TY - JOUR A1 - Krupkova, Olga A1 - Zvick, Johannes A1 - Würtz-Kozak, Karin T1 - The role of transient receptor potential channels in joint diseases JF - European cells & materials N2 - Transient receptor potential channels (TRP channels) are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous as well as exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD) cells is largely unexplored. Osteoarthritis (OA) and degenerative disc disease (DDD) are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation as well as activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells and the significance of TRP channels as therapeutic targets for the treatment of OA and DDD. KW - Transient receptor potential channels KW - degenerative disc disease KW - osteoarthritis KW - nociception KW - mechanosensing KW - osmosensing KW - inflammation KW - calcium Y1 - 2017 U6 - https://doi.org/10.22203/eCM.v034a12 SN - 1473-2262 VL - 34 SP - 180 EP - 201 PB - Univ. of Wales CY - Aberystwyth ER - TY - JOUR A1 - Krupkova, Olga A1 - Smolders, Lucas A1 - Würtz-Kozak, Karin A1 - Cook, James A1 - Pozzi, Antonio T1 - The pathobiology of the meniscus BT - a comparison between the human and dog JF - Frontiers in veterinary science N2 - Serious knee pain and related disability have an annual prevalence of approximately 25% on those over the age of 55 years. As curative treatments for the common knee problems are not available to date, knee pathologies typically progress and often lead to osteoarthritis (OA). While the roles that the meniscus plays in knee biomechanics are well characterized, biological mechanisms underlying meniscus pathophysiology and roles in knee pain and OA progression are not fully clear. Experimental treatments for knee disorders that are successful in animal models often produce unsatisfactory results in humans due to species differences or the inability to fully replicate disease progression in experimental animals. The use of animals with spontaneous knee pathologies, such as dogs, can significantly help addressing this issue. As microscopic and macroscopic anatomy of the canine and human menisci are similar, spontaneous meniscal pathologies in canine patients are thought to be highly relevant for translational medicine. However, it is not clear whether the biomolecular mechanisms of pain, degradation of extracellular matrix, and inflammatory responses are species dependent. The aims of this review are (1) to provide an overview of the anatomy, physiology, and pathology of the human and canine meniscus, (2) to compare the known signaling pathways involved in spontaneous meniscus pathology between both species, and (3) to assess the relevance of dogs with spontaneous meniscal pathology as a translational model. Understanding these mechanisms in human and canine meniscus can help to advance diagnostic and therapeutic strategies for painful knee disorders and improve clinical decision making. KW - meniscus KW - inflammation KW - oxidative stress KW - pain KW - dog Y1 - 2018 U6 - https://doi.org/10.3389/fvets.2018.00073 SN - 2297-1769 VL - 5 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Sadowska, Aleksandra A1 - Kameda, Takuya A1 - Krupkova, Olga A1 - Wuertz-Kozak, Karin T1 - Osmosensing, osmosignalling and inflammation BT - how intervertebral disc cells respond to altered osmolarity JF - European cells & materials N2 - Intervertebral disc (IVD) cells are naturally exposed to high osmolarity and complex mechanical loading, which drive microenvironmental osmotic changes. Age- and degeneration-induced degradation of the IVD’s extracellular matrix causes osmotic imbalance, which, together with an altered function of cellular receptors and signalling pathways, instigates local osmotic stress. Cellular responses to osmotic stress include osmoadaptation and activation of pro-inflammatory pathways. This review summarises the current knowledge on how IVD cells sense local osmotic changes and translate these signals into physiological or pathophysiological responses, with a focus on inflammation. Furthermore, it discusses the expression and function of putative membrane osmosensors (e.g. solute carrier transporters, transient receptor potential channels, aquaporins and acid-sensing ion channels) and osmosignalling mediators [e.g. tonicity response-element-binding protein/nuclear factor of activated T-cells 5 (TonEBP/NFAT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)] in