO28-1675 and Compound A on glucose utilisation in bovine aortic endothelial cells (BAEC) and rat MIN6 was observed by culturing the cells at high and low glucose concentration in the presence and absence of the GKAs and measuring glucose consumption. The effect of RO28-1675 at various concentrations on glucose-dependent signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-β stimulated proteoglycan synthesis was measured by 35S-SO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28-1675 on TGF-β mediated Smad2C phosphorylation in BAEC was observed by measurement of pSmad2C levels. The direct actions of RO28-1675 on vascular reactivity were observed by measuring arteriole tone and lumen diameter.ResultsGKAs were demonstrated to increase glucose utilisation in pancreatic but not endothelial cells. Glucose-activated Smad2 phosphorylation was decreased in a dose-dependent fashion in the presence of RO28-1675. No effect of RO28-1675 was observed on TGF-β stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity.ConclusionsGKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells. No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs. If reflected in vivo, these drugs are unlikely to have their use compromised by direct cardiovascular toxicity.

Endothelin receptor transactivates the TGFβ receptor to stimulate proteoglycan synthesis in human vascular smooth muscle cells

Endothelin receptor transactivates the TGFβ receptor to stimulate proteoglycan synthesis in human vascular smooth muscle cells

Best Paper NASS 2013: Link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs

Intervertebral disc (IVD) degeneration is the most common cause of back pain. Presently there is no medical treatment, leaving surgery as the only offered option. Here we evaluate the potential of Link-N to promote extracellular matrix regeneration in human IVDs. Human disc cells cultured in alginate and intact human discs were exposed to a combination of Link-N and ³⁵SO₄ in the presence or absence of interleukin (IL)-1, and the effect on proteoglycan synthesis was evaluated. In addition, message levels of aggrecan, matrix metalloproteinase (MMP)-3, MMP-13, a Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS)-4 and ADAMTS-5 were evaluated in alginate cultures. Human disc cells responded in a dose dependent manner with maximal proteoglycan synthesis at 1 µg/mL Link-N. Link-N treatment also induced proteoglycan synthesis in intact human discs, and a prolonged effect was found up to one week after Link-N treatment. Message levels of proteinases were decreased by Link-N in the presence of IL-1. Thus, Link-N can promote proteoglycan synthesis and deplete proteinase expression in adult human discs. Link-N could therefore be a promising candidate for biologically-induced disc repair, and could provide an alternative to surgical intervention for early stage disc degeneration.

Lactoferricin enhances BMP7-stimulated anabolic pathways in intervertebral disc cells

Bone-morphogenetic protein-7 (BMP7) is a well-known anabolic and anti-catabolic growth factor on intervertebral disc (IVD) matrix and cell homeostasis. Similarly, Lactoferricin B (LfcinB) has recently been shown to have pro-anabolic, anti-catabolic, anti-oxidative and/or anti-inflammatory effects in bovine disc cells in vitro. In this study, we investigated the potential benefits of using combined peptide therapy with LfcinB and BMP7 for intervertebral disc matrix repair and to understand cellular and signaling mechanisms controlled by these factors. We studied the effects of BMP7 and LfcinB as individual treatments and combined therapy on bovine nucleus pulposus (NP) cells by assessing proteoglycan (PG) accumulation and synthesis, and the gene expression of matrix protein aggrecan and transcription factor SOX-9. We also analyzed the role of Noggin, a BMP antagonist, in IVD tissue and examined its effect after stimulation with LfcinB. To understand the molecular mechanisms by which LfcinB synergizes with BMP7, we investigated the ERK-SP1 axis as a downstream intracellular signaling regulator involved in BMP7 and LfcinB-mediated activities. Treatment of bovine NP cells cultured in alginate with LfcinB plus BMP7 synergistically stimulates PG synthesis and accumulation in part by upregulation of aggrecan gene expression. The synergism results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (i) phosphorylation of SMAD 1/5/8; (ii) downregulation of SMAD inhibitory factors [i.e., noggin and SMAD6 (inhibitory SMAD)]; and (iii) upregulation of SMAD4 (universal co-SMAD). These data indicate that LfcinB-suppression of Noggin may eliminate the negative feedback of BMP7, thereby maximizing biological activity of BMP7 and ultimately shifting homeostasis to a pro-anabolic state in disc cells. We propose that combination growth factor therapy using BMP7 and LfcinB may be beneficial for treatment of disc degeneration.

