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1.
Arterioscler Thromb Vasc Biol ; 43(9): 1700-1712, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37409530

RESUMO

BACKGROUND: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. METHODS: Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbß3. RESULTS: We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. CONCLUSIONS: Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.


Assuntos
Artérias , Plaquetas , Quimiocinas , Ativação de Neutrófilo , Neutrófilos , Trombose , Plaquetas/imunologia , Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Quimiocinas/metabolismo , Trombose/imunologia , Ligante de CD40 , Neutrófilos/imunologia , Neutrófilos/metabolismo , Adesão Celular , Humanos
2.
Stem Cells Transl Med ; 11(8): 861-875, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35716044

RESUMO

Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings; hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings.


Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Proliferação de Células , Cicatriz/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Células-Tronco
3.
Rheumatology (Oxford) ; 61(3): 992-1004, 2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-34080609

RESUMO

OBJECTIVES: In pre-clinical studies, pinolenic acid (PNLA), an omega-6-polyunsaturated fatty acid from pine nuts, has shown anti-inflammatory effects. We aimed to investigate the effect of PNLA in human cell lines and peripheral blood mononuclear cells (PBMCs) from RA patients and healthy controls (HCs). METHODS: A modified Boyden chamber was used to assess chemokine-induced migration of THP-1 monocytes. Macropinocytosis was assessed using Lucifer yellow and oxidized low-density lipoprotein (oxLDL) uptake using DiI-labelled oxLDL in THP-1 macrophages and human monocyte-derived macrophages (HMDMs). IL-6, TNF-α and prostaglandin E2 (PGE2) release by lipopolysaccharide (LPS)-stimulated PBMCs from RA patients and HCs was measured by ELISA. The transcriptomic profile of PNLA-treated, LPS-activated PBMCs was investigated by RNA-sequencing. RESULTS: PNLA reduced THP-1 cell migration by 55% (P < 0.001). Macropinocytosis and DiI-oxLDL uptake were reduced by 50% (P < 0.001) and 40% (P < 0.01), respectively, in THP-1 macrophages and 40% (P < 0.01) and 25% (P < 0.05), respectively, in HMDMs. PNLA reduced IL-6 and TNF-α release from LPS-stimulated PBMCs from RA patients by 60% (P < 0.001) and from HCs by 50% and 35%, respectively (P < 0.01). PNLA also reduced PGE2 levels in such PBMCs from RA patients and HCs (P < 0.0001). Differentially expressed genes whose expression was upregulated included pyruvate dehydrogenase kinase-4, plasminogen activator inhibitor-1, fructose bisphosphatase1 and N-Myc downstream-regulated gene-2, which have potential roles in regulating immune and metabolic pathways. Pathway analysis predicted upstream activation of the nuclear receptors peroxisome proliferator-activated receptors involved in anti-inflammatory processes, and inhibition of nuclear factor-κB and signal transducer and activator of transcription 1. CONCLUSIONS: PNLA has immune-metabolic effects on monocytes and PBMCs that are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation may be beneficial in RA.


Assuntos
Leucócitos Mononucleares/efeitos dos fármacos , Ácidos Linolênicos/farmacologia , Artrite Reumatoide , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Dinoprostona/metabolismo , Perfilação da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/efeitos dos fármacos , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo
4.
J Lipid Res ; 62: 100094, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34171322

RESUMO

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx). The precise structure and localization of LOX-oxidized EOx in the human epidermis is unknown, as is their regulation in diseases such as psoriasis, one of the most common inflammatory diseases affecting the skin. Here, using precursor LC/MS/MS, we characterized multiple intermediates of EOx, including 9-HODE, 9,10-epoxy-13-HOME, and 9,10,13-TriHOME, in healthy human epidermis likely to be formed via the epidermal LOX pathways. The top layers of the skin contained more LA, 9-HODE, and 9,10,13-TriHOME EOSs, whereas 9,10-epoxy-13-HOME EOS was more prevalent deeper in the stratum corneum. In psoriatic lesions, levels of native EOx and free HODEs and HOMEs were significantly elevated, whereas oxidized species were generally reduced. A transcriptional network analysis of human psoriatic lesions identified significantly elevated expression of the entire biosynthetic/metabolic pathway for oxygenated ceramides, suggesting a regulatory function for EOx lipids in reconstituting epidermal integrity. The role of these new lipids in progression or resolution of psoriasis is currently unknown. We also discovered the central coordinated role of the zinc finger protein transcription factor, ZIC1, in driving the phenotype of this disease. In summary, long-chain oxygenated ceramide metabolism is dysregulated at the lipidomic level in psoriasis, likely driven by the transcriptional differences also observed, and we identified ZIC1 as a potential regulatory target for future therapeutic interventions.


