Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis
© de Munter et al.; licensee BioMed Central Ltd. 2013
Received: 16 July 2013
Accepted: 10 October 2013
Published: 4 November 2013
Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.
LDL receptor deficient (LDLr−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.
A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr−/− mice). Synovial wash-outs of LDLr−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.
LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.
Osteoarthritis (OA) is a common disease of unknown etiology. The association of OA with metabolic syndrome has long been established but the exact mechanism remains unclear [1, 2]. The idea that obesity enhances OA development solely due to increased loading  is obsolete and more often studies show the association between obesity and OA development in non-weight-bearing joints [4–7].
Decreased levels of high-density lipoprotein (HDL) and increased levels of low-density lipoprotein (LDL) particles are, amongst other features, part of the metabolic syndrome . In a comparative analysis of serological parameters, several studies demonstrated that OA patients have significantly higher serum levels of LDL compared to healthy controls [9, 10]. Studies focusing on cardiovascular diseases, such as atherosclerosis, show pro-inflammatory capacities of LDL and modified LDL [11, 12]. LDL particles form the main transport vehicle of cholesterol from the liver to the tissues. LDL can be oxidized in an inflammatory milieu and, therefore, high levels of LDL result in enhanced oxidized LDL (oxLDL) levels in pathological conditions where free radicals are present [13, 14]. OxLDL is taken up by macrophages via scavenger receptor class A, B (CD36) and E (lectin-like oxLDL receptor-1; LOX-1), resulting in a phenotype shift into a more inflammatory cell type [15–19].
A substantial population of OA patients develops a thickened lining layer comprising macrophages that exhibit an activated phenotype. Macrophages derived from biopsies with early OA produce elevated amounts of pro-inflammatory mediators . Depletion of macrophages from OA synovium using anti-CD14–conjugated magnetic beads led to decreased levels of TNF-α, IL-1β, IL-6 and IL-8 . In previous studies we have shown that synovial macrophages are crucial in the development of joint pathology in experimental OA. Selective depletion of lining macrophages using the clodronate-suicide technique prior to induction of collagenase-induced OA strongly inhibited development of cartilage destruction and osteophyte formation, probably regulated by a strong decrease in metalloproteinase (MMP)-3 and −9 expression .
Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMP) are important growth factors involved in the formation of new cartilage or bone in ligaments (enthesophyte formation) or along the bone surface (ectopic bone formation or osteophyte formation) . In previous studies we showed that multiple injections of members of the TGF-super family, such as TGF-β or BMP-2, directly into the knee joint of the mouse caused abundant enthesophyte/osteophyte formation [24, 25]. Moreover, we postulated that local depletion of synovial macrophages prior to injections of these growth factors significantly inhibited new formation of cartilage/bone, suggesting that macrophage factors highly contribute to this process [26, 27].
The presence of high levels of LDL in OA joints with an enhanced inflammatory environment may lead to uptake of oxLDL by synovial lining macrophages, thereby contributing to development of OA pathology. LDL receptor deficient (LDLr−/−) mice, which are generally used in atherosclerotic research , are unable to clear and metabolize cholesterol-rich intermediate and low density lipoproteins, causing hypercholesterolemia that can be enhanced by a cholesterol-rich diet . In this study, we investigated the effect of increased serum LDL levels on OA development in experimental collagenase-induced OA. We focused on synovial thickening/activation, cartilage damage and enthesophyte/osteophyte formation in both LDLr−/− mice and mice receiving a cholesterol-rich diet.
Female mice homozygous for the Ldlr tm1Her mutation (LDLr−/−) and their wild type (WT) control C57BL/6 J were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were 10 to 13 weeks old when used in the experiments, were housed in filter-top cages and received food and water ad libitum. Animal studies were approved by the Institutional Review Board (Animal Experiment Committee Radboud University Medical Center) and were performed according to the related codes of practice.
Mice were fed a standard diet or a cholesterol-rich diet (+0.15% cholesterol; AB Diets, Woerden, the Netherlands). Instability OA was induced by intra-articular injection of 1 unit bacterial collagenase (Sigma-Aldrich, St. Louis, MO, USA) into the right knee on days 84 and 86 after start of the diet . On day 120, mice were weighed and sacrificed, after which both left and right knee joints were isolated and processed for histological analysis. Serum samples were obtained to determine cholesterol levels.
