RESUMEN
Osteoarthritis (OA) treatment is limited by the lack of effective nonsurgical interventions to slow disease progression. Here, we examined the contributions of the subchondral bone properties to OA development. We used parathyroid hormone (PTH) to modulate bone mass before OA initiation and alendronate (ALN) to inhibit bone remodeling during OA progression. We examined the spatiotemporal progression of joint damage by combining histopathological and transcriptomic analyses across joint tissues. The additive effect of PTH pretreatment before OA initiation and ALN treatment during OA progression most effectively attenuated load-induced OA pathology. Individually, PTH directly improved cartilage health and slowed the development of cartilage damage, whereas ALN primarily attenuated subchondral bone changes associated with OA progression. Joint damage reflected early transcriptomic changes. With both treatments, the structural changes were associated with early modulation of immunoregulation and immunoresponse pathways that may contribute to disease mechanisms. Overall, our results demonstrate the potential of subchondral bone-modifying therapies to slow the progression of OA.
Asunto(s)
Cartílago Articular , Osteoartritis , Hormona Paratiroidea , Animales , Ratones , Alendronato/farmacología , Alendronato/uso terapéutico , Huesos , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Hormona Paratiroidea/farmacología , Hormona Paratiroidea/uso terapéutico , Remodelación Ósea/efectos de los fármacos , Soporte de PesoRESUMEN
The extensive use of opioids for chronic pain management has contributed significantly to the current opioid epidemic. While many alternative nonopioid analgesics are available, opioids remain the most potent analgesics for moderate to severe pain management. In addition to the implementation of multimodal analgesia, there is a pressing need for the development of more effective and safer opioids. In this study, we developed a thermoresponsive N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based hydromorphone (HMP) prodrug (ProGel-HMP, HMP content = 16.2 wt %, in base form). The aqueous solution of ProGel-HMP was free-flowing at 4 °C but became a hydrogel when the temperature was raised to ≥37 °C, allowing sustained local retention when administered in vivo. When tested in the destabilization of the medial meniscus (DMM) mouse model of osteoarthritis (OA), ProGel-HMP was retained after intra-articular injection in the OA knee joint for at least 2 weeks postinjection, with low extra-articular distribution. ProGel-HMP was not detected in the central nervous system (CNS). A single dose of ProGel-HMP produced rapid and sustained joint pain resolution for greater than 14 days when compared to saline and dose-equivalent HMP controls, likely mediated through peripheral µ-opioid receptors in the knee joint. Systemic analgesia effect was absent in the DMM mice treated with ProGel-HMP, as evident in the lack of difference in tail flick response between the ProGel-HMP-treated mice and the controls (i.e., Healthy, Saline, and Sham). Repeated dosing of ProGel-HMP did not induce tolerance. Collectively, these data support the further development of ProGel-HMP as a potent, safe, long-acting and nonaddictive analgesic for better clinical pain management.
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Analgesia , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Osteoartritis , Profármacos , Ratones , Animales , Hidromorfona , Manejo del Dolor , Profármacos/uso terapéutico , Dolor/tratamiento farmacológico , Analgésicos Opioides/efectos adversos , Analgésicos/uso terapéuticoRESUMEN
In this study, we aimed to assess the analgesic efficacy of a thermoresponsive polymeric dexamethasone (Dex) prodrug (ProGel-Dex) in a mouse model of osteoarthritis (OA). At 12 weeks post model establishment, the OA mice received a single intra-articular (IA) injection of ProGel-Dex, dose-equivalent Dex, or Saline. Comparing to Saline and Dex controls, ProGel-Dex provided complete and sustained pain relief for >15 weeks according to incapacitance tests. In vivo optical imaging confirmed the continuous presence of ProGel-Dex in joints for 15 weeks post-injection. According to micro-CT analysis, ProGel-Dex treated mice had significantly lower subchondral bone thickness and medial meniscus bone volume than Dex and Saline controls. Except for a transient delay of body weight increase and slightly lower endpoint liver and spleen weights, no other adverse effect was observed after ProGel-Dex treatment. These findings support ProGel-Dex's potential as a potent and safe analgesic candidate for management of OA pain.
