Intensive cholesterol-lowering treatment reduces synovial inflammation during early collagenase-induced osteoarthritis, but not pathology at end-stage disease in female dyslipidemic E3L.CETP mice.

INTRODUCTION: The association between metabolic syndrome (MetS) and osteoarthritis (OA) development has become increasingly recognized. In this context, the exact role of cholesterol and cholesterol-lowering therapies in OA development has remained elusive. Recently, we did not observe beneficial effects of intensive cholesterol-lowering treatments on spontaneous OA development in E3L.CETP mice. We postulated that in the presence of local inflammation caused by a joint lesion, cholesterol-lowering therapies may ameliorate OA pathology. MATERIALS AND METHODS: Female ApoE3∗Leiden.CETP mice were fed a cholesterol-supplemented Western type diet. After 3 weeks, half of the mice received intensive cholesterol-lowering treatment consisting of atorvastatin and the anti-PCSK9 antibody alirocumab. Three weeks after the start of the treatment, OA was induced via intra-articular injections of collagenase. Serum levels of cholesterol and triglycerides were monitored throughout the study. Knee joints were analyzed for synovial inflammation, cartilage degeneration, subchondral bone sclerosis and ectopic bone formation using histology. Inflammatory cytokines were determined in serum and synovial washouts. RESULTS: Cholesterol-lowering treatment strongly reduced serum cholesterol and triglyceride levels. Mice receiving cholesterol-lowering treatment showed a significant reduction in synovial inflammation (P = 0.008, WTD: 95% CI: 1.4- 2.3; WTD + AA: 95% CI: 0.8- 1.5) and synovial lining thickness (WTD: 95% CI: 3.0-4.6, WTD + AA: 95% CI: 2.1-3.2) during early-stage collagenase-induced OA. Serum levels of S100A8/A9, MCP-1 and KC were significantly reduced after cholesterol-lowering treatment (P = 0.0005, 95% CI: -46.0 to -12.0; P = 2.8 × 10-10, 95% CI: -398.3 to -152.1; P = 2.1 × 10-9, -66.8 to -30.4, respectively). However, this reduction did not reduce OA pathology, determined by ectopic bone formation, subchondral bone sclerosis and cartilage damage at end-stage disease. CONCLUSION: This study shows that intensive cholesterol-lowering treatment reduces joint inflammation after induction of collagenase-induced OA, but this did not reduce end stage pathology in female mice.

IL-1ß inhibition combined with cholesterol-lowering therapies decreases synovial lining thickness and spontaneous cartilage degeneration in a humanized dyslipidemia mouse model.

INTRODUCTION: Both systemic inflammation and dyslipidemia contribute to osteoarthritis (OA) development and have been suggested as a possible link between metabolic disease and OA development. Recently, the CANTOS trial showed a reduction in knee and hip replacements after inhibition of IL-1ß in patients with a history of cardiovascular disease and high inflammatory risk. In this light, we investigated whether inhibition of IL-1ß combined with cholesterol-lowering therapies can reduce OA development in dyslipidemic APOE∗3Leiden mice under pro-inflammatory dietary conditions. MATERIALS AND METHODS: Female ApoE3∗Leiden mice were fed a cholesterol-supplemented Western-Type diet (WTD) for 38 weeks. After 14 weeks, cholesterol-lowering and anti-inflammatory treatments were started. Treatments included atorvastatin alone or with an anti-IL1ß antibody, and atorvastatin combined with proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor alirocumab without or with the anti-IL1ß antibody. Knee joints were analyzed for cartilage degradation, synovial inflammation and ectopic bone formation using histology at end point. RESULTS: Cholesterol-lowering treatment successfully decreased systemic inflammation in dyslipidemic mice, which was not further affected by inhibition of IL-1ß. Synovial thickening and cartilage degeneration were significantly decreased in mice that received cholesterol-lowering treatment combined with inhibition of IL-1ß (P < 0.01, P < 0.05, respectively) compared to mice fed a WTD alone. Ectopic bone formation was comparable between all groups. CONCLUSION: These results indicate that inhibition of IL-1ß combined with cholesterol-lowering therapy diminishes synovial thickening and cartilage degeneration in mice and may imply that this combination therapy could be beneficial in patients with metabolic inflammation.

Novel high-intensive cholesterol-lowering therapies do not ameliorate knee OA development in humanized dyslipidemic mice.

OBJECTIVE: High systemic cholesterol levels have been associated with osteoarthritis (OA) development. Therefore, cholesterol lowering by statins has been suggested as a potential treatment for OA. We investigated whether therapeutic high-intensive cholesterol-lowering attenuated OA development in dyslipidemic APOE∗3Leiden.CETP mice. METHODS: Female mice (n = 13-16 per group) were fed a Western-type diet (WTD) for 38 weeks. After 13 weeks, mice were divided into a baseline group and five groups receiving WTD alone or with treatment: atorvastatin alone, combined with PCSK9 inhibitor alirocumab and/or ANGPTL3 inhibitor evinacumab. Knee joints were analysed for cartilage degradation, synovial inflammation and ectopic bone formation using histology. Aggrecanase activity in articular cartilage and synovial S100A8 expression were determined as markers of cartilage degradation/regeneration and inflammation. RESULTS: Cartilage degradation and active repair were significantly increased in WTD-fed mice, but cholesterol-lowering strategies did not ameliorate cartilage destruction. This was supported by comparable aggrecanase activity and S100A8 expression in all treatment groups. Ectopic bone formation was comparable between groups and independent of cholesterol levels. CONCLUSIONS: Intensive therapeutic cholesterol lowering per se did not attenuate progression of cartilage degradation in dyslipidemic APOE∗3Leiden.CETP mice, with minor joint inflammation. We propose that inflammation is a key feature in the disease and therapeutic cholesterol-lowering strategies may still be promising for OA patients presenting both dyslipidemia and inflammation.