Assuntos
Ceramidas/biossíntese , Ácido Linoleico/biossíntese , Lipidômica , Psoríase/metabolismo , Ceramidas/química , Ceramidas/genética , Humanos , Ácido Linoleico/química , Ácido Linoleico/genética , Estrutura Molecular , Psoríase/genética
5.
Pain Rep ; 6(4): e956, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35128295

RESUMO

INTRODUCTION: Negative affect, including anxiety and depression, is prevalent in chronic pain states such as osteoarthritis (OA) and associated with greater use of opioid analgesics, potentially contributing to present and future opioid crises. OBJECTIVES: We tested the hypothesis that the interaction between anxiety, chronic pain, and opioid use results from altered endogenous opioid function. METHODS: A genetic model of negative affect, the Wistar-Kyoto (WKY) rat, was combined with intra-articular injection of monosodium iodoacetate (MIA; 1 mg) to mimic clinical presentation. Effects of systemic morphine (0.5-3.5 mg·kg-1) on pain behaviour and spinal nociceptive neuronal activity were compared in WKY and normo-anxiety Wistar rats 3 weeks after MIA injection. Endogenous opioid function was probed by the blockade of opioid receptors (0.1-1 mg·kg-1 systemic naloxone), quantification of plasma ß-endorphin, and expression and phosphorylation of spinal mu-opioid receptor (MOR). RESULTS: Monosodium iodoacetate-treated WKY rats had enhanced OA-like pain, blunted morphine-induced analgesia, and greater mechanical hypersensitivity following systemic naloxone, compared with Wistar rats, and elevated plasma ß-endorphin levels compared with saline-treated WKY controls. Increased MOR phosphorylation at the master site (serine residue 375) in the spinal cord dorsal horn of WKY rats with OA-like pain (P = 0.0312) indicated greater MOR desensitization. CONCLUSIONS: Reduced clinical analgesic efficacy of morphine was recapitulated in a model of high anxiety and OA-like pain, in which endogenous opioid tone was altered, and MOR function attenuated, in the absence of previous exogenous opioid ligand exposure. These findings shed new light on the mechanisms underlying the increased opioid analgesic use in high anxiety patients with chronic pain.

6.
PLoS One ; 15(9): e0239663, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32991618

RESUMO

The relationship between osteoarthritis (OA) structural change and pain is complex. Surgical models of OA in rodents are often rapid in onset, limiting mechanistic utility and translational validity. We aimed to investigate the effect of refining surgical small rodent models of OA on both joint pathology and pain behaviour. Adult male C57BL/6 mice (n = 76, 10-11 weeks of age at time of surgery) underwent either traditional (transection of the medial meniscotibial ligament [MMTL]) or modified (MMTL left intact, transection of the coronary ligaments) DMM surgery, or sham surgery. Adult male Sprague Dawley rats (n = 76, weight 175-199g) underwent either modified meniscal transection (MMNX) surgery (transection of the medial meniscus whilst the medial collateral ligament is left intact) or sham surgery. Pain behaviours (weight bearing asymmetry [in mice and rats] and paw withdrawal thresholds [in rats]) were measured pre-surgery and weekly up to 16 weeks post-surgery. Post-mortem knee joints were scored for cartilage damage, synovitis, and osteophyte size. There was a significant increase in weight bearing asymmetry from 13 weeks following traditional, but not modified, DMM surgery when compared to sham operated mice. Both traditional and modified DMM surgery led to similar joint pathology. There was significant pain behaviour from 6 weeks following MMNX model compared to sham operated control rats. Synovitis was significant 4 weeks after MMNX surgery, whereas significant chondropathy was first evident 8 weeks post-surgery, compared to sham controls. Pain behaviour is not always present despite significant changes in medial tibial plateau cartilage damage and synovitis, reflecting the heterogeneity seen in human OA. The development of a slowly progressing surgical model of OA pain in the rat suggests that synovitis precedes pain behaviour and that chondropathy is evident later, providing the foundations for future mechanistic studies into the disease.