Histological and immunohistochemical preparation
Isolated knee joints were fixed in 4% buffered formalin and subsequently decalcified in formic acid and embedded in paraffin. Eight representative sections (7 μm) of each joint from various depths were stained with H & E or Safranin O-fast green for histological analysis. For immunohistochemistry, sections were deparaffinized and endogenous peroxidase activity was blocked using 1% H2O2 in methanol. Next, sections were incubated in buffered citrate (pH 6.0) for antigen retrieval. Incubation with 0.1% Triton X-100 was used for cell membrane permeabilization, followed by incubation with rabbit anti-mouse apolipoprotein B (ApoB; Abcam, Cambridge, UK), rabbit anti-mouse S100A8 (kindly provided by Dr. Vogl, Institute of Immunology, University of Muenster, Germany), or normal rabbit immunoglobulin G (IgG) (R&D Systems, Minneapolis, MN, USA) as control. Subsequently, sections were incubated with the secondary antibody, biotinylated goat anti-rabbit IgG, and binding was detected using the ABC-HRP kit (Elite kit; Vector Laboratories, Burlingame, CA, USA). Peroxidase was developed with diaminobenzidine (DAB; Sigma-Aldrich) and sections were counter-stained with hematoxylin. Sections were randomly coded and scored in a blinded way by two independent investigators.
Synovial thickening and immunohistochemical staining were measured using an arbitrary scoring system (0 to 3, where 0 = no thickening/staining and 3 = most observed thickening/staining). Three sections of various depths were scored per knee joint.
Cartilage damage in the tibial-femoral joint was scored using the OA score developed by Pritzker et al., adapted by us for mice (from 0 = no damage, to 30 = maximal damage) . Five sections were scored per knee joint.
Size of ectopic bone formation was measured by the use of image analysis (Leica Qwin, Leica Microsystems, Rijswijk, The Netherlands). Osteophytes and enthesophytes were manually circled by an investigator blinded for the experimental condition, after which the surface areas were calculated by Leica-software. The mean cross-sectional surface area in three sections per knee joint was determined, including knee joints without ectopic bone formation.
Synovium was isolated from murine knee joints 7 and 36 days after collagenase injection. Tissue was weighed and put in Roswell Park Memorial Institute medium (RPMI; Gibco, Invitrogen, Carlsbad, CA, USA), enriched with 48 μg/mL gentamicin (Centrafarm, Etten-Leur, The Netherlands) and 0.1% BSA (Sigma-Aldrich) for one hour at room temperature.
Bone marrow derived macrophages
Macrophages were differentiated out of bone marrow cells using 15 ng/mL recombinant murine macrophage colony stimulating factor (M-CSF; R&D Systems). For this, tibiae from WT or LDLr−/− mice were removed and bone marrow cells were flushed with (D)MEM; Gibco) supplemented with 10% FCS (Thermo Scientific, Waltham, MA, USA), 1 mM pyruvate (Gibco) and 48 μg/mL gentamicin.
Oxidized LDL preparation and macrophage stimulation
OxLDL was prepared from a large batch of LDL which was first isolated by single-spin density-gradient ultracentrifugation from ethylenediaminetetraacetic acid (EDTA)-treated blood donated by healthy volunteers and frozen in 10 mM phosphate buffer (pH 7.4) containing 0.9% NaCl, 10% (w/v) saccharose and 0.1 mM EDTA. LDL was thawed and dialyzed for seven hours against saline in a Slide-A-Lyzer cassette (Pierce Chemical Company, Rockford, IL, USA). Subsequently, LDL was oxidized with 0.5 mg/mL copper sulfate (Merck, Darmstadt, Germany) for 24 hours at room temperature, after which oxLDL was dialyzed again for one hour . OxLDL concentration was determined by the use of a bicinchoninic acid assay (Thermo Scientific) and a Sunrise microplate reader (Tecan Group, Männedorf, Switzerland). OxLDL was pre-incubated for 10 minutes with 10 μg/mL polymixine B sulfate (Sigma-Aldrich) before experimental use in order to rule out lipopolysaccharide (LPS) interference. Macrophages were stimulated with 50 μg/mL oxLDL, or an equal volume of saline (also pre-incubated with 10 μg/mL polymixine B sulfate) for 24 or 48 hours at 37°C. Stimulations were performed in 5% non heat-inactivated FCS. Supernatant was used for functional TGF-β and BMP analyses and cells were lysed in 500 μL TRI-reagent (Sigma-Aldrich) for quantitative detection of messenger RNA levels. All reactions were performed in quadruple.