Variable cartilage degradation in mice with diet-induced metabolic dysfunction: food for thought.

OBJECTIVE: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.

APOE*3Leiden.CETP transgenic mice as model for pharmaceutical treatment of the metabolic syndrome.

AIMS: This study aimed to investigate systematically (i) the appropriate dietary conditions to induce the features of the MetS in APOE*3Leiden.humanCholesteryl Ester Transfer Protein (E3L.CETP) mice and (ii) whether the response of this model to different antidiabetic and hypolipidemic drugs is similar as in humans. METHODS: Male obese, IR and dyslipidemic E3L.CETP mice were treated with antidiabetic drugs rosiglitazone, liraglutide or an experimental 11ß-hydroxysteroid-dehydrogenase-1 (HSD-1) inhibitor, or with hypolipidemic drugs atorvastatin, fenofibrate or niacin for 4-6 weeks. The effects on bw, IR and plasma and liver lipids were assessed. RESULTS: Rosiglitazone, liraglutide and HSD-1 inhibitor significantly decreased glucose and insulin levels or IR. Liraglutide and HSD-1 inhibitor also decreased bw. Atorvastatin, fenofibrate and niacin improved the dyslipidemia and fenofibrate and niacin increased high-density lipoprotein (HDL) cholesterol. In addition, hepatic triglycerides were significantly decreased by treatment with rosiglitazone and liraglutide, while hepatic cholesterol esters were significantly decreased by rosiglitazone and atorvastatin. CONCLUSIONS: We conclude that the E3L.CETP mouse is a promising novel translational model to investigate the effects of new drugs, alone or in combination, that affect IR, diabetic dyslipidemia and non-alcoholic fatty liver disease (NAFLD).

Osteoarthritis development is induced by increased dietary cholesterol and can be inhibited by atorvastatin in APOE*3Leiden.CETP mice--a translational model for atherosclerosis.

OBJECTIVE: Hypercholesterolaemia, a risk factor for atherosclerosis (ATH), has been suggested to have a role in the development of osteoarthritis (OA). To test this hypothesis, the effect of cholesterol and different cholesterol-lowering treatments on OA was investigated in a mouse model resembling human lipoprotein metabolism. METHODS: Female ApolipoproteinE*3Leiden.human Cholesteryl Ester Transfer Protein mice received a western-type diet with 0.1% (w/w) cholesterol (LC), 0.3% (w/w) cholesterol alone (HC) or treated with 3 mg/kg/day atorvastatin or 0.3 mg/kg/day ezetimibe. One group remained on chow (control). After 39 weeks, OA grades of the knees and the extent of ATH were determined. Plasma cholesterol levels were measured throughout the study. RESULTS: LC and HC groups developed significantly more OA at the medial side than the control group in a dose-dependent manner. Atorvastatin but not ezetimibe treatment significantly suppressed OA development. As expected, features of ATH were significantly increased in the LC and HC groups compared with the control group and suppressed by atorvastatin (48%) and ezetimibe (55%) treatment. There were significant correlations between the development of OA on the medial side of the joint and cholesterol exposure (r=0.4) or ATH features (r=0.3). CONCLUSIONS: Dietary cholesterol and accordingly increased plasma levels play a role in the development of OA. The correlation found between OA, cholesterol and ATH demonstrates that these variables are connected, but indicates the contribution of other ongoing processes in the development of OA. The suppressive effect on OA development of atorvastatin but not of ezetimibe, which had similar cholesterol exposure levels, corroborates these findings.

Association of the -2849 interleukin-10 promoter polymorphism with autoantibody production and joint destruction in rheumatoid arthritis.

OBJECTIVE: To analyze the -2849 A/G interleukin-10 (IL-10) promoter polymorphism, which is associated with high (AG/GG) and low (AA) IL-10 production, in a cohort of rheumatoid arthritis (RA) patients and controls in order to gain a better understanding of its role in the incidence and progression of RA. METHODS: Allele frequencies of the promoter polymorphism -2849 A/G and carriage rates were compared in 283 RA patients, 413 patients with other rheumatic diseases, and 1,220 healthy controls. The rate of joint damage and baseline levels of IgG and IgM rheumatoid factors and anti-citrullinated peptide antibodies were measured and were correlated with the IL-10 gene polymorphism. Furthermore, the correlation between the invasiveness of fibroblast-like synoviocytes (FLS) and the -2849 IL-10 genotype was tested. RESULTS: The IL-10 genotype was not associated with the incidence of RA, but instead, correlated with disease progression, as determined by the extent of joint destruction. A higher rate of joint destruction was observed in patients with the genotype associated with high IL-10 production. Since FLS are thought to be involved in joint destruction, we analyzed IL-10 genotypes in conjunction with FLS invasiveness. Although adenoviral gene transfer of IL-10 to FLS inhibited their invasiveness, no differences were observed in vitro in the FLS from RA patients who were -2849 non-G carriers compared with those who were G carriers. Instead, patients with the -2849 AG/GG genotype, which is associated with high IL-10 production, had higher autoantibody titers at baseline. CONCLUSION: The -2849 IL-10 promoter polymorphism is associated with autoantibody production and subsequent joint damage in RA.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.