Assuntos
Articulação do Joelho/patologia , Osteoartrite do Joelho/cirurgia , Dor/patologia , Animais , Comportamento Animal , Modelos Animais de Doenças , Articulação do Joelho/metabolismo , Masculino , Meniscectomia/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite do Joelho/patologia , Dor/etiologia , Fenótipo , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Medula Espinal/patologia , Sinovite/diagnóstico , Sinovite/etiologia
7.
Proc Natl Acad Sci U S A ; 116(16): 8038-8047, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30944221

RESUMO

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.


Assuntos
Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal , Fosfolipídeos , Angiotensinas/metabolismo , Animais , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/fisiopatologia , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , Modelos Animais de Doenças , Feminino , Lipoxigenase/genética , Lipoxigenase/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoE , Fosfolipídeos/genética , Fosfolipídeos/metabolismo
8.
Sci Rep ; 9(1): 4696, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886197

RESUMO

Clinically, osteoarthritis (OA) pain is significantly associated with synovial inflammation. Identification of the mechanisms driving inflammation could reveal new targets to relieve this prevalent pain state. Herein, a role of polyadenylation in OA synovial samples was investigated, and the potential of the polyadenylation inhibitor cordycepin (3' deoxyadenosine) to inhibit inflammation as well as to reduce pain and structural OA progression were studied. Joint tissues from people with OA with high or low grade inflammation and non-arthritic post-mortem controls were analysed for the polyadenylation factor CPSF4 and inflammatory markers. Effects of cordycepin on pain behavior and joint pathology were studied in models of OA (intra-articular injection of monosodium iodoacetate in rats and surgical destabilisation of the medial meniscus in mice). Human monocyte-derived macrophages and a mouse macrophage cell line were used to determine effects of cordycepin on nuclear localisation of the inflammatory transcription factor NFĸB and polyadenylation factors (WDR33 and CPSF4). CPSF4 and NFκB expression were increased in synovia from OA patients with high grade inflammation. Cordycepin reduced pain behaviour, synovial inflammation and joint pathology in both OA models. Stimulation of macrophages induced nuclear localisation of NFĸB and polyadenylation factors, effects inhibited by cordycepin. Knockdown of polyadenylation factors also prevented nuclear localisation of NFĸB. The increased expression of polyadenylation factors in OA synovia indicates a new target for analgesia treatments. This is supported by the finding that polyadenylation factors are required for inflammation in macrophages and by the fact that the polyadenylation inhibitor cordycepin attenuates pain and pathology in models of OA.


Assuntos
Artrite Experimental/tratamento farmacológico , Inflamação/tratamento farmacológico , Articulações/patologia , Osteoartrite/tratamento farmacológico , Dor/tratamento farmacológico , Animais , Desoxiadenosinas/uso terapêutico , Modelos Animais de Doenças , Humanos , Articulações/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Poliadenilação , Ratos , Transdução de Sinais
9.
Pain ; 160(3): 658-669, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30779717

RESUMO

Anxiety and depression are associated with increased pain responses in chronic pain states. The extent to which anxiety drives chronic pain, or vice versa, remains an important question that has implications for analgesic treatment strategies. Here, the effect of existing anxiety on future osteoarthritis (OA) pain was investigated, and potential mechanisms were studied in an animal model. Pressure pain detection thresholds, anxiety, and depression were assessed in people with (n = 130) or without (n = 100) painful knee OA. Separately, knee pain and anxiety scores were also measured twice over 12 months in 4730 individuals recruited from the general population. A preclinical investigation of a model of OA pain in normo-anxiety Sprague-Dawley (SD) and high-anxiety Wistar Kyoto (WKY) rats assessed underlying neurobiological mechanisms. Higher anxiety, independently from depression, was associated with significantly lower pressure pain detection thresholds at sites local to (P < 0.01) and distant from (P < 0.05) the painful knee in patients with OA. Separately, high anxiety scores predicted increased risk of knee pain onset in 3274 originally pain-free people over the 1-year period (odds ratio = 1.71; 95% confidence interval = 1.25-2.34, P < 0.00083). Similarly, WKY rats developed significantly lower ipsilateral and contralateral hind paw withdrawal thresholds in the monosodium iodoacetate model of OA pain, compared with SD rats (P = 0.0005). Linear regressions revealed that baseline anxiety-like behaviour was predictive of lowered paw withdrawal thresholds in WKY rats, mirroring the human data. This augmented pain phenotype was significantly associated with increased glial fibrillary acidic protein immunofluorescence in pain-associated brain regions, identifying supraspinal astrocyte activation as a significant mechanism underlying anxiety-augmented pain behaviour.