(S)-[6]-Gingerol inhibits TGF-β-stimulated biglycan synthesis but not glycosaminoglycan hyperelongation in human vascular smooth muscle cells

(S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)-β stimulates proteoglycan synthesis, leading to increased binding of low-density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)-[6]-gingerol on these TGF-β-mediated proteoglycan changes to explore its potential as an anti-atherosclerotic agent. Purified (S)-[6]-gingerol was assessed for its effects on proteoglycan synthesis by [(35) S]-sulfate incorporation into glycosaminoglycan chains and [(35) S]-Met/Cys incorporation into proteoglycans and total proteins in human vascular smooth muscle cells. Biglycan level was assessed by real-time quantitative polymerase chain reactions and the effects of (S)-[6]-gingerol on TGF-β signalling by assessment of the phosphorylation of Smads and Akt by western blotting. (S)-[6]-Gingerol concentration-dependently inhibited TGF-β-stimulated proteoglycan core protein synthesis, and this was not secondary to inhibition of total protein synthesis. (S)-[6]-Gingerol inhibited biglycan mRNA expression. (S)-[6]-Gingerol did not inhibit TGF-β-stimulated glycosaminoglycan hyperelongation or phosphorylation of Smad 2, in either the carboxy terminal or linker region, or Akt phosphorylation. The activity of (S)-[6]-gingerol to inhibit TGF-β-stimulated biglycan synthesis suggests a potential role for ginger in the prevention of atherosclerosis or other lipid-binding diseases. The signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-β responses.

Ionizing radiation causes active degradation and reduces matrix synthesis in articular cartilage

Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1.Materials and methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours.Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy.Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.

Effect of Intervertech-1 Levels on Proteoglycan Synthesis by Intervertebral Disk Cells

IntroductionBack pain is a fairly common problem, which affects a large portion of the population across all ages and has an impact on quality of life. It has a major financial impact on the health care system and industry due to loss of productive labor hours. Intervertebral disk degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Intevertech 1(IVT-1), currently being patented by Intervertech Inc, represents a novel peptide produced in mammals. It has the ability to heal injured tissues and increase cell resistance to hypoxic episodes. The aim of this study was to determine how nucleus pulposus (NP) and annulus fibrosus (AF) cells respond to different Intervertech 1 concentrations when cultured in a three dimensional system consisting of an alginate scaffold.Materials and MethodsIsolation and culture of Bovine intervertebral disk cells: Adult bovine tails (2 to 3 years old) were obtained 2 to 3 hours after slaughter. The IVDs were dissected from their adjacent vertebral bodies and separated into NP and AF. AF and NP cells were isolated by digestion with collagenase, and were resuspended in 1.2% alginate in 0.15M sodium chloride at a concentration of 2 million cells per mL. Droplets of cell suspension were released through a 18-gauge needle into 102 mM calcium chloride solution and left to polymerize for 5 minutes.1 The beads were placed in 48 well plates at a density of 5 beads/well (in 1 mL of DMEM high glucose supplemented with 10% fetal bovine serum and antibiotics). Analysis of proteoglycan (aggrecan) synthesis:Alginate beads were stabilized for 5 days in 1 mL of DMEM and then exposed to 5 nM-400 nM of IVT-1 in DMEM supplemented with 25µCi of 35S-sulfate/mL for 48 hours to determine aggrecan synthesis. At the end of the culture period, media was collected and dialyzed exhaustively against RO water (Microdialysis chamber, Life Technologies with Spectr/Por 3, 3-kDa molecular weight cut-off dialysis membrane) followed by cold chase with 1M MgSO4 for 2 hours to remove unincorporated 35S-sulfate. Counts per minutes (CPM) reads were normalized to control and expressed as an arbitrary value.All experiments were performed in triplicates. Results were considered significant with a p < 0.05.ResultsIn both cell types proteoglycan synthesis increased with dosage in culture up to 10 nM, then tended to plateau between 25 nM and 50 nM but increased at 100 n M and 200 nM (Fig. 1). Maximal response was at 200 nM but declining thereafter. At all time points, the levels of proteoglycan synthesis by AF cells was greater than by NP cells.ConclusionThe role of Intevertech 1 in disk metabolism is not understood. Intevertech 1 is a small peptide involved in a wide variety of physiological functions. This study indicates that IVT-1 is able to stimulate proteoglycan synthesis in bovine disk cells cultured in a 3D system. Perhaps surprisingly, intervertech 1 stimulates AF cells to produce more proteoglycan than NP cells. This difference may relate to receptor usage on AF and NP cells by intervertech 1. The most significant biochemical change to occur in disk degeneration is loss of proteoglycan. Promotion of proteoglycan synthesis is the main strategy for disk regeneration therapy and this peptide shows promise in this direction and can be established as a potential therapeutic agent for annular repair especially in later stages of disk degeneration when the disc's load bearing behavior shifts to the annulus. One major advantage of Intevertech 1 over recombinant growth factors for therapeutic use is the large saving in cost.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredMwale, F, Ciobanu, I, Giannitsios, D, Roughley, P, Steffen, T, and Antoniou, J. Effect of oxygen levels on proteoglycan synthesis by intervertebral disk cells. Spine 2011;36:E131-E138