Quantitative real-time polymerase chain reaction
RNA was extracted using the single step RNA isolation method described by Chomczynski and Sacchi . Isopropanol (Merck) was used for precipitation, after which the RNA pellet was washed twice with 75% ethanol. The pellet was reconstituted in RNAse free water and subsequently treated with DNase (Invitrogen). RNA was reversed transcribed into complementary DNA (cDNA) using reverse transcriptase, oligo(dT) primers and dNTPs (Invitrogen).
Quantitative real-time PCR (qPCR) was performed using StepOnePlus Real-Time PCR system and StepOne software v2.2 (Applied Biosystems, Foster City, CA, USA), under the following conditions: 10 minutes 95°C, followed by 40 cycles of 15 seconds 95°C and 1 minute 60°C. Data were collected during the last 30 seconds of each cycle. Product specificity was confirmed by assessment of dissociation-characteristics. The reaction was performed in a total volume of 10 μL, containing 3 μL diluted cDNA, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM) and 5 μL SYBR Green Master Mix (Applied Biosystems). The following primers were designed with Primer Express 2.0 (Applied Biosystems) and manufactured by Biolegio (Nijmegen, the Netherlands): GAPDH: 5′-GGCAAATTCAACGGCACA-3′ (forward) and 5′-GTTAGTGGGGTCTCGCTCCTG-3′ (reverse); IL-12p40: 5′-AGCTAACCATCTCCTGGTTTGC-3′ (forward) and 5′-CCACCTCTACAACATAAACGTCTTTC-3′ (reverse); TGF-β1: 5′-GCAGTGGCTGAACCAAGGA-′3 (forward) and 5′-AAGAGCAGTGAGCGCTGAATC-3′ (reverse); BMP2: 5′-CGCAGCTTCCATCAC-3′ (forward) and 5′-GCCGGGCCGTTTTCC-3′ (reverse); BMP4: 5′-CCGCTTCTGCAGGAACCA-3′ (forward) and 5′-AGTGCGTCGCTCCGAATG-3′ (reverse); BMP7: 5′-ACGGACAGGGCTTCTCCT-3′ (forward) and 5′-ATGGTGGTATCGAGGGTG-3′ (reverse). Relative expression levels were presented as ΔΔCt, being the threshold cycle (Ct) value corrected for GAPDH and unstimulated control.
CAGA-Luc and BRE-Luc reporter constructs
3T3 cells (ATCC, Manassas, VA, USA) were transduced with a CAGA-Luciferase reporter plasmid (CAGA-Luc) or a BMP responsive-luciferace reporter plasmid (BRE-Luc) for two and a half hours with a multiplicity of infection of 10. Both plasmids were kindly provided by Dr. Ten Dijke (Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands). After 24 hours, the transduction efficiency of CAGA-Luc was checked with fluorescence microscopy, confirming the presence of GFP that is constitutively active in this construct. The transduced cells were then stimulated overnight with macrophage supernatant or synovial wash-outs under serum-free conditions, after which luminescence was measured.
Statistical differences between two values were calculated using a student’s t-test or Mann Whitney U test depending on Gaussian distribution. Statistical differences between more than two values were tested using a one-way analysis of variance (ANOVA) or Kruskal-Wallis test, followed by a Bonferoni or Dunns post test, respectively. All analyses were performed using Graph Pad Prism 5 (GraphPad Software, La Jolla, CA, USA) and P-values less than 0.05 were considered to be significant. Data are depicted as mean ± standard error of the mean (SEM).
A cholesterol-rich diet increased serum cholesterol levels and enhanced ApoB uptake by LDLr deficient synovial lining cells
High serum cholesterol levels did not alter synovial thickening but increased the activation status of the macrophage particularly in wild type mice during collagenase-induced osteoarthritis
High serum cholesterol levels caused by a cholesterol-rich diet did not alter cartilage destruction in collagenase-induced osteoarthritis
Increased serum cholesterol levels result in enhanced bone formation in ligaments during experimental osteoarthritis
High serum cholesterol levels result in increased osteophyte formation and presence of active TGF-β in collagenase-induced osteoarthritis
OxLDL stimulated macrophages activate TGF-β in vitro
In the present study, we show that enhanced serum LDL cholesterol levels increase ectopic bone formation in mice with collagenase-induced OA. By using both a cholesterol-rich diet and LDLr deficiency, we demonstrated that an enhanced LDL cholesterol level in particular, rather than LDLr deficiency or a specific food element, is responsible for the enhancement of ectopic bone formation.