Assuntos
Ansiedade/etiologia , Astrócitos/fisiologia , Dor Crônica/complicações , Dor Musculoesquelética/complicações , Dor Musculoesquelética/patologia , Idoso , Animais , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Escalas de Graduação Psiquiátrica , Ratos Endogâmicos WKY , Ratos Sprague-Dawley
10.
J Pharmacol Exp Ther ; 366(3): 509-518, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29945931

RESUMO

The immunomodulatory prodrug 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), which acts as an agonist for sphingosine-1-phosphate (S1P) receptors (S1PR) when phosphorylated, is proposed as a novel pain therapeutic. In this study, we assessed FTY720-mediated antinociception in the radiant heat tail-flick test and in the chronic constriction injury (CCI) model of neuropathic pain in mice. FTY720 produced antinociception and antiallodynia, respectively, and these effects were dose-dependent and mimicked by the S1PR1-selective agonist CYM-5442. Repeated administration of FTY720 for 1 week produced tolerance to acute thermal antinociception, but not to antiallodynia in the CCI model. S1PR-stimulated [35S]GTPγS autoradiography revealed apparent desensitization of G protein activation by S1P or the S1PR1 agonist 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole (SEW-2871) throughout the brain. Similar results were seen in spinal cord membranes, whereby the Emax value of S1PR-stimulated [35S]GTPγS binding was greatly reduced in repeated FTY720-treated mice. These results suggest that S1PR1 is a primary target of FTY720 in alleviating both acute thermal nociception and chronic neuropathic nociception. Furthermore, the finding that tolerance develops to antinociception in the tail-flick test but not in chronic neuropathic pain suggests a differential mechanism of FTY720 action between these models. The observation that repeated FTY720 administration led to desensitized S1PR1 signaling throughout the central nervous system suggests the possibility that S1PR1 activation drives the acute thermal antinociceptive effects, whereas S1PR1 desensitization mediates the following: 1) tolerance to thermal antinociceptive actions of FTY720 and 2) the persistent antiallodynic effects of FTY720 in neuropathic pain by producing functional antagonism of pronociceptive S1PR1 signaling.


Assuntos
Cloridrato de Fingolimode/farmacologia , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Peptídeos Opioides/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/metabolismo , Temperatura , Animais , Modelos Animais de Doenças , Cloridrato de Fingolimode/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neuralgia/fisiopatologia , Receptores de Lisoesfingolipídeo/agonistas , Nociceptina
11.
Pain Rep ; 3(6): e685, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30706033

RESUMO

INTRODUCTION: The mounting evidence that osteoclasts play an important role in osteoarthritis (OA) pain lead us to investigate the effects of L-006235, a potent and selective inhibitor of cathepsin K, on pain behaviour and joint pathology in a model of OA pain. METHODS: Effects of preventative (30 and 100 mg/kg) and therapeutic (100 mg/kg) oral dosing with L-006235 on weight-bearing asymmetry, hind paw withdrawal thresholds, cartilage and bone pathology, synovial inflammation, and drug exposure were studied in the monosodium iodoacetate rat model of OA pain. RESULTS: Preventative L-006235 inhibited weight-bearing asymmetry from day 14, with this measure nearly abolished by the higher dose. In the same treatment setting, L-006235 prevented lowering of hind paw withdrawal thresholds from day 7. Exposure to L-006235 in plasma was higher for the 100 mg/kg dose, compared with 30 mg/kg. Therapeutic dosing with L-006235 from day 14 significantly inhibited weight-bearing asymmetry, compared with monosodium iodoacetate vehicle rats. Regression analysis revealed a significant interaction coefficient of the effects of L-006235 on weight-bearing asymmetry and synovitis score, but not for cartilage damage nor osteophyte scores. CONCLUSION: Our novel finding that cathepsin K inhibition is analgesic in a clinically relevant model of OA pain provides new evidence for the therapeutic potential of this target.