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Osteoartritis , Profármacos , Ratones , Animales , Dexametasona/farmacología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Profármacos/farmacología , Profármacos/uso terapéutico , Osteoartritis/tratamiento farmacológico , Artralgia/inducido químicamente , Artralgia/tratamiento farmacológico , Analgésicos/farmacología , Analgésicos/uso terapéuticoRESUMEN
BACKGROUND: The aim of the study was to examine the relationship among patients' characteristics, intraoperative pathology and pre/post-operative symptoms in a cohort of patients undergoing arthroscopic partial meniscectomy for symptomatic meniscal tears. METHODS: Clinical data were collected (age, sex, body mass index, time to surgery, trauma). Intraoperative cartilage pathology was assessed with Outerbridge score. Meniscal tears were graded with the ISAKOS classification. Synovial inflammation was scored using the Macro-score. Patient symptoms were assessed pre/post-operatively using the KOOS instrument. RESULTS: In the series of 109 patients (median age 47 years), 50% of the meniscal tears were traumatic; 85% of patients showed mild to moderate synovitis; 52 (47.7%) patients had multiple cartilage defects and 31 (28.4%) exhibited a single focal chondral lesion. Outerbridge scores significantly correlated with patient age, BMI and synovial inflammation. There was a correlation between severity of chondral pathology and high-grade synovial hyperplasia. Pre-operative KOOS correlated with BMI, meniscal degenerative changes and symptom duration. Obesity, time to surgery, presence of high-grade synovial hyperplasia and high-grade cartilage lesions were independent predictors of worse post-operative pain and function. CONCLUSION: We demonstrated that pre-operative symptoms and post-operative outcomes correlate with synovitis severity and cartilage pathology, particularly in old and obese patients that underwent arthroscopic partial meniscectomy. Importantly, patients with a degenerative meniscal pattern and with longer time to surgery experienced more severe cartilage damage and, consequentially, pain and dysfunction. These findings are fundamental to identify patients suitable for earlier interventions.
RESUMEN
Objective: Clinical evidence suggests that abnormal mechanical forces play a major role in the initiation and progression of osteoarthritis (OA). However, few studies have examined the mechanical environment that leads to disease. Thus, using a mouse tibial loading model, we quantified the cartilage contact stresses and examined the effects of altering tissue material properties on joint stresses during loading. Design: Using a discrete element model (DEA) in conjunction with joint kinematics data from a murine knee joint compression model, the magnitude and distribution of contact stresses in the tibial cartilage during joint loading were quantified at levels ranging from 0 to 9 N in 1 N increments. In addition, a simplified finite element (FEA) contact model was developed to simulate the knee joint, and parametric analyses were conducted to investigate the effects of altering bone and cartilage material properties on joint stresses during compressive loading. Results: As loading increased, the peak contact pressures were sufficient to induce fibrillations on the cartilage surfaces. The computed areas of peak contact pressures correlated with experimentally defined areas of highest cartilage damage. Only alterations in cartilage properties and geometry caused large changes in cartilage contact pressures. However, changes in both bone and cartilage material properties resulted in significant changes in stresses induced in the bone during compressive loading. Conclusions: The level of mechanical stress induced by compressive tibial loading directly correlated with areas of biological change observed in the mouse knee joint. These results, taken together with the parametric analyses, are the first to demonstrate both experimentally and computationally that the tibial loading model is a useful preclinical platform with which to predict and study the effects of modulating bone and/or cartilage properties on attenuating OA progression. Given the direct correlation between computational modeling and experimental results, the effects of tissue-modifying treatments may be predicted prior to in vivo experimentation, allowing for novel therapeutics to be developed.
RESUMEN
Due to their potent immunosuppressive and anti-inflammatory effects, glucocorticoids (GCs) are the most widely used medications in treating lupus nephritis (LN). Long-term use of GCs, however, is associated with numerous off-target adverse effects. To reduce GCs' adverse effects, we previously developed two polymeric dexamethasone prodrug nanomedicines: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based dexamethasone prodrug (P-Dex), and micelle-forming polyethylene glycol (PEG)-based dexamethasone prodrug (ZSJ-0228). Both P-Dex and ZSJ-0228 provided sustained amelioration of LN in lupus-prone NZB/W F1 mice with reduced GC-associated adverse effects. Here, we have extended our investigation to the MRL/lpr mouse model of LN. Compared to dose equivalent daily dexamethasone sodium phosphate (Dex) treatment, monthly P-Dex or ZSJ-0228 treatments were more effective in reducing proteinuria and extending the lifespan of MRL/lpr mice. Unlike the daily Dex treatment, ZSJ-0228 was not associated with measurable GC-associated adverse effects. In contrast, adrenal gland atrophy was observed in P-Dex treated mice.
