Vivian de Oliveira Nunes Teixeira

Mecanismos de perda muscular da sarcopenia

Approximately 66% of the patients with rheumatoid arthritis (RA) have significant loss of cell mass (rheumatoid cachexia), mainly of skeletal muscle (rheumatoid sarcopenia). Sarcopenia is defined as muscle wasting associated with functional impairment. Patients with RA possess significant reduction in muscle strength, caused by muscle protein wasting, and loss of functionality. Various conditions leading to muscle wasting involve different pathways of intracellular signaling that trigger: (i) programmed cell death (apoptosis); (ii) increased protein degradation through autophagy, calcium-dependent proteases (calpains and caspases), and proteasome system; (iii) decreased satellite cell activation, responsible for muscle regeneration. This article aimed at reviewing these general mechanisms of sarcopenia and their involvement in RA. Greater knowledge of these mechanisms may lead to the development of innovative therapies to this important comorbidity.

Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females

The aim of the present study was to address the involvement of Rhipicephalus microplus larval cysteine endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading cysteine endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic cysteine endopeptidase activity was detected in larval gut. It was shown that cysteine and aspartic endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic endopeptidase Boophilus yolk cathepsin (BYC) is associated with a cysteine endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development.

Muscle wasting in collagen-induced arthritis and disuse atrophy

The mechanisms of muscle wasting and decreased mobility have a major functional effect in rheumatoid arthritis, but they have been poorly studied. The objective of our study is to describe muscular involvement and the pathways in an experimental model of arthritis compared to the pathways in disuse atrophy. Female Wistar rats were separated into three groups: control (CO), collagen-induced arthritis (CIA), and immobilized (IM). Spontaneous locomotion and weight were evaluated weekly. The gastrocnemius muscle was evaluated by histology and immunoblotting to measure the expression of myostatin (a negative regulator), LC3 (autophagy), MuRF-1 (proteasome-mediated proteolysis), MyoD, and myogenin (satellite-cell activation). The significance level was set at P < 0.05, and histological analysis of joints confirmed the severity of the arthropathy. There was a significant difference in spontaneous locomotion in the CIA group. Animal body weight, gastrocnemius muscle weight, and relative muscle weight decreased 20%, 30%, and 20%, respectively, in the CIA rats. Inflammatory infiltration and swelling were present in the gastrocnemius muscles of the CIA rats. The mean cross-sectional area was reduced by 30% in the CIA group and by 60% in the IM group. The expressions of myostatin and LC3 between the groups were similar. There was increased expression of MuRF-1 in the IM (1.9-fold) and CIA (3.1-fold) groups and of myogenin in the muscles of the CIA animals (1.7-fold), while MyoD expression was decreased in the IM (20%) rats. This study demonstrated that the development of experimental arthritis is associated with decreased mobility, body weight, and muscle loss. Both IM and CIA animal models presented muscle atrophy, but while proteolysis and the regeneration pathways were activated in the CIA model, there was no activation of regeneration in the IM model. We can assume that muscle atrophy in experimental arthritis is associated with the disease itself and not simply with decreased mobility.

Gastrin-releasing peptide and its receptor increase arthritis fibroblast-like synoviocytes invasiveness through activating the PI3K/AKT pathway

&NA; Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. The fibroblast‐like synoviocytes (FLS) has a central role on the disease pathophysiology. The present study aimed to examine the role of gastrin‐releasing peptide (GRP) and its receptor (GRPR) on invasive behavior of mice fibroblast‐like synoviocytes (FLS), as well as to evaluate GRP‐induced signaling on PI3K/AKT pathway. The expression of GRPR in FLS was investigated by immunocytochemistry, western blot (WB) and qRT‐PCR. The proliferation and invasion were assessed by SRB and matrigel‐transwell assay after treatment with GRP and/or RC‐3095 (GRPR antagonist), and/or Ly294002 (inhibitor of PI3K/AKT pathway). Finally, AKT phosphorylation was assessed by WB. GRPR protein was detected in FLS and the exposure to GRP increased FLS invasion by nearly two‐fold, compared with untreated cells (p < 0.05), while RC‐3095 reversed that effect (p < 0.001). GRP also increased phosphorylated AKT expression in FLS. When Ly294002 was added with GRP, it prevented the GRP‐induced increased cell invasiveness (p < 0.001). These data suggest that GRPR expression in FLS and that exogenous GRP are able to activate FLS invasion. This effect occurs at least in part through the AKT activation. Therefore, understanding of the GRP/GRPR pathway could be relevant in the development of FLS‐targeted therapy for RA. HighlightsFibroblast‐like synoviocytes express gastrin‐releasing receptor protein.GRP increased FLS invasiveness and RC‐3095 reverse that effect.The effect of GRP could be through activation of PI3K/AKT pathway.GRP/GRPR signaling could be a new target for the treatment of arthritis.