12.
Stem Cells Int ; 2017: 2905104, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29434641

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. METHODS: Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5 × 106 late passage MSCs labelled with 10 µg/ml SiMAG, 1.5 × 106 late passage mesenchymal stem cells, the steroid Kenalog (200 µg/20 µL), 1.5 × 106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. RESULTS: Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. CONCLUSION: Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain.

13.
Arthritis Rheumatol ; 69(5): 996-1008, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27860453

RESUMO

OBJECTIVE: Pain is a major symptom of osteoarthritis (OA); currently available analgesics either do not provide adequate pain relief or are associated with serious side effects. The aim of this study was to investigate the therapeutic potential of targeting the resolvin receptor system to modify OA pain and pathology. METHODS: Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)-hydroxy-docosahexaenoic acid (17[R]-HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)-HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry. RESULTS: There was a positive correlation between resolvin receptor and interleukin-6 (IL-6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL-1ß, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)-HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate-induced OA rat model. CONCLUSION: Our preclinical data demonstrate the robust analgesic effects of activation of the D series resolvin pathways in 2 different animal models of OA. Our data support a predominant central mechanism of action in clinically relevant models of OA pain.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Comportamento Animal/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Osteoartrite do Joelho/genética , Receptores de Quimiocinas/genética , Animais , Artralgia/induzido quimicamente , Cartilagem Articular/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/toxicidade , Expressão Gênica , Humanos , Ácido Iodoacético/toxicidade , Meniscos Tibiais/cirurgia , Neuroglia/citologia , Neuroglia/metabolismo , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Quimiocinas/efeitos dos fármacos , Receptores de Lipoxinas/efeitos dos fármacos , Receptores de Lipoxinas/genética , Medula Espinal/citologia , Medula Espinal/metabolismo , Membrana Sinovial/patologia
14.
J Neuroinflammation ; 13(1): 233, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27589850

RESUMO

BACKGROUND: Harnessing the actions of the resolvin pathways has the potential for the treatment of a wide range of conditions associated with overt inflammatory signalling. Aspirin-triggered resolvin D1 (AT-RvD1) has robust analgesic effects in behavioural models of pain; however, the potential underlying spinal neurophysiological mechanisms contributing to these inhibitory effects in vivo are yet to be determined. This study investigated the acute effects of spinal AT-RvD1 on evoked responses of spinal neurones in vivo in a model of acute inflammatory pain and chronic osteoarthritic (OA) pain and the relevance of alterations in spinal gene expression to these neurophysiological effects. METHODS: Pain behaviour was assessed in rats with established carrageenan-induced inflammatory or monosodium iodoacetate (MIA)-induced OA pain, and changes in spinal gene expression of resolvin receptors and relevant enzymatic pathways were examined. At timepoints of established pain behaviour, responses of deep dorsal horn wide dynamic range (WDR) neurones to transcutaneous electrical stimulation of the hind paw were recorded pre- and post direct spinal administration of AT-RvD1 (15 and 150 ng/50 µl). RESULTS: AT-RvD1 (15 ng/50 µl) significantly inhibited WDR neurone responses to electrical stimuli at C- (29 % inhibition) and Aδ-fibre (27 % inhibition) intensities. Both wind-up (53 %) and post-discharge (46 %) responses of WDR neurones in carrageenan-treated animals were significantly inhibited by AT-RvD1, compared to pre-drug response (p < 0.05). These effects were abolished by spinal pre-administration of a formyl peptide receptor 2 (FPR2/ALX) antagonist, butoxy carbonyl-Phe-Leu-Phe-Leu-Phe (BOC-2) (50 µg/50 µl). AT-RvD1 did not alter evoked WDR neurone responses in non-inflamed or MIA-treated rats. Electrophysiological effects in carrageenan-inflamed rats were accompanied by a significant increase in messenger RNA (mRNA) for chemerin (ChemR23) receptor and 5-lipoxygenase-activating protein (FLAP) and a decrease in 15-lipoxygenase (15-LOX) mRNA in the ipsilateral spinal cord of the carrageenan group, compared to controls. CONCLUSIONS: Our data suggest that peripheral inflammation-mediated changes in spinal FLAP expression may contribute to the novel inhibitory effects of spinal AT-RvD1 on WDR neuronal excitability, which are mediated by FPR2/ALX receptors. Inflammatory-driven changes in this pathway may offer novel targets for inflammatory pain treatment.