Asunto(s)
Nefritis Lúpica , Profármacos , Animales , Dexametasona/farmacología , Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Riñón , Nefritis Lúpica/tratamiento farmacológico , Ratones , Ratones Endogámicos MRL lpr , Ratones Endogámicos NZB , Polímeros/farmacología , Profármacos/farmacología , Profármacos/uso terapéuticoRESUMEN
The menisci exert a prominent role in joint stabilization and in the distribution of mechanical loading. Meniscal damage is associated with increased risk of knee OA. The aim of this study was to characterize the synovial membrane and meniscal tissues in patients undergoing arthroscopic partial meniscectomy for meniscal tear and to evaluate association with clinical outcomes. A total of 109 patients were recruited. Demographic and clinical data were collected. Visual Analogic Scale (VAS) measuring pain and Knee injury and Osteoarthritis Outcome Score (KOOS) were recorded at baseline and at 2-years follow-up. Histological and immunohistochemical characterizations were performed on synovial membranes and meniscal tissues. More than half of the patients demonstrated synovial mononuclear cell infiltration and hyperplasia. Synovial fibrosis was present in most of the patients; marked vascularity and CD68 positivity were observed. Inflammation had an impact on both pain and knee symptoms. Patients with synovial inflammation had higher values of pre-operative VAS and inflammation. Higher pre-operative pain was observed in patients with meniscal MMP-13 production. In conclusion, multivariate analysis showed that synovial inflammation was associated with pre-operative total KOOS scores, knee symptoms, and pain. Moreover, meniscal MMP-13 expression was found to be associated with pre-operative pain in multivariate analysis. Thus, targeting inflammation of the synovial membrane and meniscus might reduce clinical symptoms and dysfunction at the time of surgery.
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Menisco , Lesiones de Menisco Tibial , Humanos , Inflamación/patología , Metaloproteinasa 13 de la Matriz , Meniscectomía/efectos adversos , Meniscos Tibiales/patología , Meniscos Tibiales/cirugía , Menisco/cirugía , Dolor/patología , Lesiones de Menisco Tibial/complicaciones , Lesiones de Menisco Tibial/cirugíaRESUMEN
Intra-articular (IA) glucocorticoids (GC) are commonly used for clinical management of both osteoarthritis and rheumatoid arthritis, but their efficacy is limited by the relatively short duration of action and associated side effects. To provide sustained efficacy and to improve the safety of GCs, we previously developed a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based dexamethasone (Dex) prodrug. Serendipitously, we discovered that, by increasing the Dex content of the prodrug to unusually high levels, the aqueous solution of the polymeric prodrug becomes thermoresponsive, transitioning from a free-flowing liquid at 4 °C to a hydrogel at 30 °C or greater. Upon IA injection, the prodrug solution forms a hydrogel (ProGel-Dex) that is retained in the joint for more than 1 month, where it undergoes gradual dissolution, releasing the water-soluble polymeric prodrug. The released prodrug is swiftly internalized and intracellularly processed by phagocytic synoviocytes to release free Dex, resulting in sustained amelioration of joint inflammation and pain in rodent models of inflammatory arthritis and osteoarthritis. The low molecular weight (6.8 kDa) of the ProGel-Dex ensures rapid renal clearance once it escapes the joint, limiting systemic GC exposure and risk of potential off-target side effects. The present study illustrates the translational potential of ProGel-Dex as a potent opioid-sparing, locally delivered adjuvant analgesic for sustained clinical management of arthritis pain and inflammation. Importantly, the observed thermoresponsive properties of the prodrug establishes ProGel as a platform technology for the local delivery of a broad spectrum of therapeutic agents to treat a diverse array of pathological conditions.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Profármacos , Animales , Artritis Experimental/tratamiento farmacológico , Dexametasona , DolorRESUMEN