Muscle wasting in osteoarthritis model induced by anterior cruciate ligament transection

This study aimed to investigate the molecular pathways involved in muscle wasting in an animal model of osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT) in rats. Reduction of protein syntheses, increased proteolysis and impaired muscle regeneration are important pathways related to muscle wasting, and myogenin, MyoD, myostatin and MuRF-1 are some of their markers. Female Wistar rats were allocated into two groups: OA (submitted to the ACLT) and SHAM (submitted to surgery without ACLT). Nociception, spontaneous exploratory locomotion and body weight of animals were evaluated weekly. Twelve weeks after the disease induction, animals were euthanized, and the right knee joints were collected. Gastrocnemius muscle of the right hind paw were dissected and weighed. Gastrocnemius was used for evaluation of muscle atrophy and expression of IL-1β, TNF-α, Pax7, myogenin, MyoD, myostatin and MuRF-1. Histopathology of the knee confirmed the development of the disease in animals of OA group. Gastrocnemius of OA animals showed a reduction of about 10% in area and an increased IL-1β expression compared to animals of SHAM group. Expression of myostatin was increased in OA group, while myogenin expression was decreased. TNF-α, Pax7, MuRF-1 and MyoD expression was similar in both OA and SHAM groups. Nociception was significantly elevated in OA animals in the last two weeks of experimental period. Spontaneous exploratory locomotion, body weight and weight of gastrocnemius showed no difference between OA and SHAM groups. Gastrocnemius atrophy in OA induced by ACLT involves elevated expression of IL-1β within the muscle, as well as increased expression of myostatin and decreased expression of myogenin. Therefore, muscle wasting may be linked to impaired muscle regeneration.

Collagen-induced arthritis as an animal model of rheumatoid cachexia: CIA as an animal model of RA

Rheumatoid arthritis is characterized by chronic polyarticular synovitis and presents systemic changes that impact quality of life, such as impaired muscle function, seen in up to 66% of the patients. This can progress to severely debilitating state known as rheumatoid cachexia—without loss of fat mass and body weight—for which there is little consensus in terms of diagnosis or treatment. This study aims to evaluate whether the collagen‐induced arthritis (CIA) animal model also develops clinical and functional features characteristic of rheumatoid cachexia.

AB0110 The Proteasome Is Related To Muscle Wasting in Experimental Arthritis and Is Altered by Etanercept Treatment

Background Rheumatoid arthritis is an autoimmune inflammatory disease associated with systemic complications like fatigue and muscle wasting. Muscle wasting could be related to the activation of the ubiquitin-proteasome system. Objectives To evaluate muscle loss and involvement of the proteasome in collagen-induced arthritis (CIA), with or without treatment with methotrexate or a TNF inhibitor (etanercept). Methods Male DBA1/J mice were divided into 4 groups (n=8 each): CIA (saline); ETN (etanercept, 5.5 mg/kg) and MTX (methotrexate, 35 mg/kg), treated twice a week for 6 weeks, and a healthy control group (CO). Treatments started one week after booster injection. Clinical score, hind paw oedema, and body weight were analysed during the experimental period. Gastrocnemius muscles (GA) were weighted after death and used to quantify proteasome activity, protein levels and mRNA expression of its subunits by Western blot and rtPCR. Significance was considered when p≤0.05. Results Treatments slowed disease development, observed through smaller clinical score and hindpaw edema in ETN and MTX. ETN presented higher body weight (21±1.0g) compared to MTX (19±1.3) at weeks 5 and 7. GA weight was heavier in ETN (105±12g) than CIA and MTX (80±10 and 79±10g, respectively), a result also observed after normalization of muscle with body weight. Of note, the catalytic properties of 26S proteasome showed an increase of caspase-like activity in CIA and MTX groups (150 and 200% of activity, respectively). Furthermore, muscles of MTX treated animals showed higher protein levels for proteasomal subunits PSMB8 and PSMB9 and increased gene expression for Psmb5, Psmb8 and Psmb9. In contrast, expression of Psmb6 was decreased and of Psmb9 was enhanced in CIA. Conclusions Although both drugs improved the disease score, ETN presented a stronger anti-arthritic effect and was the only treatment able to partially prevent muscle wasting. In contrast to ETN, MTX treatment did not prevent muscle loss due to CIA accompanied by persistent up-regulation of proteasome expression and activity. Acknowledgement Financial support: CAPES, CNPq, FIPE-HCPA. Disclosure of Interest None declared

AB0136 Can Nitric Oxide (NO) Regulator Drugs be Used as Treatment for Muscle Loss in Collagen-Induced Arthritis (CIA)?