Assuntos
Anti-Inflamatórios/uso terapêutico , Aspirina/uso terapêutico , Dor Crônica/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/uso terapêutico , Medula Espinal/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Carragenina/toxicidade , Dor Crônica/etiologia , Dor Crônica/fisiopatologia , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Inibidores Enzimáticos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/complicações , Ácido Iodoacético/toxicidade , Masculino , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Osteoartrite/induzido quimicamente , Osteoartrite/complicações , Limiar da Dor/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/fisiologia
15.
Br J Pharmacol ; 173(21): 3134-3144, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27501482

RESUMO

BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. EXPERIMENTAL APPROACH: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight-bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. KEY RESULTS: Acute MJN110 (5 mg·kg-1 ) significantly reversed MIA-induced weight-bearing asymmetry (MIA/vehicle: 68 ± 6 g; MIA/MJN110: 35 ± 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 ± 0.8 g; MIA/MJN110: 11 ± 0.6 g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg-1 ) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg-1 ) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane-associated PGE synthase-1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle-treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-arachidonoylglycerol. CONCLUSIONS AND IMPLICATIONS: Our data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain.


Assuntos
Analgésicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ácido Iodoacético/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Osteoartrite/tratamento farmacológico , Dor/tratamento farmacológico , Analgésicos/administração & dosagem , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Injeções Intra-Articulares , Ácido Iodoacético/administração & dosagem , Masculino , Monoacilglicerol Lipases/metabolismo , Medição da Dor , Ratos , Ratos Sprague-Dawley
16.
Handb Exp Pharmacol ; 227: 119-43, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25846617

RESUMO

Preparations of the Cannabis sativa plant have been used to analgesic effect for millenia, but only in recent decades has the endogenous system responsible for these effects been described. The endocannabinoid (EC) system is now known to be one of the key endogenous systems regulating pain sensation, with modulatory actions at all stages of pain processing pathways. The EC system is composed of two main cannabinoid receptors (CB1 and CB2) and two main classes of endogenous ligands or endocannabinoids (ECs). The receptors have distinct expression profiles, with CB1 receptors found at presynaptic sites throughout the peripheral and central nervous systems (PNS and CNS, respectively), whilst CB2 receptor is found principally (but not exclusively) on immune cells. The endocannabinoid ligands are lipid neurotransmitters belonging to either the N-acyl ethanolamine (NAEs) class, e.g. anandamide (AEA), or the monoacylglycerol class, e.g. 2-arachidonoyl glycerol (2-AG). Both classes are short-acting transmitter substances, being synthesised on demand and with signalling rapidly terminated by specific enzymes. ECs acting at CB1 negatively regulate neurotransmission throughout the nervous system, whilst those acting at CB2 regulate the activity of CNS immune cells. Signalling through both of these receptor subtypes has a role in normal nociceptive processing and also in the development resolution of acute pain states. In this chapter, we describe the general features of the EC system as related to pain and nociception and discuss the wealth of preclinical and clinical data involving targeting the EC system with focus on two areas of particular promise: modulation of 2-AG signalling via specific enzyme inhibitors and the role of spinal CB2 in chronic pain states.