Glucocorticoids (GCs) are widely used in the clinical management of lupus nephritis (LN). Their long-term use, however, is associated with the risk of significant systemic side effects. We have developed a poly(ethylene glycol) (PEG)-based dexamethasone (Dex) prodrug (i.e., ZSJ-0228) and in a previous study, demonstrated its potential therapeutic efficacy in mice with established LN, while avoiding systemic GC-associated toxicity. In the present study, we have employed a dose-escalation design to establish the optimal dose-response relationships for ZSJ-0228 in treating LN and further investigated the safety of ZSJ-0228 in lupus-prone NZB/W F1 mice with established nephritis. ZSJ-0228 was intravenously (i.v.) administered monthly at four levels: 0.5 (L1), 1.0 (L2), 3.0 (L3), and 8.0 (L4) mg/kg/day Dex equivalent. For controls, mice were treated with i.v. saline every 4 weeks. In addition, a group of mice received intraperitoneal injections (i.p.) of Dex every day or i.v. injections of Dex every four weeks. Treatment of mice with LN with ZSJ-0228 dosed at L1 resulted in the resolution of proteinuria in 14% of the mice. Mice treated with ZSJ-0228 dosed at L2 and L3 levels resulted in the resolution of proteinuria in â¼60% of the mice in both groups. Treatment with ZSJ-0228 dosed at L4 resulted in the resolution of proteinuria in 30% of the mice. The reduction and/or resolution of the proteinuria, improvement in renal histological scores, and survival data indicate that the most effective dose range for ZSJ-0228 in treating LN in NZB/W F1 mice is between 1.0 and 3.0 mg/kg/day Dex equivalent. Typical GC-associated side effects (e.g., osteopenia, adrenal glands atrophy, etc.) were not observed in any of the ZSJ-0228 treatment groups, confirming its excellent safety profile.
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Dexametasona/administración & dosificación , Nefritis Lúpica/tratamiento farmacológico , Animales , Dexametasona/efectos adversos , Dexametasona/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Nefritis Lúpica/inmunología , Ratones , Polietilenglicoles , Profármacos/administración & dosificación , Profármacos/químicaRESUMEN
Osteoarthritis is increasingly viewed as a heterogeneous disease with multiple phenotypic subgroups. Obesity enhances joint degeneration in mouse models of posttraumatic osteoarthritis (PTOA). Most models of PTOA involve damage to surrounding tissues caused by surgery/fracture; it is unclear if obesity enhances cartilage degeneration in the absence of surgery/fracture. We used a nonsurgical animal model of load-induced PTOA to determine the effect of obesity on cartilage degeneration 2 weeks after loading. Cartilage degeneration was caused by a single bout of cyclic tibial loading at either a high or moderate load magnitude in adult male mice with severe obesity (C57Bl6/J + high-fat diet), mild obesity (toll-like receptor 5 deficient mouse [TLR5KO]), or normal adiposity (C57Bl6/J mice + normal diet and TLR5KO mice in which obesity was prevented by manipulation of the gut microbiome). Two weeks after loading, cartilage degeneration occurred in limbs loaded at a high magnitude, as determined by OARSI scores (P < .001). However, the severity of cartilage damage did not differ among groups. Osteophyte width and synovitis of loaded limbs did not differ among groups. Furthermore, obesity did not enhance cartilage damage in limbs evaluated 6 weeks after loading. Constituents of the gut microbiota differed among groups. Our findings suggest that, in the absence of surgery/fracture, obesity may not influence cartilage loss after a single mechanical insult, suggesting that either damage to surrounding tissues or repeated mechanical insult is necessary for obesity to influence cartilage degeneration. These findings further illustrate heterogeneity in PTOA phenotypes and complex interactions between mechanical/metabolic factors in cartilage loss.
