Amanda Villalvilla

TLR4 signalling in osteoarthritis--finding targets for candidate DMOADs.

Osteoarthritis (OA), the most common rheumatic disease, is characterized by joint-space narrowing due to progressive cartilage degradation and alterations in subchondral bone and the synovial membrane. These articular disturbances can have severe consequences, including pain, disability and loss of joint architectural integrity. Although the aetiology of OA is not understood, chondrocyte-mediated inflammatory responses triggered by the activation of innate immune receptors by damage-associated molecules are thought to be involved. In this Review, we examine the relationship between Toll-like receptor 4 (TLR4) and OA in cartilage as well as in other OA-affected tissues, such as subchondral bone and synovium. We also discuss the different TLR4 agonists associated with OA and their effects in joint tissues. Finally, we describe existing and novel strategies that might be used to develop TLR4-specific disease-modifying OA drugs (DMOADs).

Lipid Transport and Metabolism in Healthy and Osteoarthritic Cartilage

Cartilage is an avascular tissue and cartilage metabolism depends on molecule diffusion from synovial fluid and subchondral bone. Thus, nutrient availability is limited by matrix permeability according to the size and charge of the molecules. Matrix composition limits the access of molecules to chondrocytes, determining cell metabolism and cartilage maintenance. Lipids are important nutrients in chondrocyte metabolism and are available for these cells through de novo synthesis but also through diffusion from surrounding tissues. Cartilage status and osteoarthritis development depend on lipid availability. This paper reviews lipid transport and metabolism in cartilage. We also analyze signalling pathways directly mediated by lipids and those that involve mTOR pathways, both in normal and osteoarthritic cartilage.

SDF-1 signaling: a promising target in rheumatic diseases

Introduction: Stromal cell-derived factor 1 (SDF-1) is a potent chemoattractant cytokine with various biological functions such as stem cell mobilization, inflammatory cell infiltration and angiogenesis. Therefore, it has also been implicated in several pathological processes, from ischemic conditions to cancer. Remarkably, SDF-1 and its receptors, CXCR4 and CXCR7, are also present in joint tissues, where they play a role in the pathogenesis of rheumatoid arthritis (RA) and osteoarthritis (OA). Areas covered: This review summarizes the physiological and pathological role of SDF-1 signaling and its involvement in RA and OA. That includes synovial inflammation, bone erosion, cartilage degradation and increased bone turnover. Although this cytokine could play different roles in these rheumatic diseases, specific and differentiated therapeutic targets in each process can be identified. Current therapeutic strategies to block SDF-1 signaling in several diseases and their possible use in rheumatic diseases are also discussed. Expert opinion: Emerging drugs that block CXCR4 or CXCR7 in different disorders may represent promising therapies for rheumatic disease via inhibition of key pathological events involved in the progression of RA and OA.

Selective estrogen receptor modulators (SERMs): New alternatives for osteoarthritis?

The dramatic rise in the prevalence rate of osteoarthritis (OA) after the menopause and the presence of estrogen receptors in joint tissues suggest that estrogen may help protect against the development of OA. Trials of estrogen therapy have produced inconclusive results, however, partly because of flaws in study design and partly because of the complexity of the mechanisms underlying estrogens effects on joint tissues. Initial studies of the use of selective estrogen receptor modulators (SERMs) have reported beneficial effects in OA. These agents may exert both a direct effect upon joint cartilage and indirect effects on subchondral bone, synovium, muscle, tendons and ligaments. SERMs may be particularly beneficial for postmenopausal patients with osteoporotic OA, a phenotype defined by decreased bone density, associated with high remodeling in subchondral bone. More research is needed, though, before SERMs can become a therapeutic option for OA.

6-Shogaol inhibits chondrocytes’ innate immune responses and cathepsin-K activity

SCOPE Ginger has long been used in traditional Asian medicine to treat osteoarthritis. Indeed, scientific research has reported that ginger derivatives (GDs) have the potential to control innate immune responses. Given the widespread use and demonstrated properties of GDs, we set out to study their anti-inflammatory and anticatabolic properties in chondrocytes. METHODS AND RESULTS 6-shogaol (6-S), the most active GD, was obtained from ginger. 6-S was not toxic as measured by MTT assay, and inhibited NO production and IL-6 and MCP-1 induced gene expression in LPSbut not in IL-1β-stimulated chondrocytes. 6-S also inhibited LPS-mediated ERK1/2 activation as well as NOS2 and MyD88 induced expression as determined by Western blot. Moreover, zymography revealed that 6-S inhibited matrix metalloproteinases (MMP) 2/9 induction in LPS-treated cells. Hydrated 6-S was modified to obtain a compound (SSi6) without 6-S potential anti-inflammatory properties. Both 6-S and SSi6 inhibited cathepsin-K activity. CONCLUSION 6-S blocked TLR4-mediated innate immune responses and MMP induction in chondrocytes. These results, together with GDs-mediated cathepsin-K inhibition, suggest the potential for GDs use against cartilage and bone degradation. Therefore, considering that clinical trials involving oral administration of ginger achieved relevant nontoxic GDs serum concentrations, we suggest that a ginger-supplemented diet might reduce OA symptoms.