healthy and degenerated IVDs. Finally, an overview of the potential therapeutic targets for modifying osmosensing and osmosignalling in degenerated IVDs is provided. KW - Intervertebral disc degeneration KW - degenerative disc disease KW - osmolarity KW - hyper-osmolarity KW - hypo-osmolarity KW - osmotic KW - inflammatory KW - transient receptor potential channel KW - aquaporin KW - tonicity-responsive enhancer binding protein Y1 - 2018 U6 - https://doi.org/10.22203/eCM.v036a17 SN - 1473-2262 VL - 36 SP - 231 EP - 250 PB - Ao research institute davos-Ari CY - Davos ER - TY - JOUR A1 - Krupkova, Olga A1 - Sadowska, Aleksandra A1 - Kameda, Takuya A1 - Hitzl, Wolfgang A1 - Hausmann, Oliver Nic A1 - Klasen, Jürgen A1 - Wuertz-Kozak, Karin T1 - p38 MaPK Facilitates crosstalk Between endoplasmic reticulum stress and IL-6 release in the intervertebral Disc JF - Frontiers in Immunology N2 - Degenerative disc disease is associated with increased expression of pro-inflammatory cytokines in the intervertebral disc (IVD). However, it is not completely clear how inflammation arises in the IVD and which cellular compartments are involved in this process. Recently, the endoplasmic reticulum (ER) has emerged as a possible modulator of inflammation in age-related disorders. In addition, ER stress has been associated with the microenvironment of degenerated IVDs. Therefore, the aim of this study was to analyze the effects of ER stress on inflammatory responses in degenerated human IVDs and associated molecular mechanisms. Gene expression of ER stress marker GRP78 and pro-inflammatory cytokines IL-6, IL-8, IL-1 beta, and TNF-alpha was analyzed in human surgical IVD samples (n = 51, Pfirrmann grade 2-5). The expression of GRP78 positively correlated with the degeneration grade in lumbar IVDs and IL-6, but not with IL-1 beta and TNF-alpha. Another set of human surgical IVD samples (n = 25) was used to prepare primary cell cultures. ER stress inducer thapsigargin (Tg, 100 and 500 nM) activated gene and protein expression of IL-6 and induced phosphorylation of p38 MAPK. Both inhibition of p38 MAPK by SB203580 (10 mu M) and knockdown of ER stress effector CCAAT-enhancer-binding protein homologous protein (CHOP) reduced gene and protein expression of IL-6 in Tg-treated cells. Furthermore, the effects of an inflammatory microenvironment on ER stress were tested. TNF-alpha (5 and 10 ng/mL) did not activate ER stress, while IL-1 beta (5 and 10 ng/mL) activated gene and protein expression of GRP78, but did not influence [Ca2+](i) flux and expression of CHOP, indicating that pro-inflammatory cytokines alone may not induce ER stress in vivo. This study showed that IL-6 release in the IVD can be initiated following ER stress and that ER stress mediates IL-6 release through p38 MAPK and CHOP. Therapeutic targeting of ER stress response may reduce the consequences of the harsh microenvironment in degenerated IVD. KW - intervertebral disc KW - inflammation KW - endoplasmic reticulum stress KW - p38 MAPK KW - CHOP KW - GADD153 KW - GRP78 KW - IL-6 Y1 - 2018 U6 - https://doi.org/10.3389/fimmu.2018.01706 SN - 1664-3224 VL - 9 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Kameda, Takuya A1 - Zvick, Joel A1 - Vuk, Miriam A1 - Sadowska, Aleksandra A1 - Tam, Wai Kit A1 - Leung, Victor Y. A1 - Bölcskei, Kata A1 - Helyes, Zsuzsanna A1 - Applegate, Lee Ann A1 - Hausmann, Oliver N. A1 - Klasen, Juergen A1 - Krupkova, Olga A1 - Würtz-Kozak, Karin T1 - Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc BT - Association with Inflammation and Matrix Remodeling JF - International journal of molecular sciences N2 - Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1) or tumor necrosis factor alpha (TNF-) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 mu M), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 mu M AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease. KW - low back pain KW - TRP channels KW - pro-inflammatory cytokines KW - aggrecanases KW - collagen KW - TRPA1 KW - TRPV1 KW - TRPV2 KW - TRPV4 KW - TRPC6 Y1 - 2019 U6 - https://doi.org/10.3390/ijms20071767 SN - 1422-0067 VL - 20 IS - 7 PB - MDPI CY - Basel ER - TY - GEN A1 - Krupkova, Olga A1 - Sadowska, Aleksandra A1 - Kameda, Takuya A1 - Hitzl, Wolfgang A1 - Hausmann, Oliver Nic A1 - Klasen, Jürgen A1 - Wuertz-Kozak, Karin T1 - p38 MaPK facilitates crosstalk between endoplasmic reticulum stress and IL-6 release in the intervertebral