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Cartílago Articular/patología , Obesidad/complicaciones , Osteoartritis/etiología , Tibia/lesiones , Animales , Microbioma Gastrointestinal , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis/patología , Soporte de PesoRESUMEN
Despite their notorious adverse effects, glucocorticoids (GC, potent anti-inflammatory drugs) are used extensively in clinical management of rheumatoid arthritis (RA) and other chronic inflammatory diseases. To achieve a sustained therapeutic efficacy and reduced toxicities, macromolecular GC prodrugs have been developed with promising outcomes for the treatment of RA. Fine-tuning the activation kinetics of these prodrugs may further improve their therapeutic efficacy and minimize the off-target adverse effects. To assess the feasibility of this strategy, five different dexamethasone (Dex, a potent GC)-containing monomers with distinctively different linker chemistries were designed, synthesized, and copolymerized with N-(2-hydroxypropyl) methacrylamide (HPMA) to obtain 5 macromolecular Dex prodrugs. Their Dex releasing rates were analyzed in vitro and shown to display a wide spectrum of activation kinetics. Their therapeutic efficacy and preliminary toxicology profiles were assessed and compared in vivo in an adjuvant-induced arthritis (AA) rat model in order to identify the ideal prodrug design for the most effective and safe treatment of inflammatory arthritis. The in vivo data demonstrated that the C3 hydrazone linker-containing prodrug design was the most effective in preserving joint structural integrity. The results from this study suggest that the design and screening of different activation mechanisms may help to identify macromolecular prodrugs with the most potent therapeutic efficacy and safety for the management of inflammatory arthritis.
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Artritis Experimental , Profármacos , Animales , Artritis Experimental/tratamiento farmacológico , Dexametasona , Metacrilatos , RatasRESUMEN
BACKGROUND: Hip osteoarthritis (HOA) is the most common hip disorder and a major cause of disability in the adult population, with an estimated prevalence of end-stage disease and total hip replacement. Thus, the diagnosis, prevention, and treatment of the early stages of the disease in young adults are crucial to reduce the incidence of end-stage HOA. The purpose of this study was to determine whether (1) a relationship among the inflammatory status of labrum and synovium collected from patients with femoroacetabular impingement (FAI) would exist; and (2) to investigate the associations among the histopathological features of joint tissues, the pre-operative symptoms and the post-operative outcomes after arthroscopic surgery. METHODS: Joint tissues from 21 patients undergoing hip arthroscopy for FAI were collected and their histological and immunohistochemical features were correlated with clinical parameters. RESULTS: Synovial mononuclear cell infiltration was observed in 25% of FAI patients, inversely correlated with the hip disability and osteoarthritis outcome score (HOOS) pain and function subscales and with the absolute and relative change in total HOOS. All the labral samples showed some pattern of degeneration and 67% of the samples showed calcium deposits. The total labral score was associated with increased CD68 positive cells in the synovium. The presence of labral calcifications, along with the chondral damage worsened the HOOS post-op symptoms (adjusted R-square = 0.76 p = 0.0001). CONCLUSIONS: Our study reveals a relationship between the histologic labral features, the synovial inflammation, and the cartilage condition at the time of FAI. The presence of labral calcifications, along with the cartilage damage and the synovitis negatively affects the post-operative outcomes in patients with FAI.
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Artralgia/cirugía , Artroplastia de Reemplazo de Cadera/métodos , Calcinosis/cirugía , Pinzamiento Femoroacetabular/cirugía , Articulación de la Cadera/cirugía , Osteoartritis de la Cadera/cirugía , Adulto , Artralgia/patología , Artroplastia de Reemplazo de Cadera/tendencias , Calcinosis/patología , Femenino , Pinzamiento Femoroacetabular/patología , Articulación de la Cadera/patología , Humanos , Masculino , Osteoartritis de la Cadera/patologíaRESUMEN
The human meniscus plays a crucial role for transmission and distribution of load across the knee, as well as shock absorption, joint stability, lubrication, and congruity. The aim of this study was to compare the complex geometry, and unique ultrastructure and tissue composition of the meniscus in healthy (control) and pathological conditions to provide understanding of structural changes that could be helpful in the future design of targetted therapies and improvement of treatment indications. We analyzed meniscus samples collected from 3 healthy multi-organ donors (median age, 66 years), 5 patients with traumatic meniscal tear (median age, 41 years) and 3 patients undergoing total knee replacement (TKR) for end-stage osteoarthritis (OA) (median age, 72 years). We evaluated the extracellular matrix (ECM) organization, the appearance and distribution of areas of calcification, and modifications of cellular organization and structure by electron microscopy and histology. The ECM structure was similar in specimens from traumatic meniscus tears compared to those from patients with late-stage OA, showing disorganization of collagen fibers and increased proteoglycan content. Cells of healthy menisci showed mainly diffuse chromatin and well preserved organelles. Both in traumatic and in OA menisci, we observed increased chromatin condensation, organelle degeneration, and cytoplasmic vacuolization, a portion of which contained markers of autophagic vacuoles. Areas of calcification were also observed in both traumatic and OA menisci, as well as apoptotic-like features that were particularly prominent in traumatic meniscal tear samples. We conclude that meniscal tissue from patients with traumatic meniscal injury demonstrate pathological alterations characteristic of tissue from older patients undergoing TKR, suggesting that they have high susceptibility to develop OA.