From previous studies we know that synovial lining cells are important for OA pathology. Selective elimination of synovial macrophages from the intimal lining of the knee joint prior to induction of collagenase-induced OA ameliorated the development of both cartilage damage and ectopic bone formation. In mice that lack the LDLr, a cholesterol-rich diet led to a strong increase in ApoB staining within the lining layer. Every LDL particle contains a single ApoB molecule that can be used as a marker for LDL and oxLDL uptake . The accumulation of ApoB in synovial lining cells of LDLr deficient mice suggests that lining cells in an OA joint take up oxLDL in a cholesterol-rich environment. Macrophages can phagocytize LDL via the LDLr which is suppressed when LDL serum levels rise, leading to regulation of LDL uptake into the cell . Under inflammatory conditions, LDL is oxidized and phagocytized by scavenger type A and B receptors lacking this negative feedback loop . A macrophage is a very plastic cell type which can differentiate into activated M1 or suppressive M2 subtypes. Recently we found in in vitro studies that M2 macrophages express higher levels of SR-A and CD36 receptors compared to M1 macrophages and that enhanced uptake of oxLDL by M2 macrophages changed the suppressive character of this cell type into an M1-like macrophage releasing enhanced levels of IL-1 and IL-6 . Although uptake of oxLDL in the lining layer of the LDLr−/− mice receiving a cholesterol-rich diet was strongly enhanced, no elevation of S100A8 staining was observed. S100A8 is a member of the alarmin family which is particularly produced by activated macrophages but not by non-activated macrophages and has been described as a marker for macrophage activation . The lack of S100A8 staining in LDLr−/− mice suggests that uptake of large amounts of oxLDL by lining macrophages does not enhance the production of pro-inflammatory molecules and chemokines and, therefore, does not alter synovial thickening and cartilage destruction. In contrast, high cholesterol levels in WT mice induced only minor uptake of oxLDL by lining macrophages, but strongly enhanced S100A8 levels. This accumulation of S100A8 within the lining layer might explain the trend of increased cartilage destruction observed in WT mice on a cholesterol-rich diet. S100A8 production by synovial macrophages may be driven by moderate uptake of oxLDL or LDL, while excessive uptake of oxLDL may inhibit production of pro-inflammatory factors by synovial macrophages. Another explanation of the slight increase in cartilage damage observed in WT mice on a cholesterol-rich diet (Figure 3) is that it might also be due to the increased body weight (Figure 1A). Nevertheless, by using a cholesterol-rich diet rather than a high-fat diet, we minimized weight gain, preventing amelioration of the OA process via increased loading . Lack of cartilage damage in cholesterol-rich conditions may also be explained by the increased presence of active TGF-β which protects the cartilage from proteoglycan depletion and matrix breakdown, as described in an earlier study by Van Beuningen et al. . From our own experience, we know that the experimental model which is used here is less severe in female mice compared to males. Although all mice in this experiment developed cartilage lesions, the OA scores were relatively low. It would be interesting to repeat our experiment in a more severe model, investigating whether increased cholesterol-associated cartilage damage is detected.
Apart from cartilage destruction, new formation of cartilage and bone is also observed in ligaments and locations along the bone surface during collagenase-induced OA. Focusing on ligaments (enthesophyte formation), we noticed that an increase in LDL levels by a cholesterol-rich diet markedly enhanced new cartilage and bone formation. OxLDL might directly induce proliferation and differentiation of stem cells present within the ligaments, or indirectly by enhancing growth factor production in synovium. Several studies have shown that oxLDL has a toxic effect on proliferation and differentiation of stem cells, and, in line with that, our own findings show that oxLDL (50 μg/mL) is highly toxic for stem cells (data not shown), whereas