The adipokine lipocalin-2 in the context of the osteoarthritic osteochondral junction

Obesity and osteoarthritis (OA) form a vicious circle in which obesity contributes to cartilage destruction in OA, and OA-associated sedentary behaviour promotes weight gain. Lipocalin-2 (LCN2), a novel adipokine with catabolic activities in OA joints, contributes to the obesity and OA pathologies and is associated with other OA risk factors. LCN2 is highly induced in osteoblasts in the absence of mechanical loading, but its role in osteoblast metabolism is unclear. Therefore, because osteochondral junctions play a major role in OA development, we investigated the expression and role of LCN2 in osteoblasts and chondrocytes in the OA osteochondral junction environment. Our results showed that LCN2 expression in human osteoblasts and chondrocytes decreased throughout osteoblast differentiation and was induced by catabolic and inflammatory factors; however, TGF-β1 and IGF-1 reversed this induction. LCN2 reduced osteoblast viability in the presence of iron and enhanced the activity of MMP-9 released by osteoblasts. Moreover, pre-stimulated human osteoblasts induced LCN2 expression in human chondrocytes, but the inverse was not observed. Thus, LCN2 is an important catabolic adipokine in osteoblast and chondrocyte metabolism that is regulated by differentiation, inflammation and catabolic and anabolic stimuli, and LCN2 expression in chondrocytes is regulated in a paracrine manner after osteoblast stimulation.

Compensatory anabolic signaling in the sarcopenia of experimental chronic arthritis

Inflammatory activity in rheumatoid arthritis may alter the regulation of muscle mass leading to a secondary sarcopenia, commonly termed rheumatoid cachexia (RC). We characterized alterations to muscle structure and various pro-inflammatory, catabolic and regenerative markers in an animal model of RC. Antigen induced arthritis (AiA) was performed in 20 male adult rabbits. AiA animals exhibited significantly less weight gain, a markedly elevated serum C-reactive protein (CRP), lighter muscles with shorter cross-sectional diameter and increased myonuclei when compared to controls. Atrogin-1 and MuRF-1 were up-regulated alongside an increase in IL-1β, active NF-κB and a higher ratio of phosphorylated to inactive p38 MAPK. CCL-2 and TNF levels were reduced and IL-6 was unchanged between groups. We observed decreased pSTAT3, unchanged pSTAT1 and Myf5, but increased Pax7, MyoD and myogenin. AiA rabbits had a reduction in myostatin from gastrocnemii and synovium with a congruent decrease in serum myostatin compared to controls. Chronic arthritis induced an RC-like secondary sarcopenia with increased muscle protein breakdown. Elevated IL-1β may trigger proteolysis via elevated NF-κB and p38 MAPK signaling with a compensatory anabolic response suggested by myonuclear expansion, increased Pax7, MyoD and myogenin, reduced pSTAT3 as well as reduced serum, synovial and muscular myostatin.

Corrigendum: The adipokine lipocalin-2 in the context of the osteoarthritic osteochondral junction.

Scientific Reports 6: Article number: 2924310.1038/srep29243; published online: July 07 2016; updated: August 19 2016.

Aromatase expression in human chondrocytes: An induction due to culture.