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Traumatismos de la Rodilla/patología , Articulación de la Rodilla/anatomía & histología , Menisco/citología , Menisco/patología , Osteoartritis de la Rodilla/patología , Adulto , Anciano , Calcinosis/patología , Matriz Extracelular/patología , Matriz Extracelular/ultraestructura , Femenino , Humanos , Articulación de la Rodilla/patología , Masculino , Menisco/lesiones , Menisco/ultraestructura , Microscopía Electrónica de Transmisión , Persona de Mediana EdadRESUMEN
The aim of this study was to identify the molecules and pathways involved in the cross-talk between meniscus and synovium that may play a critical role in osteoarthritis (OA) pathophysiology. Samples of synovium and meniscus were collected from patients with early and end-stage OA and cultured alone or cocultured. Cytokines, chemokines, metalloproteases, and their inhibitors were evaluated at the gene and protein levels. The extracellular matrix (ECM) changes were also investigated. In early OA cultures, higher levels of interleukin-6 (IL-6) and IL-8 messenger RNA were expressed by synovium and meniscus in coculture compared with meniscus cultured alone. RANTES release was significantly increased when the two tissues were cocultured compared with meniscus cultured alone. Increased levels of matrix metalloproteinase-3 (MMP-3) and MMP-10 proteins, as well as increased release of glycosaminoglycans and aggrecan CS846 epitope, were observed when synovium was cocultured with meniscus. In end-stage OA cultures, increased levels of IL-8 and monocyte chemoattractant protein-1 (MCP-1) proteins were released in cocultures compared with cultures of meniscus alone. Chemokine (C-C motif) ligand 21 (CCL21) protein release was higher in meniscus cultured alone and in coculture compared with synovium cultured alone. Increased levels of MMP-3 and 10 proteins were observed when tissues were cocultured compared with meniscus cultured alone. Aggrecan CS846 epitope release was increased in cocultures compared with cultures of either tissue cultured alone. Our study showed the production of inflammatory molecules by synovium and meniscus which could trigger inflammatory signals in early OA patients, and induce ECM loss in the progressive and final stages of OA pathology.
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Matriz Extracelular/patología , Menisco/metabolismo , Osteoartritis de la Rodilla/patología , Membrana Sinovial/metabolismo , Anciano , Anciano de 80 o más Años , Agrecanos/metabolismo , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL21/metabolismo , Quimiocina CCL5/metabolismo , Técnicas de Cocultivo , Femenino , Glicosaminoglicanos/metabolismo , Humanos , Inflamación/patología , Interleucina-6/genética , Interleucina-8/genética , Masculino , Metaloproteinasa 10 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana EdadRESUMEN
While highly efficacious in treating rheumatoid arthritis (RA), the approved Janus kinase (JAK) inhibitor, Tofacitinib (Tofa, CP-690 550), has dose-dependent toxicities that limit its clinical application. In this study, we have examined whether a prodrug design that targets arthritic joints would enhance Tofa's therapeutic efficacy, which may provide an opportunity for future development of safer Tofa dosing regimens. A prodrug of Tofa (P-Tofa) was synthesized by conjugating the drug to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an acid cleavable carbamate linker. The therapeutic efficacy of a single dose of P-Tofa was compared to the dose-equivalent daily oral administration of Tofa in an adjuvant-induced arthritis (AA) rat model. Saline treated AA rats and age-matched healthy rats were used as controls. Observational analyses support the superior and sustained efficacy of a single dose P-Tofa treatment compared to the dose-equivalent daily Tofa administration in ameliorating joint inflammation. Micro-CT and histological analyses demonstrated that the P-Tofa treatment provided a structural preservation of the joints better than that of the dose-equivalent Tofa. Optical imaging, immunohistochemistry, and fluorescence-activated cell sorting analyses attribute P-Tofa's superior therapeutic efficacy to its passive targeting to arthritic joints and inflammatory cell-mediated sequestration. In vitro cell culture studies reveal that the P-Tofa treatment produced sustained the inhibition of JAK/STAT6 signaling in IL-4-treated murine bone marrow macrophages, consistent with a gradual subcellular release of Tofa. Collectively, a HPMA-based nanoscale prodrug of P-Tofa has the potential to enhance the therapeutic efficacy and widen the therapeutic window of Tofa therapy in RA.