Background Rheumatoid arthritis is an autoimmune disease of unknown etiology associated with progressive disability, systemic complications like fatigue and muscle weakness, early death, and socioeconomic costs [1,2]. Nitric oxide (NO) have been related with inflammation and its regulation can lead to anti-inflammatory and anti-arthritic effects in CIA as well as induce muscle repair in muscle injury [3,4]. The role of NO in CIA muscle loss has not yet been studied. Objectives How NO synthase inhibitor (NG-nitroL-arginie methy ester: L-NAME) and the NO donor (3-morpholinosydnonimine: SIN-1) affects CIA muscle loss? Methods Female Wistar rats with CIA [5] were separated in four groups: CIA (saline, n=10); L-NAME (30mg kg-1 n=10); and SIN-1 (0.3 mg kg-1, n=13), treated twice a day for 10 days after the onset of the disease, and a wildtype group (WT, n=8). Clinical score (arbitrary units – au), hind paw edema (mm), spontaneous locomotion (cm), and body weight (g) were analyzed. Ankle was collected and used for histological confirmation of the disease. Tibialis anterior (TA), gastrocnemious and soleus muscles were weighted (g). TA was used for histological analysis and immune stained for TNF-alfa, TGF-beta and IL-1beta. Serum was collected for albumin (g dL-1), total iron (μg dL-1) and ionized calcium (mg dL-1) analysis. Proper statistics were performed and p<.05 was set for critical limit. Data are in Mean ± SEM. Results Ankle histology confirmed that all CIA groups developed arthritis. In vivo analysis of hindpaw edema, clinical score, body and muscle weight, and spontaneous locomotion showed no difference among CIA groups. On the other hand, both L-NAME (48±6,5) and SIN-1 (48±6,5) groups have shown statistically decreased clinical score than saline (77±9,2) when analyzed by the area under curve. Muscle cross sectional area were higher in L-NAME (1074±315 μm) and SIN-1 (1115±303 μm) than saline (786±243 μm), however it did not reach WT diameter (1755±278 μm). Blood vessels diameter were smaller in L-NAME (287±121 μm) group compared to SIN-1 (524±169 μm) and saline (445±165 μm). Albumin was lower in all CIA groups (saline 3,8±0,2; L-NAME 3,9±0,1; SIN-1 3,7±0,1; WT 4,2±0,1), and ionized calcium had no difference among all groups. Iron was decreased in L-NAME (229±42) and SIN-1 (226±62) than WT (353±53). All CIA groups had shown increased immune cells infiltration shown by TNF-alfa, TGF-beta and IL-1beta immune staining. Conclusions The data above mentioned suggests that nitric oxide regulator drugs show good prospects as intervention for muscle loss. As was observed, even a simply drug that have main influence in vessel pressure shows preventive clinical score development and ameliorates muscle cross sectional area. The mechanism behind such findings will soon be depicted by molecular analysis. References McInnes IB, Schett G. NEJM 2011;365(23):2205-19. Alver A, et al. Clinical Biochemistry 2011;44(17-18):1385-9. Gomaa A, et al. BJP 2009;158(7):1835-47. Filippin LI, et al. Nitric Oxide 2009;21(3-4):157-63. Rosloniec EF, et al. Curr Protoc Immunol 2010; Chapter 15: Unit 15.5.1-25. Acknowledgements Financial support: CAPES, CNPq, FAPERGS, FIPE-HCPA. Disclosure of Interest None declared

AB0135 Aerobic Exercise in Inclined Treadmill Reduce Fatigue in Collagen-Induced Arthritis