Assuntos
Endocanabinoides/fisiologia , Dor/fisiopatologia , Animais , Humanos , Receptores de Canabinoides/fisiologia , Medula Espinal/fisiologia
17.
Neurosci Lett ; 576: 51-5, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-24909619

RESUMO

Mechanisms that may underlie age and sex differences in the pharmacological effects of cannabinoids are relatively unexplored. The purpose of the present study was to determine whether sex differences in metabolism of Δ(9)-tetrahydrocannabinol (THC), similar to those observed previously in adult rats, also occurred in adolescent rats and might contribute to age and sex differences in its in vivo pharmacology. Male and female adolescent rats were exposed to THC acutely or repeatedly for 10 days. Subsequently, some of the rats were sacrificed and blood and brain levels of THC and one of its metabolites, 11-hydroxy-Δ(9)-THC (11-OH-THC), were measured. Other rats were evaluated in a battery of in vivo tests that are sensitive to cannabinoids. Concentrations of 11-OH-THC in the brains of female adult and adolescent rats exceeded those observed in male conspecifics, particularly after repeated THC administration. In contrast, brain levels of THC did not differ between the sexes. In vivo, acute THC produced dose-related hypothermia, catalepsy and suppression of locomotion in adolescent rats of both sexes, with tolerance developing after repeated administration. With a minor exception, sex differences in THC's effects in the in vivo assays were not apparent. Together with previous findings, the present results suggest that sex differences in pharmacokinetics cannot fully explain the patterns of sex differences (and lack of sex differences) in cannabinoid effects across behaviors. Hormonal and/or pharmacodynamic factors are also likely to play a role.


Assuntos
Dronabinol/metabolismo , Dronabinol/farmacologia , Psicotrópicos/metabolismo , Psicotrópicos/farmacologia , Fatores Etários , Animais , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Dronabinol/administração & dosagem , Feminino , Masculino , Atividade Motora/efeitos dos fármacos , Psicotrópicos/administração & dosagem , Ratos Long-Evans , Fatores Sexuais
18.
Eur J Pharmacol ; 737: 97-105, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24858366

RESUMO

The mechanism through which marijuana produces its psychoactive effects is Δ(9)-tetrahydrocannabinol (THC)-induced activation of cannabinoid CB1 receptors. These receptors are normally activated by endogenous lipids, including anandamide and 2-arachidonoyl glycerol (2-AG). A logical "first step" in determination of the role of these endocannabinoids in THC׳s psychoactive effects is to investigate the degree to which pharmacologically induced increases in anandamide and/or 2-AG concentrations through exogenous administration and/or systemic administration of inhibitors of their metabolism, fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), respectively, share THC׳s discriminative stimulus effects. To this end, adult male mice and rats were trained to discriminate THC (5.6 and 3mg/kg, respectively). In Experiment 1, exogenous administration of anandamide or 2-AG did not substitute for THC in mice nor was substitution enhanced by co-administration of the FAAH or MAGL inhibitors, URB597 and N-arachidonyl maleimide (NAM), respectively. Significant decreases in responding may have prevented assessment of adequate endocannabinoid doses. In mice trained at higher baseline response rates (Experiment 2), the FAAH inhibitor PF3845 (10mg/kg) enhanced anandamide substitution for THC without producing effects of its own. The MAGL inhibitor JZL184 increased brain levels of 2-AG in vitro and in vivo, increased THC-like responding without co-administration of 2-AG. In rats, neither URB597 nor JZL184 engendered significant THC-appropriate responding, but co-administration of these two enzyme inhibitors approached full substitution. The present results highlight the complex interplay between anandamide and 2-AG and suggest that endogenous increases of both endocannabinoids are most effective in elicitation of THC-like discriminative stimulus effects.


Assuntos
Ácidos Araquidônicos/metabolismo , Discriminação Psicológica/efeitos dos fármacos , Dronabinol/farmacologia , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Amidoidrolases/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Monoacilglicerol Lipases/antagonistas & inibidores , Ratos , Receptor CB1 de Canabinoide/metabolismo
19.
Ann Rheum Dis ; 73(8): 1558-65, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23723320

