Ranjan Kc

Altered Spinal MicroRNA-146a and the MicroRNA-183 Cluster Contribute to Osteoarthritic Pain in Knee Joints

The objective of this study was to examine whether altered expression of microRNAs in central nervous system components is pathologically linked to chronic knee joint pain in osteoarthritis. A surgical animal model for knee joint OA was generated by medial meniscus transection in rats followed by behavioral pain tests. Relationships between pathological changes in knee joint and development of chronic joint pain were examined by histology and imaging analyses. Alterations in microRNAs associated with OA‐evoked pain sensation were determined in bilateral lumbar dorsal root ganglia (DRG) and the spinal dorsal horn by microRNA array followed by individual microRNA analyses. Gain‐ and loss‐of‐function studies of selected microRNAs (miR‐146a and miR‐183 cluster) were conducted to identify target pain mediators regulated by these selective microRNAs in glial cells. The ipsilateral hind leg displayed significantly increased hyperalgesia after 4 weeks of surgery, and sensitivity was sustained for the remainder of the 8‐week experimental period (F = 341, p < 0.001). The development of OA‐induced chronic pain was correlated with pathological changes in the knee joints as assessed by histological and imaging analyses. MicroRNA analyses showed that miR‐146a and the miR‐183 cluster were markedly reduced in the sensory neurons in DRG (L4/L5) and spinal cord from animals experiencing knee joint OA pain. The downregulation of miR‐146a and/or the miR‐183 cluster in the central compartments (DRG and spinal cord) are closely associated with the upregulation of inflammatory pain mediators. The corroboration between decreases in these signature microRNAs and their specific target pain mediators were further confirmed by gain‐ and loss‐of‐function analyses in glia, the major cellular component of the central nervous system (CNS). MicroRNA therapy using miR‐146a and the miR‐183 cluster could be powerful therapeutic intervention for OA in alleviating joint pain and concomitantly regenerating peripheral knee joint cartilage.

MicroRNA-146a reduces IL-1 dependent inflammatory responses in the intervertebral disc

Because miR-146a expression in articular chondrocytes is associated with osteoarthritis (OA), we assessed whether miR-146a is linked to cartilage degeneration in the spine. Monolayer cultures of nucleus pulposus (NP) cells from the intervertebral discs (IVD) of bovine tails were transfected with a miR-146a mimic. To provoke inflammatory responses and catabolic extracellular matrix (ECM) degradation, cells were co-treated with interleukin-1 (IL-1). Transfection of miR-146a decreases IL-1 induced mRNA levels of inflammatory genes and catabolic proteases in NP cells based on quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis. Similarly, miR146a suppresses IL-1 induced protein levels of matrix metalloproteinases and aggrecanases as revealed by immunoblotting. Disc segments from wild type (WT) and miR-146a knockout (KO) mice were cultured ex vivo in the presence or absence of IL-1 for 3days. Histological and immuno-histochemical (IHC) analyses of disc organ cultures revealed that IL-1 mediates changes in proteoglycan (PG) content and in-situ levels of catabolic proteins (MMP-13 and ADAMTS-5) in the nucleus pulposus of the disc. However, these IL-1 effects are more pronounced in miR-146a KO discs compared to WT discs. For example, absence of miR-146a increases the percentage of MMP-13 and ADAMTS-5 positive cells after treatment with IL-1. Thus, miR-146a appears to protect against IL-1 induced IVD degeneration and inflammation. Stimulation of endogenous miR-146a expression or exogenous delivery of miRNA-146a are viable therapeutic strategies that may decelerate disc degeneration and regain a normal homeostatic balance in extracellular matrix production and turn-over.

PAIN ASSESSMENT IN ANIMAL MODELS OF OSTEOARTHRITIS

Assessment of pain in animal models of osteoarthritis is integral to interpretation of a models utility in representing the clinical condition, and enabling accurate translational medicine. Here we describe behavioral pain assessments available for small and large experimental osteoarthritic pain animal models.