similar concentrations do not affect macrophage viability (Figure 6A) [43–47]. For that reason, we assume that enthesophyte formation may be more related to the effects of oxLDL on synovial lining activity. In previous studies in our lab it was found that intra-articular injection of the growth factors TGF-β and BMP2 resulted in formation of osteophytes [24, 25]. Moreover, in the absence of synovial macrophages, injection of TGF-β was less able to induce new formation of cartilage and bone, suggesting that macrophages are crucial in mediating TGF-β effects . Furthermore, we found that spheroid formation in a mesenchymal cell line was increased when these cells were co-cultured with macrophages, whereas total TGF-β levels in supernatant did not increase . This fits with the present study where we show that there is increased activation of TGF-β, rather than production after stimulation of macrophages with oxLDL. Using supernatants of oxLDL stimulated macrophages, strongly elevated levels of active TGF-β and, to a lesser extent, BMP were measured. Also in vivo, LDLr−/− mice show an increased presence of TGF-β compared to WT mice, suggesting that this in vitro observation could very well reflect the in vivo mechanism. TGF-β was measured using a CAGA-Luc assay in which the CAGA promoter is coupled to a luciferase gene which becomes activated by active TGF-β via TGF-β receptor 1. TGF-β is released in an inactive form and can be activated by enzymes such as MMP2 and MMP9 . These enzymes may be released either by activated synovial macrophages or by synovial fibroblasts in response to pro-inflammatory factors released by the macrophage. Recently, Ishikawa et al. demonstrated that apart from macrophages in rheumatoid arthritis fibroblast-like synoviocytes can also accumulate oxLDL via LOX-1, resulting in massive MMP production . Studies are now in progress to define the factors involved in TGF-β activation. In addition to TGF-β activation, increased BMP activation was found after stimulation of macrophages with oxLDL. Enhanced BMP presence can also contribute to the observed bone formation, since previous studies showed that TGF-β is important for initiating osteophyte formation, whereas BMP are mainly involved at later stages in the process of endochondral ossification . Also osteoarthritic chondrocytes have been shown to express LOX-1 and could, therefore, be affected by oxLDL directly . Although our data support that (ox)LDL affects chondrocytes indirectly via synovial activation, it would be interesting to investigate how hypercholesterolemia could influence the chondrocytes directly and how this affects OA development.
We did not find osteophyte or enthesophyte formation in naïve knee joints of mice with an LDLr deficiency receiving a cholesterol-rich diet (data not shown). This would suggest that systemically enhanced LDL levels alone are not sufficient to induce joint pathology. Only when there are pro-inflammatory or damage associated stimuli present (that is, during OA or instability), may enhanced LDL levels result in increased osteophyte/enthesophyte formation.
This study shows for the first time that increased LDL cholesterol levels in an OA milieu are able to enhance ectopic bone formation. Our experimental data points towards a potential mechanism in which uptake of oxLDL by synovial lining macrophages results in activation of TGF-β and to a lesser extent BMP. Further research is needed in order to elucidate what factors in particular are responsible for the enhanced levels of active TGF-β and BMP, since direct production does not seem to increase according to mRNA expression levels. Nevertheless, this mechanism provides a firm step forward to unraveling possible factors affecting etiopathology of OA and osteophyte formation in particular.
Bone morphogenetic proteins
Bone morphogenetic protein responsive element luciferase reporter plasmid
Bovine serum albumin
CAGA-box luciferase reporter plasmid
(Dulbecco’s) Modified Eagle’s Medium
Fetal calf serum
Green fluorescent protein
- H & E:
hematoxylin and eosin
Low-density lipoprotein receptor deficient
Lectin-like oxidized low-density lipoprotein receptor-1
Macrophage colony stimulating factor
Oxidized low-density lipoprotein
Quantitative real-time polymerase chain reaction