RESUMO

BACKGROUND: Increased subchondral bone turnover may contribute to pain in osteoarthritis (OA). OBJECTIVES: To investigate the analgesic potential of a modified version of osteoprotegerin (osteoprotegerin-Fc (OPG-Fc)) in the monosodium iodoacetate (MIA) model of OA pain. METHODS: Male Sprague Dawley rats (140-260 g) were treated with either OPG-Fc (3 mg/kg, subcutaneously) or vehicle (phosphate-buffered saline) between days 1 and 27 (pre-emptive treatment) or days 21 and 27 (therapeutic treatment) after an intra-articular injection of MIA (1 mg/50 µl) or saline. A separate cohort of rats received the bisphosphonate zoledronate (100 µg/kg, subcutaneously) between days 1 and 25 post-MIA injection. Incapacitance testing and von Frey (1-15 g) hind paw withdrawal thresholds were used to assess pain behaviour. At the end of the study, rats were killed and the knee joints and spinal cord removed for analysis. Immunohistochemical studies using Iba-1 and GFAP quantified levels of activation of spinal microglia and astrocytes, respectively. Joint sections were stained with haematoxylin and eosin or Safranin-O fast green and scored for matrix proteoglycan and overall joint morphology. The numbers of tartrate-resistant acid phosphatase-positive osteoclasts were quantified. N=10 rats/group. RESULTS: Pre-emptive treatment with OPG-Fc significantly attenuated the development of MIA-induced changes in weightbearing, but not allodynia. OPG-Fc decreased osteoclast number, inhibited the formation of osteophytes and improved structural pathology within the joint similarly to the decrease seen after pretreatment with the bisphosphonate, zoledronate. Therapeutic treatment with OPG-Fc decreased pain behaviour, but did not improve pathology in rats with established joint damage. CONCLUSIONS: Our data suggest that early targeting of osteoclasts may reduce pain associated with OA.


Assuntos
Artralgia/tratamento farmacológico , Artralgia/patologia , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Osteoprotegerina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Remodelação Óssea/efeitos dos fármacos , Difosfonatos/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Ácido Iodoacético/farmacologia , Articulações/efeitos dos fármacos , Articulações/patologia , Masculino , Nociceptores/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteófito/tratamento farmacológico , Osteófito/patologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Ácido Zoledrônico
20.
Ann Rheum Dis ; 73(9): 1710-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23852764

RESUMO

OBJECTIVES: Nerve growth factor (NGF) is a promising analgesic target, particularly in osteoarthritis (OA) where existing therapies are inadequate. We hypothesised that pain responses to NGF are increased in OA joints. Here, NGF-evoked pain behaviour was compared in two rodent models of OA, and possible mechanisms underlying altered pain responses were examined. METHODS: OA was induced in rat knees by meniscal transection (MNX) or intra-articular monosodium iodoacetate injection (MIA). Once OA pathology was fully established (day 20), we assessed pain behaviour (hindlimb weight-bearing asymmetry and hindpaw mechanical withdrawal thresholds) evoked by intra-articular injection of NGF (10 µg). Possible mechanisms underlying alterations in NGF-induced pain behaviour were explored using indomethacin pretreatment, histopathological evaluation of synovitis, and rtPCR for NGF receptor (tropomyosin receptor kinase (Trk)-A) expression in dorsal root ganglia (DRG). RESULTS: Both the MIA and MNX models of OA displayed reduced ipsilateral weight bearing and hindpaw mechanical withdrawal thresholds, mild synovitis and increased TrkA expression in DRG. NGF injection into OA knees produced a prolonged augmentation of weight-bearing asymmetry, compared to NGF injection in non-osteoarthritic knees. However, hindpaw mechanical withdrawal thresholds were not further decreased by NGF. Pretreatment with indomethacin attenuated NGF-facilitated weight-bearing asymmetry and reversed OA-induced ipsilateral TrkA mRNA up-regulation. CONCLUSIONS: OA knees were more sensitive to NGF-induced pain behaviour compared to non-osteoarthritic knees. Cyclo-oxygenase products may contribute to increased TrkA expression during OA development, and the subsequent increased NGF sensitivity. Treatments that reduce sensitivity to NGF have potential to improve OA pain.


Assuntos
Artrite Experimental/complicações , Fator de Crescimento Neural/toxicidade , Osteoartrite/complicações , Dor/etiologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gânglios Espinais/metabolismo , Indometacina/uso terapêutico , Injeções Intra-Articulares , Masculino , Fator de Crescimento Neural/administração & dosagem , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Dor/induzido quimicamente , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptor trkA/biossíntese , Receptor trkA/genética , Sinovite/induzido quimicamente , Regulação para Cima/efeitos dos fármacos , Suporte de Carga/fisiologia
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