Environmental Disruption of Circadian Rhythm Predisposes Mice to Osteoarthritis-Like Changes in Knee Joint

Circadian rhythm dysfunction is linked to many diseases, yet pathophysiological roles in articular cartilage homeostasis and degenerative joint disease including osteoarthritis (OA) remains to be investigated in vivo. Here, we tested whether environmental or genetic disruption of circadian homeostasis predisposes to OA‐like pathological changes. Male mice were examined for circadian locomotor activity upon changes in the light:dark (LD) cycle or genetic disruption of circadian rhythms. Wild‐type (WT) mice were maintained on a constant 12 h:12 h LD cycle (12:12 LD) or exposed to weekly 12 h phase shifts. Alternatively, male circadian mutant mice (ClockΔ19 or Csnk1etau mutants) were compared with age‐matched WT littermates that were maintained on a constant 12:12 LD cycle. Disruption of circadian rhythms promoted osteoarthritic changes by suppressing proteoglycan accumulation, upregulating matrix‐degrading enzymes and downregulating anabolic mediators in the mouse knee joint. Mechanistically, these effects involved activation of the PKCδ‐ERK‐RUNX2/NFκB and β‐catenin signaling pathways, stimulation of MMP‐13 and ADAMTS‐5, as well as suppression of the anabolic mediators SOX9 and TIMP‐3 in articular chondrocytes of phase‐shifted mice. Genetic disruption of circadian homeostasis does not predispose to OA‐like pathological changes in joints. Our results, for the first time, provide compelling in vivo evidence that environmental disruption of circadian rhythms is a risk factor for the development of OA‐like pathological changes in the mouse knee joint. J. Cell. Physiol. 230: 2174–2183, 2015.

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.

PKCδ null mutations in a mouse model of osteoarthritis alter osteoarthritic pain independently of joint pathology by augmenting NGF/TrkA-induced axonal outgrowth

Objectives A key clinical paradox in osteoarthritis (OA), a prevalent age-related joint disorder characterised by cartilage degeneration and debilitating pain, is that the severity of joint pain does not strictly correlate with radiographic and histological defects in joint tissues. Here, we determined whether protein kinase Cδ (PKCδ), a key mediator of cartilage degeneration, is critical to the mechanism by which OA develops from an asymptomatic joint-degenerative condition to a painful disease. Methods OA was induced in 10-week-old PKCδ null (PKCδ−/−) and wild-type mice by destabilisation of the medial meniscus (DMM) followed by comprehensive examination of the histology, molecular pathways and knee-pain-related-behaviours in mice, and comparisons with human biopsies. Results In the DMM model, the loss of PKCδ expression prevented cartilage degeneration but exacerbated OA-associated hyperalgesia. Cartilage preservation corresponded with reduced levels of inflammatory cytokines and of cartilage-degrading enzymes in the joints of PKCδ-deficient DMM mice. Hyperalgesia was associated with stimulation of nerve growth factor (NGF) by fibroblast-like synovial cells and with increased synovial angiogenesis. Results from tissue specimens of patients with symptomatic OA strikingly resembled our findings from the OA animal model. In PKCδ null mice, increases in sensory neuron distribution in knee OA synovium and activation of the NGF-tropomyosin receptor kinase (TrkA) axis in innervating dorsal root ganglia were highly correlated with knee OA hyperalgesia. Conclusions Increased distribution of synovial sensory neurons in the joints, and augmentation of NGF/TrkA signalling, causes OA hyperalgesia independently of cartilage preservation.

Osteoarthritis-like pathologic changes in the knee joint induced by environmental disruption of circadian rhythms is potentiated by a high-fat diet

A variety of environmental factors contribute to progressive development of osteoarthritis (OA). Environmental factors that upset circadian rhythms have been linked to various diseases. Our recent work establishes chronic environmental circadian disruption - analogous to rotating shiftwork-associated disruption of circadian rhythms in humans - as a novel risk factor for the development of OA. Evidence suggests shift workers are prone to obesity and also show altered eating habits (i.e., increased preference for high-fat containing food). In the present study, we investigated the impact of chronic circadian rhythm disruption in combination with a high-fat diet (HFD) on progression of OA in a mouse model. Our study demonstrates that when mice with chronically circadian rhythms were fed a HFD, there was a significant proteoglycan (PG) loss and fibrillation in knee joint as well as increased activation of the expression of the catabolic mediators involved in cartilage homeostasis. Our results, for the first time, provide the evidence that environmental disruption of circadian rhythms plus HFD potentiate OA-like pathological changes in the mouse joints. Thus, our findings may open new perspectives on the interactions of chronic circadian rhythms disruption with diet in the development of OA and may have potential clinical implications.