Effect of Intervertech-1 Levels on Proteoglycan Synthesis by Intervertebral Disk Cells

Introduction Back pain is a fairly common problem, which affects a large portion of the population across all ages and has an impact on quality of life. It has a major financial impact on the health care system and industry due to loss of productive labor hours. Intervertebral disk degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Intevertech 1(IVT-1), currently being patented by Intervertech Inc, represents a novel peptide produced in mammals. It has the ability to heal injured tissues and increase cell resistance to hypoxic episodes. The aim of this study was to determine how nucleus pulposus (NP) and annulus fibrosus (AF) cells respond to different Intervertech 1 concentrations when cultured in a three dimensional system consisting of an alginate scaffold. Materials and Methods Isolation and culture of Bovine intervertebral disk cells: Adult bovine tails (2 to 3 years old) were obtained 2 to 3 hours after slaughter. The IVDs were dissected from their adjacent vertebral bodies and separated into NP and AF. AF and NP cells were isolated by digestion with collagenase, and were resuspended in 1.2% alginate in 0.15M sodium chloride at a concentration of 2 million cells per mL. Droplets of cell suspension were released through a 18-gauge needle into 102 mM calcium chloride solution and left to polymerize for 5 minutes.1 The beads were placed in 48 well plates at a density of 5 beads/well (in 1 mL of DMEM high glucose supplemented with 10% fetal bovine serum and antibiotics). Analysis of proteoglycan (aggrecan) synthesis:Alginate beads were stabilized for 5 days in 1 mL of DMEM and then exposed to 5 nM-400 nM of IVT-1 in DMEM supplemented with 25µCi of 35S-sulfate/mL for 48 hours to determine aggrecan synthesis. At the end of the culture period, media was collected and dialyzed exhaustively against RO water (Microdialysis chamber, Life Technologies with Spectr/Por 3, 3-kDa molecular weight cut-off dialysis membrane) followed by cold chase with 1M MgSO4 for 2 hours to remove unincorporated 35S-sulfate. Counts per minutes (CPM) reads were normalized to control and expressed as an arbitrary value. All experiments were performed in triplicates. Results were considered significant with a p &lt; 0.05. Results In both cell types proteoglycan synthesis increased with dosage in culture up to 10 nM, then tended to plateau between 25 nM and 50 nM but increased at 100 n M and 200 nM (Fig. 1). Maximal response was at 200 nM but declining thereafter. At all time points, the levels of proteoglycan synthesis by AF cells was greater than by NP cells. Conclusion The role of Intevertech 1 in disk metabolism is not understood. Intevertech 1 is a small peptide involved in a wide variety of physiological functions. This study indicates that IVT-1 is able to stimulate proteoglycan synthesis in bovine disk cells cultured in a 3D system. Perhaps surprisingly, intervertech 1 stimulates AF cells to produce more proteoglycan than NP cells. This difference may relate to receptor usage on AF and NP cells by intervertech 1. The most significant biochemical change to occur in disk degeneration is loss of proteoglycan. Promotion of proteoglycan synthesis is the main strategy for disk regeneration therapy and this peptide shows promise in this direction and can be established as a potential therapeutic agent for annular repair especially in later stages of disk degeneration when the disc's load bearing behavior shifts to the annulus. One major advantage of Intevertech 1 over recombinant growth factors for therapeutic use is the large saving in cost. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mwale, F, Ciobanu, I, Giannitsios, D, Roughley, P, Steffen, T, and Antoniou, J. Effect of oxygen levels on proteoglycan synthesis by intervertebral disk cells. Spine 2011;36:E131-E138

The Growing Promiscuity of G Protein Coupled Receptors

The Growing Promiscuity of G Protein Coupled Receptors