Transforming growth factor-β
Tumor necrosis factor-alpha
This study was financially supported by the Dutch Arthritis Foundation (grant number 10-1-410).
- Felson DT, Chaisson CE: Understanding the relationship between body weight and osteoarthritis. Baillieres Clin Rheumatol. 1997, 11: 671-681. 10.1016/S0950-3579(97)80003-9.View ArticlePubMedGoogle Scholar
- Velasquez MT, Katz JD: Osteoarthritis: another component of metabolic syndrome?. Metab Syndr Relat Disord. 2010, 8: 295-305. 10.1089/met.2009.0110.View ArticlePubMedGoogle Scholar
- Hunter DJ, Felson DT: Osteoarthritis. BMJ. 2006, 332: 639-642. 10.1136/bmj.332.7542.639.PubMed CentralView ArticlePubMedGoogle Scholar
- Yusuf E, Nelissen RG, Ioan-Facsinay A, Stojanovic-Susulic V, DeGroot J, van Osch G, Middeldorp S, Huizinga TW, Kloppenburg M: Association between weight or body mass index and hand osteoarthritis: a systematic review. Ann Rheum Dis. 2010, 69: 761-765. 10.1136/ard.2008.106930.View ArticlePubMedGoogle Scholar
- Katz JD, Agrawal S, Velasquez M: Getting to the heart of the matter: osteoarthritis takes its place as part of the metabolic syndrome. Curr Opin Rheumatol. 2010, 22: 512-519. 10.1097/BOR.0b013e32833bfb4b.View ArticlePubMedGoogle Scholar
- Carman WJ, Sowers M, Hawthorne VM, Weissfeld LA: Obesity as a risk factor for osteoarthritis of the hand and wrist: a prospective study. Am J Epidemiol. 1994, 139: 119-129.PubMedGoogle Scholar
- Grotle M, Hagen KB, Natvig B, Dahl FA, Kvien TK: Obesity and osteoarthritis in knee, hip and/or hand: an epidemiological study in the general population with 10 years follow-up. BMC Musculoskelet Disord. 2008, 9: 132-10.1186/1471-2474-9-132.PubMed CentralView ArticlePubMedGoogle Scholar
- Koskinen J, Magnussen CG, Wurtz P, Soininen P, Kangas AJ, Viikari JS, Kahonen M, Loo BM, Jula A, Ahotupa M, Lehtimäki T, Ala-Korpela M, Juonala M, Raitakari OT: Apolipoprotein B, oxidized low-density lipoprotein, and LDL particle size in predicting the incidence of metabolic syndrome: the Cardiovascular risk in Young Finns study. Eur J Prev Cardiol. 2012, 19: 1296-1303. 10.1177/1741826711425343.View ArticlePubMedGoogle Scholar
- Mishra R, Singh A, Chandra V, Negi MP, Tripathy BC, Prakash J, Gupta V: A comparative analysis of serological parameters and oxidative stress in osteoarthritis and rheumatoid arthritis. Rheumatol Int. 2012, 32: 2377-2382. 10.1007/s00296-011-1964-1.View ArticlePubMedGoogle Scholar
- Oliviero F, Lo Nigro A, Bernardi D, Giunco S, Baldo G, Scanu A, Sfriso P, Ramonda R, Plebani M, Punzi L: A comparative study of serum and synovial fluid lipoprotein levels in patients with various arthritides. Clin Chim Acta. 2012, 413: 303-307. 10.1016/j.cca.2011.10.019.View ArticlePubMedGoogle Scholar
- Badimon L, Storey RF, Vilahur G: Update on lipids, inflammation and atherothrombosis. Thromb Haemost. 2011, 105: S34-S42. 10.1160/THS10-11-0717.View ArticlePubMedGoogle Scholar
- Badimon L, Vilahur G: LDL-cholesterol versus HDL-cholesterol in the atherosclerotic plaque: inflammatory resolution versus thrombotic chaos. Ann N Y Acad Sci. 2012, 1254: 18-32. 10.1111/j.1749-6632.2012.06480.x.View ArticlePubMedGoogle Scholar
- Morel DW, Hessler JR, Chisolm GM: Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res. 1983, 24: 1070-1076.PubMedGoogle Scholar
- James MJ, van Reyk D, Rye KA, Dean RT, Cleland LG, Barter PJ, Jessup W: Low density lipoprotein of synovial fluid in inflammatory joint disease is mildly oxidized. Lipids. 1998, 33: 1115-1121. 10.1007/s11745-998-0313-8.View ArticlePubMedGoogle Scholar
- Groeneweg M, Kanters E, Vergouwe MN, Duerink H, Kraal G, Hofker MH, de Winther MP: Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL. J Lipid Res. 2006, 47: 2259-2267. 10.1194/jlr.M600181-JLR200.View ArticlePubMedGoogle Scholar
- van Tits LJ, Stienstra R, van Lent PL, Netea MG, Joosten LA, Stalenhoef AF: Oxidized LDL enhances pro-inflammatory responses of alternatively activated M2 macrophages: a crucial role for Kruppel-like factor 2. Atherosclerosis. 2011, 214: 345-349. 10.1016/j.atherosclerosis.2010.11.018.View ArticlePubMedGoogle Scholar