Bovine Lactoferricin-induced Anti-inflammation Is, in Part, via Up-regulation of Interleukin-11 by Secondary Activation of STAT3 in Human Articular Cartilage

Background: Bovine lactoferricin (LfcinB) promotes anti-catabolism and anti-inflammation in articular cartilage. Results: LfcinB induces IL-11 via AP-1, which in turn induces TIMP-1 via STAT3. Conclusion: LfcinB sequentially regulates IL-11 and TIMP-1 expression through distinct mechanisms in articular chondrocytes. Significance: These findings further suggest the potential of LfcinB as a novel therapeutic agent in osteoarthritis. Bovine lactoferricin (LfcinB), a multifunctional peptide, was recently demonstrated to be anti-catabolic and anti-inflammatory in human articular cartilage. LfcinB blocks IL-1-mediated proteoglycan depletion, matrix-degrading enzyme expression, and pro-inflammatory mediator induction. LfcinB selectively activates ERK1/2, p38 (but not JNK), and Akt signaling. However, the relationship between these pathways and LfcinB target genes has never been explored. In this study, we uncovered the remarkable ability of LfcinB in the induction of an anti-inflammatory cytokine, IL-11. LfcinB binds to cell surface heparan sulfate to initiate ERK1/2 signaling and activate AP-1 complexes composed of c-Fos and JunD, which transactivate the IL-11 gene. The induced IL-11 functions as an anti-inflammatory and chondroprotective cytokine in articular chondrocytes. Our data show that IL-11 directly attenuates IL-1-mediated catabolic and inflammatory processes ex vivo and in vitro. Moreover, IL-11 activates STAT3 signaling pathway to critically up-regulate TIMP-1 expression, as a consecutive secondary cellular response after IL-11 induction by LfcinB-ERK-AP-1 axis in human adult articular chondrocytes. The pathological relevance of IL-11 signaling to osteoarthritis is evidenced by significant down-regulation of its cognate receptor expression in osteoarthritic chondrocytes. Together, our results suggest a two-step mechanism, whereby LfcinB induces TIMP-1 through an IL-11-dependent pathway involving transcription factor AP-1 and STAT3.

Induction of Osteoarthritis‐like Pathologic Changes by Chronic Alcohol Consumption in an Experimental Mouse Model