OBJECTIVES Despite the high prevalence of osteoarthritis (OA) in postmenopausal women, a relationship between circulating estrogen levels and the development of OA has not been found. Therefore, the purpose of this study was to evaluate the expression and activity of aromatase, a key enzyme in local production of estrogens, in human OA cultured articular chondrocytes, and to determine the physiological relevance of this enzyme in cartilage. METHODS Human OA articular chondrocytes were isolated and cultured. Local production of estradiol was measured after incubation with 100 ng/ml testosterone for 8 and 24h. Furthermore, chondrocytes were culture for 2h, 48 h, 7 days or 15 days, or in alginate beads for 10 days. Aromatase, type II and X collagen, aggrecan, alkaline phosphatase, and Runx2 expression were evaluated in cartilage, freshly isolated chondrocytes and cultured chondrocytes. RESULTS Aromatase was expressed and active in cultured human chondrocytes. Human cartilage, freshly isolated chondrocytes, and chondrocytes cultured for 2h expressed an insignificant amount of aromatase; however, expression arose after 48 h of culture and remained increased thereafter. Aromatase expression was not related to estrogen deprivation and was inversely correlated with differentiation. Re-differentiation did not reduce its expression. CONCLUSIONS Aromatase presents an almost undetectable expression in human cartilage but is induced in cultured chondrocytes. Therefore, human cartilage might act as a mere target for estrogens rather than a producer, and researchers using cell expansion in culture for latter therapies should consider these changes in estrogen metabolism which may not be reverted after re-differentiation.

OP0077 Muscle Alterations in an Experimental Model of Chronic Arthritis

Background Rheumatoid arthritis (RA) is an inflammatory arthropathy with a range of additional systemic consequences. Rheumatoid cachexia (RC), the condition of decreased muscle mass with stable or increased fat mass, is one such phenomenon. Muscle atrophy in RC appears to be driven by circulating pro-inflammatory cytokines1. However, alterations to pro-inflammatory gene expression within muscle may also contribute to the wasting process. Objectives Characterise alterations to muscle structure and gene expression in an animal model of chronic inflammatory arthritis. Methods Nineteen male, New Zealand white rabbits were randomly assigned to two groups: control (n=12) and antigen-induced arthritis (AiA; n=7). Chronic arthritis was induced over six weeks via intra-dermal ovalbumin sensitization and four subsequent intra-articular injections. Nineteen gastrocnemius muscles were collected for gene expression studies and 10 tibialis anterior muscles (control n=6; AiA n=4) were collected for both gene expression and histological studies. IL-1β, IL-6, TNF, CCL-2 (MCP-1), myostatin and two atrogenes, MuRF-1 and atrogin-1, were quantified using real-time PCR. Tibialis anterior mid-belly cross-sections were stained with haematoxylin & eosin and for ATPase activity to measure whole muscle cross-sectional diameter (CSD) and type II fibre CSD, respectively. Rabbits were weighed weekly and serum CRP was measured at week 6. Group comparisons were performed using Mann-Whitney tests and associations were tested using Spearman correlation analyses. Results AiA rabbits exhibited significantly less weight gain than controls, with a maximum cumulative difference of -160g vs. +570g (p<0.0001) and a 74-fold increase in serum CRP (p=0.0003). Tibialis anterior of AiA rabbits showed a 16% reduction in type II myofibre CSD (p=0.0159) and a 27% reduction in whole muscle CSD (p=0.0095) alongside a 11-fold increase in IL-1β (p=0.0095), a 21-fold increase in IL-6 (p=0.0381) and a 14-fold increase in CCL-2 mRNA (p=0.0095). Gastrocnemii of AiA rabbits demonstrated similar, yet less pronounced inflammatory activity with a 3-fold increase in IL-1β (p=0.0012), and a 5-fold increase in both IL-6 (p=0.0221) and CCL-2 (p=0.0018) alongside a 3-fold and 2-fold increase in the atrogenes MuRF-1 (p=0.0283) and atrogin-1 (p=0.0130), respectively. Conversely, tibialis anterior and gastrocnemius muscles of AiA rabbits expressed 78% (p=0.0095) and 70% (p=0.0097) less myostatin with no alteration in TNF mRNA. Correlation analyses revealed that tibialis anterior whole muscle CSD and type II myofibre CSD were inversely associated with the expression of pro-inflammatory, muscle-derived cytokines. Conclusions Our model of chronic arthritis induced an RC-like state with reduced animal weight and muscle size, an up-regulation of atrogene expression, indicating muscle protein breakdown, and a parodixcal decrease in myostatin. Arthritis also up-regulated serum CRP and appeared to activate an inflammatory phenotype in skeletal muscle. We propose that inflamed muscle tissue may contribute to the process of RC via a mechanism of autocrine atrophy triggered by increased secretion of muscle-derived, pro-inflammatory mediators. References Summers GD, Metsios GS, Stavropoulos-Kalinoglou A, Kitas GD. Rheumatoid cachexia and cardiovascular disease. Nature Publishing Group. 2010 Aug;6(8):445–51. Disclosure of Interest None declared