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Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Inhibidores de las Cinasas Janus/uso terapéutico , Piperidinas/uso terapéutico , Profármacos/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Acrilamidas/química , Administración Oral , Animales , Articulación del Tobillo/diagnóstico por imagen , Articulación del Tobillo/efectos de los fármacos , Articulación del Tobillo/patología , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/etiología , Artritis Experimental/patología , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/etiología , Artritis Reumatoide/patología , Células Cultivadas , Inhibidores de las Cinasas Janus/química , Inhibidores de las Cinasas Janus/farmacología , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Macrófagos , Masculino , Ratones Endogámicos C57BL , Piperidinas/química , Piperidinas/farmacología , Cultivo Primario de Células , Profármacos/química , Profármacos/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Pirroles/química , Pirroles/farmacología , Ratas , Ratas Endogámicas Lew , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Distribución Tisular , Resultado del Tratamiento , Microtomografía por Rayos XRESUMEN
Interactions among risk factors for osteoarthritis (OA) are not well understood. We investigated the combined impact of two prevalent risk factors: mechanical loading and genetically abnormal cartilage tissue properties. We used cyclic tibial compression to simulate mechanical loading in the cho/+ (Col11a1 haploinsufficient) mouse, which has abnormal collagen fibrils in cartilage due to a point mutation in the Col11a1 gene. We hypothesized that the mutant collagen would not alter phenotypic bone properties and that cho/+ mice, which develop early onset OA, would develop enhanced load-induced cartilage damage compared to their littermates. To test our hypotheses, we applied cyclic compression to the left tibiae of 6-month-old cho/+ male mice and wild-type (WT) littermates for 1, 2, and 6 weeks at moderate (4.5 N) and high (9.0 N) peak load magnitudes. We then characterized load-induced cartilage and bone changes by histology, microcomputed tomography, and immunohistochemistry. Prior to loading, cho/+ mice had less dense, thinner cortical bone compared to WT littermates. In addition, in loaded and non-loaded limbs, cho/+ mice had thicker cartilage. With high loads, cho/+ mice experienced less load-induced cartilage damage at all time points and displayed decreased matrix metalloproteinase (MMP)-13 levels compared to WT littermates. The thinner, less dense cortical bone and thicker cartilage were unexpected and may have contributed to the reduced severity of load-induced cartilage damage in cho/+ mice. Furthermore, the spontaneous proteoglycan loss resulting from the mutant collagen XI was not additive to cartilage damage from mechanical loading, suggesting that these risk factors act through independent pathways. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:711-720, 2018.
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Hueso Esponjoso/fisiología , Cartílago Articular/anomalías , Colágeno Tipo XI/genética , Hueso Cortical/fisiología , Osteoartritis/genética , Animales , Hueso Esponjoso/anatomía & histología , Hueso Cortical/anatomía & histología , Masculino , Ratones Endogámicos C57BL , Osteofito/etiología , Fenotipo , Mutación Puntual , Tibia/fisiología , Soporte de PesoRESUMEN
Polymeric nanocarriers such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers deliver drugs to solid tumors and avoid the systemic toxicity of conventional chemotherapy. Because HPMA copolymers can target sites of inflammation and accumulate within innate immune cells, we hypothesized that HPMA copolymers could target tumor-associated macrophages (TAM) in both primary and metastatic tumor microenvironments. We verified this hypothesis, first in preliminary experiments with isolated bone marrow macrophage cultures in vitro and subsequently in a spontaneously metastatic murine breast cancer model generated from a well-established, cytogenetically characterized 4T1 breast cancer cell line. Using our standardized experimental conditions, we detected primary orthotopic tumor growth at 7 days and metastatic tumors at 28 days after orthotopic transplantation of 4T1 cells into the mammary fat pad. We investigated the uptake of HPMA copolymer conjugated with Alexa Fluor 647 and folic acid (P-Alexa647-FA) and HPMA copolymer conjugated with IRDye 800CW (P-IRDye), following their retroorbital injection into the primary and metastatic tumor-bearing mice. A significant uptake of P-IRDye was observed at all primary and metastatic tumor sites in these mice, and the P-Alexa647-FA signal was found specifically within CD11b+ TAMs costained with pan-macrophage marker CD68. These findings demonstrate, for the first time, a novel capacity of a P-Alexa647-FA conjugate to colocalize to CD11b+CD68+ TAMs in both primary and metastatic breast tumors. This underscores the potential of this HPMA nanocarrier to deliver functional therapeutics that specifically target tumor-promoting macrophage activation and/or polarization during tumor development. Mol Cancer Ther; 16(12); 2701-10. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/genética , Macrófagos/metabolismo , Metacrilatos/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , PolímerosRESUMEN
Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator differentially expressed in FDCP 6 homolog (Def6) as a novel inhibitor of osteoclastogenesis in physiological and inflammatory conditions. Def6 deficiency in Def6-/- mice enhanced the sensitivity of osteoclast precursors to the physiological osteoclastogenic inducer receptor activator for NF-κB ligand, and Def6-/- osteoclasts formed actin rings. Furthermore, Def6 deficiency markedly increased TNF-α-induced osteoclastogenesis in vitro and in vivo and enhanced bone resorption in an inflammatory osteolysis mouse model. TNF-α serum levels correlated negatively with Def6 expression levels in osteoclast precursors obtained from RA patients, and the osteoclastogenic capacity of the osteoclast precursors was significantly inversely correlated with their Def6 expression levels, indicating that Def6 functions as an inhibitor of excessive osteoclast formation and bone destruction in RA. Mechanistically, Def6 suppressed osteoclastogenesis and the expression of key osteoclastogenic factors NFATc1, B lymphocyte-induced maturation protein-1, and c-Fos by regulating an endogenous IFN-ß-mediated autocrine feedback loop. The Def6-dependent pathway may represent a novel therapeutic target to prevent pathological bone destruction.
Asunto(s)
Artritis Reumatoide/metabolismo , Resorción Ósea/inmunología , Proteínas de Unión al ADN/metabolismo , Macrófagos/fisiología , Proteínas Nucleares/metabolismo , Osteoclastos/fisiología , Osteogénesis , Osteólisis/inmunología , Actinas/metabolismo , Animales , Artritis Reumatoide/genética , Comunicación Autocrina , Resorción Ósea/genética , Diferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Factores de Intercambio de Guanina Nucleótido , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Osteogénesis/genética , Osteólisis/genética , Ligando RANK/inmunologíaRESUMEN
Alterations in the gut microbiome have been associated with changes in bone mass and microstructure, but the effects of the microbiome on bone biomechanical properties are not known. Here we examined bone strength under two conditions of altered microbiota: (1) an inbred mouse strain known to develop an altered gut microbiome due to deficits in the immune system (the Toll-like receptor 5-deficient mouse [TLR5KO]); and (2) disruption of the gut microbiota (ΔMicrobiota) through chronic treatment with selected antibiotics (ampicillin and neomycin). The bone phenotypes of TLR5KO and WT (C57Bl/6) mice were examined after disruption of the microbiota from 4 weeks to 16 weeks of age as well as without treatment (n = 7 to 16/group, 39 animals total). Femur bending strength was less in ΔMicrobiota mice than in untreated animals and the reduction in strength was not fully explained by differences in bone cross-sectional geometry, implicating impaired bone tissue material properties. Small differences in whole-bone bending strength were observed between WT and TLR5KO mice after accounting for differences in bone morphology. No differences in trabecular bone volume fraction were associated with genotype or disruption of gut microbiota. Treatment altered the gut microbiota by depleting organisms from the phyla Bacteroidetes and enriching for Proteobacteria, as determined from sequencing of fecal 16S rRNA genes. Differences in splenic immune cell populations were also observed; B and T cell populations were depleted in TLR5KO mice and in ΔMicrobiota mice (p < 0.001), suggesting an association between alterations in bone tissue material properties and immune cell populations. We conclude that alterations in the gut microbiota for extended periods during growth may lead to impaired whole-bone mechanical properties in ways that are not explained by bone geometry. © 2017 American Society for Bone and Mineral Research.