- Jiang Y, Wang M, Huang K, Zhang Z, Shao N, Zhang Y, Wang W, Wang S: Oxidized low-density lipoprotein induces secretion of interleukin-1beta by macrophages via reactive oxygen species-dependent NLRP3 inflammasome activation. Biochem Biophys Res Commun. 2012, 425: 121-126. 10.1016/j.bbrc.2012.07.011.View ArticlePubMedGoogle Scholar
- Yoshida H, Kondratenko N, Green S, Steinberg D, Quehenberger O: Identification of the lectin-like receptor for oxidized low-density lipoprotein in human macrophages and its potential role as a scavenger receptor. Biochem J. 1998, 334: 9-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Murphy JE, Vohra RS, Dunn S, Holloway ZG, Monaco AP, Homer-Vanniasinkam S, Walker JH, Ponnambalam S: Oxidised LDL internalisation by the LOX-1 scavenger receptor is dependent on a novel cytoplasmic motif and is regulated by dynamin-2. J Cell Sci. 2008, 121: 2136-2147. 10.1242/jcs.020917.View ArticlePubMedGoogle Scholar
- Bondeson J, Blom AB, Wainwright S, Hughes C, Caterson B, van den Berg WB: The role of synovial macrophages and macrophage-produced mediators in driving inflammatory and destructive responses in osteoarthritis. Arthritis Rheum. 2010, 62: 647-657.View ArticlePubMedGoogle Scholar
- Bondeson J, Wainwright SD, Lauder S, Amos N, Hughes CE: The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis. Arthritis Res Ther. 2006, 8: R187-10.1186/ar2099.PubMed CentralView ArticlePubMedGoogle Scholar
- Blom AB, van Lent PL, Libregts S, Holthuysen AE, van der Kraan PM, van Rooijen N, van den Berg WB: Crucial role of macrophages in matrix metalloproteinase-mediated cartilage destruction during experimental osteoarthritis: involvement of matrix metalloproteinase 3. Arthritis Rheum. 2007, 56: 147-157. 10.1002/art.22337.View ArticlePubMedGoogle Scholar
- Rogers J, Shepstone L, Dieppe P: Bone formers: osteophyte and enthesophyte formation are positively associated. Ann Rheum Dis. 1997, 56: 85-90. 10.1136/ard.56.2.85.PubMed CentralView ArticlePubMedGoogle Scholar
- van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB: Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage. 1998, 6: 306-317. 10.1053/joca.1998.0129.View ArticlePubMedGoogle Scholar
- van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB: Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections. Osteoarthritis Cartilage. 2000, 8: 25-33. 10.1053/joca.1999.0267.View ArticlePubMedGoogle Scholar
- Blom AB, van Lent PL, Holthuysen AE, van der Kraan PM, Roth J, van Rooijen N, van den Berg WB: Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage. 2004, 12: 627-635. 10.1016/j.joca.2004.03.003.View ArticlePubMedGoogle Scholar
- van Lent PL, Blom AB, van der Kraan P, Holthuysen AE, Vitters E, van Rooijen N, Smeets RL, Nabbe KC, van den Berg WB: Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation. Arthritis Rheum. 2004, 50: 103-111. 10.1002/art.11422.View ArticlePubMedGoogle Scholar
- Getz GS, Reardon CA: Animal models of atherosclerosis. Arterioscler Thromb Vasc Biol. 2012, 32: 1104-1115. 10.1161/ATVBAHA.111.237693.PubMed CentralView ArticlePubMedGoogle Scholar
- Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J: Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993, 92: 883-893. 10.1172/JCI116663.PubMed CentralView ArticlePubMedGoogle Scholar
- van der Kraan PM, Vitters EL, van Beuningen HM, van de Putte LB, van den Berg WB: Degenerative knee joint lesions in mice after a single intra-articular collagenase injection. A new model of osteoarthritis. J Exp Pathol. 1990, 71: 19-31.Google Scholar
- Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, Salter D, van den Berg WB: Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage. 2006, 14: 13-29. 10.1016/j.joca.2005.07.014.View ArticlePubMedGoogle Scholar
- Glasson SS, Chambers MG, Van Den Berg WB, Little CB: The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010, 18: S17-S23.View ArticlePubMedGoogle Scholar
- van Tits LJ, Demacker PN, de Graaf J, Hak-Lemmers HL: Stalenhoef AF: alpha-tocopherol supplementation decreases production of superoxide and cytokines by leukocytes ex vivo in both normolipidemic and hypertriglyceridemic individuals. Am J Clin Nutr. 2000, 71: 458-464.PubMedGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987, 162: 156-159.View ArticlePubMedGoogle Scholar
- van Lent PL, Blom AB, Schelbergen RF, Sloetjes A, Lafeber FP, Lems WF, Cats H, Vogl T, Roth J, van den Berg WB: Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis. Arthritis Rheum. 2012, 64: 1466-1476. 10.1002/art.34315.View ArticlePubMedGoogle Scholar