Osteoarthritis (OA) is characterized by slow and progressive deterioration of articular cartilage. OA likely arises from a combination of systemic factors (genetics, age, environment) and local factors (abnormal joint loading, overuse, or trauma) working in concert to create a condition with definable morphologic and clinical characteristics. Several risk factors for OA have been identified previously, including genetic predisposition, obesity, diabetes, hypertension, hyperuricemia, history of trauma, and aging (1). However, due in large part to an inability to control for confounding factors, the underlying pathogenesis and causative features responsible for initiation and progression of the disease remain largely unknown. There is a growing body of evidence indicating that progression of OA is correlated with up-regulation of inflammatory processes (2). Oxidative stress elicited by reactive oxygen species (ROS) further disturbs cartilage homeostasis and promotes catabolism via induction of cell death, breakdown of matrix proteoglycans (PGs), up-regulation of latent matrixdegrading enzyme production, inhibition of extracellular matrix synthesis, and oxidation of intracellular and extracellular molecules (3). Thus, environmental factors that promote oxidative stress and inflammatory states could potentially act as a risk factor for OA. Alcohol consumption could be one potential risk factor, because 1) chronic alcohol consumption, highly common in Western and industrial societies, generates ROS, leading to systemic and tissue oxidative stress in humans and rodents (4), and 2) alcohol is capable of inducing proinflammatory states in multiple organs, e.g., the liver, heart, central nervous system, and pancreas (4,5). There have been several previous studies in which investigators attempted to elucidate a relationship between alcohol consumption and inflammatory arthritis such as rheumatoid arthritis, with conflicting results (6,7). However, despite recent evidence demonstrating the importance of oxidative stress and proinflammatory states in the development and progression of degenerative joint disease (8), the impact of alcohol consumption on OA has not yet been studied. In the present study, we obtained evidence suggesting that chronic alcohol exposure may increase susceptibility to the development and/or progression of OA. Using a validated in vivo model of chronic alcohol treatment, we found that chronic alcohol consumption increases PG loss in both the knee and the shoulder joints of mice and stimulates multiple inflammatory, catabolic, and antianabolic mediators involved in cartilage. In our experimental protocol, young adult (ages 7–9 weeks) male C57BL/6 mice were provided ad libitum access to an alcohol diet (i.e., the Nanji diet), containing 4.5% (volume/volume) ethanol (29% ethanol-derived calories) or an isocaloric alcohol-free control diet for 8 weeks (n 5 14–16 per group). All animal protocols and practices were reviewed and approved in advance by the Institutional Animal Care and Use Committees at Rush University and Northwestern University. Following 8 weeks on the alcohol-containing diet or the control diet, mice were killed and joint sections were collected, fixed, paraffin embedded, and stained with Safranin O to assess cartilage structure and matrix PG content. The alcohol diet used in this study is a slight modification of the wellvalidated Lieber-DeCarli diet (9) in which the fat source comes from fish oil, and consumption of this diet by BL6 mice has been shown to produce blood alcohol levels that are in the low-to-moderate range (10). Our group and others have successfully used this alcohol diet to induce a variety of alcohol-related pathologies, including colon cancer, intestinal hyperpermeability, endotoxemia, and liver disease, in rodents (10–12). Serum alcohol levels at the time of death in the alcohol-fed mice were ;3 mg/dl, and these mice did not exhibit any overt behavioral abnormalities during the experimental protocol. Histologic examination of knee joints of control diet–fed mice demonstrated normal architecture of articular cartilage, with intense Safranin O staining. In contrast, knee joints of alcohol-fed mice displayed OA-like characteristics, with increased PG loss as indicated by reduced Safranin O staining and mild fibrillation (Figure 1A). These results were quantified using the Osteoarthritis Research Society International (OARSI) semiquantitative scoring system (13). Scores were significantly higher in alcohol-fed mice than in control mice (mean 6 SD 1.3 6 0.67 versus 0.3 6 0.27; P , 0.05) (Figure 1A), indicating more severe arthritic changes in the knee joints of alcohol-fed mice. Similar results were observed in shoulder joints (Figure 1B), with decreased PG content as shown by Safranin O staining, and an irregular cartilage surface, in the shoulders of alcohol-fed mice. As in the knee, pathologic changes in the shoulder, quantified using the OARSI scoring system, were significantly greater in alcohol-fed mice compared to control mice (0.75 6 0.28 versus 0.12 6 0.25; P , 0.05) (Figure 1B). Interestingly, the intervertebral discs of alcohol-fed mice exhibited no pathologic changes compared to control mice (Figure 1C). Our results demonstrate a pathologic effect of alcohol on specific catabolic and antianabolic mediators in the knee joints, which may increase susceptibility to OA development. We found that levels of the phosphorylated forms of protein kinase Cd (PKCd), NF-kB, and ERK-1/2 were significantly increased in the knee joints of alcohol-fed mice compared to control mice (P , 0.05) (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary. wiley.com/doi/10.1002/art.39090/abstract), suggesting that chronic alcohol consumption stimulates these catabolic signaling pathways, which may result in subsequent production of cartilagedestructive enzymes. Levels of the hypertrophic marker RUNX-2, as well as the key cartilage-destructive enzymes matrix metalloprotease 13 and ADAMTS-5, were also significantly increased in the knee joints of alcohol-fed mice (P , 0.05) (Supplementary Figure 1).

Vascular Endothelial Growth Factor in Cartilage Development and Osteoarthritis.

Genome wide studies indicate that vascular endothelial growth factor A (VEGF) is associated with osteoarthritis (OA), and increased VEGF expression correlates with increased disease severity. VEGF is also a chondrocyte survival factor during development and essential for bone formation, skeletal growth and postnatal homeostasis. This raises questions of how the important embryonic and postnatal functions of VEGF can be reconciled with an apparently destructive role in OA. Addressing these questions, we find that VEGF acts as a survival factor in growth plate chondrocytes during development but only up until a few weeks after birth in mice. It is also required for postnatal differentiation of articular chondrocytes and the timely ossification of bones in joint regions. In surgically induced knee OA in mice, a model of post-traumatic OA in humans, increased expression of VEGF is associated with catabolic processes in chondrocytes and synovial cells. Conditional knock-down of Vegf attenuates induced OA. Intra-articular anti-VEGF antibodies suppress OA progression, reduce levels of phosphorylated VEGFR2 in articular chondrocytes and synovial cells and reduce levels of phosphorylated VEGFR1 in dorsal root ganglia. Finally, oral administration of the VEGFR2 kinase inhibitor Vandetanib attenuates OA progression.