Disc T2 - Postprints der Universität Potsdam : Humanwissenschaftliche Reihe N2 - Degenerative disc disease is associated with increased expression of pro-inflammatory cytokines in the intervertebral disc (IVD). However, it is not completely clear how inflammation arises in the IVD and which cellular compartments are involved in this process. Recently, the endoplasmic reticulum (ER) has emerged as a possible modulator of inflammation in age-related disorders. In addition, ER stress has been associated with the microenvironment of degenerated IVDs. Therefore, the aim of this study was to analyze the effects of ER stress on inflammatory responses in degenerated human IVDs and associated molecular mechanisms. Gene expression of ER stress marker GRP78 and pro-inflammatory cytokines IL-6, IL-8, IL-1 beta, and TNF-alpha was analyzed in human surgical IVD samples (n = 51, Pfirrmann grade 2-5). The expression of GRP78 positively correlated with the degeneration grade in lumbar IVDs and IL-6, but not with IL-1 beta and TNF-alpha. Another set of human surgical IVD samples (n = 25) was used to prepare primary cell cultures. ER stress inducer thapsigargin (Tg, 100 and 500 nM) activated gene and protein expression of IL-6 and induced phosphorylation of p38 MAPK. Both inhibition of p38 MAPK by SB203580 (10 mu M) and knockdown of ER stress effector CCAAT-enhancer-binding protein homologous protein (CHOP) reduced gene and protein expression of IL-6 in Tg-treated cells. Furthermore, the effects of an inflammatory microenvironment on ER stress were tested. TNF-alpha (5 and 10 ng/mL) did not activate ER stress, while IL-1 beta (5 and 10 ng/mL) activated gene and protein expression of GRP78, but did not influence [Ca2+](i) flux and expression of CHOP, indicating that pro-inflammatory cytokines alone may not induce ER stress in vivo. This study showed that IL-6 release in the IVD can be initiated following ER stress and that ER stress mediates IL-6 release through p38 MAPK and CHOP. Therapeutic targeting of ER stress response may reduce the consequences of the harsh microenvironment in degenerated IVD. T3 - Zweitveröffentlichungen der Universität Potsdam : Humanwissenschaftliche Reihe - 705 KW - intervertebral disc KW - inflammation KW - endoplasmic reticulum stress KW - p38 MAPK KW - CHOP KW - GADD153 KW - GRP78 KW - IL-6 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-468698 SN - 1866-8364 IS - 705 ER - TY - GEN A1 - Sadowska, Aleksandra A1 - Kameda, Takuya A1 - Krupkova, Olga A1 - Würtz-Kozak, Karin T1 - Osmosensing, osmosignalling and inflammation BT - how intervertebral disc cells respond to altered osmolarity T2 - Postprints der Universität Potsdam : Humanwissenschaftliche Reihe N2 - Intervertebral disc (IVD) cells are naturally exposed to high osmolarity and complex mechanical loading, which drive microenvironmental osmotic changes. Age- and degeneration-induced degradation of the IVD's extracellular matrix causes osmotic imbalance, which, together with an altered function of cellular receptors and signalling pathways, instigates local osmotic stress. Cellular responses to osmotic stress include osmoadaptation and activation of pro-inflammatory pathways. This review summarises the current knowledge on how IVD cells sense local osmotic changes and translate these signals into physiological or pathophysiological responses, with a focus on inflammation. Furthermore, it discusses the expression and function of putative membrane osmosensors (e.g. solute carrier transporters, transient receptor potential channels, aquaporins and acid-sensing ion channels) and osmosignalling mediators [e.g. tonicity responseelement-binding protein/nuclear factor of activated T-cells 5 (TonEBP/NFAT5), nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kappa B)] in healthy and degenerated IVDs. Finally, an overview of the potential therapeutic targets for modifying osmosensing and osmosignalling in degenerated IVDs is provided. T3 - Zweitveröffentlichungen der Universität Potsdam : Humanwissenschaftliche Reihe - 693 KW - intervertebral disc degeneration KW - degenerative disc disease KW - osmolarity KW - hyper-osmolarity KW - hypo-osmolarity KW - osmotic KW - inflammatory KW - transient receptor potential channel KW - aquaporin KW - tonicity-responsive enhancer binding protein Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-469080 SN - 1866-8364 IS - 693 ER - TY - GEN A1 - Krupkova, Olga A1 - Smolders, Lucas A1 - Wuertz-Kozak, Karin A1 - Cook, James A1 - Pozzi, Antonio T1 - The pathobiology of the meniscus BT - a comparison between the human and dog T2 - Frontiers in Veterinary Science N2 - Serious knee pain and related disability have an annual prevalence of approximately 25% on those over the age of 55 years. As curative treatments for the common knee problems are not available to date, knee pathologies typically progress and often lead to osteoarthritis (OA). While the roles that the meniscus plays in knee biomechanics are well characterized, biological mechanisms underlying meniscus pathophysiology and roles in knee pain and OA progression are not fully clear. Experimental treatments for knee disorders that are successful in animal models often produce unsatisfactory results in humans due to species differences or the inability to fully replicate disease progression in experimental animals. The use of animals with spontaneous knee pathologies, such as dogs, can significantly help addressing this issue. As microscopic and macroscopic anatomy of the canine and human menisci are similar, spontaneous meniscal pathologies in canine patients are thought to be highly relevant for translational medicine. However, it is not clear whether the biomolecular mechanisms of pain, degradation of extracellular matrix, and inflammatory responses are species dependent. The aims of this review are (1) to provide an overview of the anatomy, physiology, and pathology of the human and canine meniscus, (2) to compare the known signaling pathways involved in spontaneous meniscus pathology between both species, and (3) to assess the relevance of dogs with spontaneous meniscal pathology as a translational model. Understanding these mechanisms in human and canine meniscus can help to advance diagnostic and therapeutic strategies for painful knee disorders and improve clinical decision making. T3 - Zweitveröffentlichungen der Universität Potsdam : Humanwissenschaftliche Reihe - 677 KW - meniscus KW - inflammation KW - oxidative stress KW - pain KW - dog Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-460868 SN - 1866-8364 IS - 677 ER - TY - JOUR A1 - Löpfe, Moira A1 - Duss, Anja A1 - Zafeiropoulou, Katerina-Alexandra A1 - Bjoergvinsdottir, Oddny A1 - Eglin, David A1 - Fortunato, Giuseppino A1 - Klasen, Jürgen A1 - Ferguson, Stephen J. A1 - Würtz-Kozak, Karin A1 - Krupkova, Olga T1 - Electrospray-Based Microencapsulation of Epigallocatechin 3-Gallate for Local Delivery into the Intervertebral Disc JF - Pharmaceutics N2 - Locally delivered anti-inflammatory compounds can restore the homeostasis of the degenerated intervertebral disc (IVD). With beneficial effects on IVD cells, epigallocatechin 3-gallate (EGCG) is a promising therapeutic candidate. However, EGCG is prone to rapid degradation and/or depletion. Therefore, the purpose of this study was to develop a method for controlled EGCG delivery in the degenerated IVD. Primary IVD cells were isolated from human donors undergoing IVD surgeries. EGCG was encapsulated into microparticles by electrospraying of glutaraldehyde-crosslinked gelatin. The resulting particles were characterized in terms of cytocompatibility and anti-inflammatory activity, and combined with a thermoresponsive carrier to produce an injectable EGCG delivery system. Subsequently, electrospraying was scaled up using the industrial NANOSPIDER (TM) technology. The produced EGCG microparticles reduced the expression of inflammatory (IL-6, IL-8, COX-2) and catabolic (MMP1, MMP3, MMP13) mediators in pro-inflammatory 3D cell cultures. Combining the EGCG microparticles with the carrier showed a trend towards modulating EGCG activity/release. Electrospray upscaling was achieved, leading to particles with homogenous spherical morphologies. In conclusion, electrospray-based encapsulation of EGCG resulted in cytocompatible microparticles that preserved the activity of EGCG and showed the potential to control EGCG release, thus favoring IVD health by downregulating local inflammation. Future studies will focus on further exploring the biological activity of the developed delivery system for potential clinical use. KW - degenerative disc disease KW - inflammation KW - drug delivery KW - EGCG KW - microparticles KW - injectable biomaterial KW - electrospraying Y1 - 2019 U6 - https://doi.org/10.3390/pharmaceutics11090435 SN - 1999-4923 VL - 11 IS - 9 PB - MDPI CY - Basel ER -