Background Rheumatoid arthritis (RA) patients suffer from joint pain and decreased physical capacity, like muscle wasting and fatigue. Fatigue is a clinical manifestation reported by 40-80% of patients with RA and is regarded as an important feature of the disease. Aerobic exercise may be beneficial for treating this feature in RA patients, however the mechanisms involved are still unclear. Objectives To evaluate the effect of aerobic exercise training on the endurance exercise performance in collagen-induced arthritis (CIA) mice. Methods Male DBA1/J mice with CIA [1] were randomly divided into 3 groups: wildtype with exercise (WT-EXE, n=4), CIA exercised (CIA-EXE, n=5) and CIA non-exercised (CIA, n=4). Endurance exercise performance test (fatigue) was analyzed in all groups prior to booster injection and each 15 days after protocol started. Eighteen days after the disease induction (booster), WT and CIA-EXE were submitted to training on an inclined treadmill (θ=5°), 45 minutes a day, 5 days per week for 6 weeks at 60% of their own endurance exercise performance. Variables analyzed were disease score, hindpaw nociception, body weight (g), fatigue (by endurance exercise performance in min) and relative muscle weight (muscle weight in mg divided by total animal weight in g). Data was analyzed with ANOVA Two-Way followed by Bonferroni and independent sample t-test and p<0.05 was considered significant. All data are represented as Mean ± SEM. Results Body weight was significantly higher in WT-EXE compared with CIA after 4 and 6 weeks of exercise. At week 6 of exercise, CIA-EXE had higher body weight than CIA. Fatigue test at 4 and 6 weeks of experiment was significantly different among all experimental groups; WT-EXE and CIA had, respectively, the highest and the lowest fatigue velocity. Gastrocnemius muscle weight was significantly heavier in control group than in CIA-EXE and CIA. Nociception and clinical score of arthritis did not differ between CIA-EXE and CIA. Conclusions Inclined aerobic exercise appears as an interesting intervention in RA to treat decreased physical capacity. Collagen-induced arthritis animals demonstrated decreased endurance, and consequently increased fatigue, characteristics of a good animal model to study fatigue. The exercise protocol used in this study was able to improve this feature, demonstrating that interventions used to treat physical disabilities in RA are also valid in this model. Further studies are necessary to clarify the mechanisms behind fatigue, especially when combining exercise training and common treatments of RA. References Rosloniec EF, et al. Curr Protoc Immunol 2010; Chapter 15: Unit 15.5.1-25. Acknowledgements Financial support: CAPES, CNPq, FAPERGS, FIPE-HCPA. Disclosure of Interest None declared

AB0117 Collagen-Induced Arthritis in Mice as a Model of Rheumatoid Cachexia

Background Rheumatoid arthritis (RA) extra-articular features frequently involved metabolism alterations which in a context are known as rheumatoid cachexia. Loss of lean muscle mass, reduction of grip strength and fatigue are the most common signs in RA. Loss of body weight and reduction in food intake are not usually part of human rheumatoid cachexia, but have been demonstrated before in animal models of arthritis. Recently, Evans et al1 published a definition for cachexia that seems to fulfil rheumatoid condition. Therefore a better elucidation of rheumatoid cachexia is needed and an animal model that mimics human condition may be useful. Objectives To explored the possibility of using collagen-induced arthritis in mice as an animal model of cachexia. Methods 8-12 weeks male DBA/1J mice were separated in two groups: control (CO) and collagen-induced arthritis (CIA). CIA animals were immunized by intradermal injection of bovine type II collagen with complete Freunds adjuvant followed by a booster. Immunization protocol and clinical signs (arthritis score and paw edema) evaluation occur as previous described by Brand et al 20072. Animals were followed by 65 days with evaluations made in day zero and at 18, 25, 35, 45, 55 and 65 days after immunization. Rheumatoid cachexia was evaluated by body weight change (%), food intake (g), fatigue (by endurance exercise performance in minutes), grip strength (g), locomotion (cm) and relative muscle weight (muscle weight in mg divided by total animal weight g). Statistical analysis includes repeated measures analysis of variance (ANOVA) for variables with variation between time and group and t-test for variables with variation only between groups. Difference was assumed when p value were lower than 0.05. Results Arthritis score and paw edema confirm disease in CIA group. Fatigue was higher in CIA group (lower time in endurance exercise) at the same time that grip strength and locomotion was lower (after 35 days until the end of the experiment). Relative gastrocnemius muscle weight was also lower in CIA group 3.9±0.57mg/g vs 5.0±0.61mg/g (CO). Body weight change and food intake were not statistical different within groups, however in days 25 and 35 CIA animals lost more weight than CO animals. Conclusions These data demonstrate firstly that CIA animals show metabolic extra articular events that mimics human pathophysiology arthritis. Even though there were no difference in body weight change and food intake it reinforces the similarity among CIA animal model and human RA once this variables have the same behavior in humans. Also considering the lack of studies involving rheumatoid cachexia and the obstacle that involve this study design in humans it is essential to have a solid animal model to evaluate prospectively mechanism of rheumatoid cachexia. References Evans WJ, et al. 2008. Cachexia: a new definition. Clinical Nutrition 27(6):793-9. Brand DD, Latham KA, Rosloniec EF. 2007. Collagen-induced arthritis. Nature protocols 2(5):1269-75. Disclosure of Interest None declared