- Tan AL, Grainger AJ, Tanner SF, Shelley DM, Pease C, Emery P, McGonagle D: High-resolution magnetic resonance imaging for the assessment of hand osteoarthritis. Arthritis Rheum. 2005, 52: 2355-2365. 10.1002/art.21210.View ArticlePubMedGoogle Scholar
- Chung SW, Kang BY, Kim SH, Pak YK, Cho D, Trinchieri G, Kim TS: Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B. J Biol Chem. 2000, 275: 32681-32687.View ArticlePubMedGoogle Scholar
- Mahley RW, Innerarity TL, Rall SC, Weisgraber KH: Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res. 1984, 25: 1277-1294.PubMedGoogle Scholar
- Goldstein JL, Brown MS: The LDL receptor. Arterioscler Thromb Vasc Biol. 2009, 29: 431-438. 10.1161/ATVBAHA.108.179564.PubMed CentralView ArticlePubMedGoogle Scholar
- Spady DK, Meddings JB, Dietschy JM: Kinetic constants for receptor-dependent and receptor-independent low density lipoprotein transport in the tissues of the rat and hamster. J Clin Invest. 1986, 77: 1474-1481. 10.1172/JCI112460.PubMed CentralView ArticlePubMedGoogle Scholar
- Mooney RA, Sampson ER, Lerea J, Rosier RN, Zuscik MJ: High-fat diet accelerates progression of osteoarthritis after meniscal/ligamentous injury. Arthritis Res Ther. 2011, 13: R198-10.1186/ar3529.PubMed CentralView ArticlePubMedGoogle Scholar
- van Beuningen HM, van der Kraan PM, Arntz OJ, van den Berg WB: In vivo protection against interleukin-1-induced articular cartilage damage by transforming growth factor-beta 1: age-related differences. Ann Rheum Dis. 1994, 53: 593-600. 10.1136/ard.53.9.593.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang H, Mohamed AS, Zhou SH: Oxidized low density lipoprotein, stem cells, and atherosclerosis. Lipids Health Dis. 2012, 11: 85-10.1186/1476-511X-11-85.PubMed CentralView ArticlePubMedGoogle Scholar
- Salvayre R, Auge N, Benoist H, Negre-Salvayre A: Oxidized low-density lipoprotein-induced apoptosis. Biochim Biophys Acta. 2002, 1585: 213-221. 10.1016/S1388-1981(02)00343-8.View ArticlePubMedGoogle Scholar
- Chu L, Hao H, Luo M, Huang Y, Chen Z, Lu T, Zhao X, Verfaillie CM, Zweier JL, Liu Z: Ox-LDL modifies the behaviour of bone marrow stem cells and impairs their endothelial differentiation via inhibition of Akt phosphorylation. J Cell Mol Med. 2011, 15: 423-432. 10.1111/j.1582-4934.2009.00948.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu T, Parthasarathy S, Hao H, Luo M, Ahmed S, Zhu J, Luo S, Kuppusamy P, Sen CK, Verfaillie CM, Tian J, Liu Z: Reactive oxygen species mediate oxidized low-density lipoprotein-induced inhibition of oct-4 expression and endothelial differentiation of bone marrow stem cells. Antioxid Redox Signal. 2010, 13: 1845-1856. 10.1089/ars.2010.3156.PubMed CentralView ArticlePubMedGoogle Scholar
- Tie G, Yan J, Yang Y, Park BD, Messina JA, Raffai RL, Nowicki PT, Messina LM: Oxidized low-density lipoprotein induces apoptosis in endothelial progenitor cells by inactivating the phosphoinositide 3-kinase/Akt pathway. J Vasc Res. 2010, 47: 519-530. 10.1159/000313879.PubMed CentralView ArticlePubMedGoogle Scholar
- Yu Q, Stamenkovic I: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Gene Dev. 2000, 14: 163-176.PubMed CentralPubMedGoogle Scholar
- Ishikawa M, Ito H, Akiyoshi M, Kume N, Yoshitomi H, Mitsuoka H, Tanida S, Murata K, Shibuya H, Kasahara T, Kakino A, Fujita Y, Sawamura T, Yasuda T, Nakamura T: Lectin-like oxidized low-density lipoprotein receptor 1 signal is a potent biomarker and therapeutic target for human rheumatoid arthritis. Arthritis Rheum. 2012, 64: 1024-1034. 10.1002/art.33452.View ArticlePubMedGoogle Scholar
- Blaney Davidson EN, Vitters EL, van Beuningen HM, van de Loo FA, van den Berg WB, van der Kraan PM: Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor beta-induced osteophytes: limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation. Arthritis Rheum. 2007, 56: 4065-4073. 10.1002/art.23034.View ArticlePubMedGoogle Scholar
- Simopoulou T, Malizos KN, Tsezou A: Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) expression in human articular chondrocytes. Clin Exp Rheumatol. 2007, 25: 605-612.PubMedGoogle Scholar
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