RESUMEN
OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RESULTS: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.
Asunto(s)
Artritis Reumatoide , Captura por Microdisección con Láser , Análisis de Secuencia de ARN , Membrana Sinovial , Humanos , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Membrana Sinovial/patología , Membrana Sinovial/metabolismo , Femenino , Masculino , Persona de Mediana Edad , Transcriptoma , Anciano , Osteoartritis/genética , Osteoartritis/patología , Análisis de Componente Principal , Perfilación de la Expresión GénicaRESUMEN
Aim: To investigate the effect of incorporating bis(monoacylglycerol)phosphate (BMP) lipid into a lipid nanoparticle and the functional transport of mRNA by the formulated nanoparticles in vivo. Materials & methods: The nanoparticles were prepared from ionizable lipid, 1,2-distearoyl-sn-glycerol-3-phosphocholine, cholesterol, 1,2-dimyristoyl-sn-glycerol PEG 2000, BMP and formulated mRNA encoding human erythropoietin. We measured the effect of BMP on physicochemical properties and impact on functional efficacy to transport mRNA to its target cells/tissue as measured by protein expression both in vitro and in vivo. Results: Lipid nanoparticles composed of BMP displayed increased endosomal membrane fusion and improved mRNA delivery to the cytosol. Conclusion: The results establish the foundation for future development of these nanoparticulated entities by designing new BMP derivatives and correlating structures to enhanced pharmacokinetic profiles.
Asunto(s)
Nanopartículas , Fosfatos , Humanos , Monoglicéridos/metabolismo , Nanopartículas/química , ARN MensajeroRESUMEN
Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.
Asunto(s)
Esclerodermia Sistémica , Fibroblastos/metabolismo , Fibrosis , Humanos , Estrés Oxidativo , Esclerodermia Sistémica/patología , Tirosina/metabolismoRESUMEN
Lipid nanoparticles (LNPs) mediated mRNA delivery has gained prominence due to the success of mRNA vaccines against Covid-19, without which it would not have been possible. However, there is little clinical validation of this technology for other mRNA-based therapeutic approaches. Systemic administration of LNPs predominantly targets the liver, but delivery to other organs remains a challenge. Local approaches remain a viable option for some disease indications, such as Cystic Fibrosis, where aerosolized delivery to airway epithelium is the preferred route of administration. With this in mind, novel cationic lipids (L1-L4) have been designed, synthesized and co-formulated with a proprietary ionizable lipid. These LNPs were further nebulized, along with baseline control DOTAP-based LNP (DOTAP+), and tested in vitro for mRNA integrity and encapsulation efficiency, as well as transfection efficiency and cytotoxicity in cell cultures. Improved biodegradability and potentially superior elimination profiles of L1-L4, in part due to physicochemical characteristics of putative metabolites, are thought to be advantageous for prospective therapeutic lung delivery applications using these lipids.
Asunto(s)
Liposomas/química , Pulmón , Nanopartículas/química , ARN Mensajero/administración & dosificación , HumanosRESUMEN
Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.
Asunto(s)
Antirreumáticos , Artritis Reumatoide , Sinoviocitos , Animales , Antirreumáticos/uso terapéutico , Células Cultivadas , Fibroblastos/metabolismo , Ratones , Sinoviocitos/metabolismo , Sinoviocitos/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: We aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFß-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity. CONCLUSION: In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Tirosina Fosfatasas no Receptoras/fisiología , Transducción de Señal/fisiología , Sinoviocitos/metabolismo , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Artritis Reumatoide/metabolismo , Proteínas de Ciclo Celular/fisiología , Humanos , Ratones , Proteínas Señalizadoras YAPRESUMEN
Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors, and loss of PTPN2 promotes T cell expansion and CD4- and CD8-driven autoimmunity. However, it remains unknown whether loss of PTPN2 in FoxP3+ regulatory T cells (Tregs) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, the presence of which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell-dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, PTPN2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired RORγt expression, at a stage when chromatin accessibility for STAT3-targeted IL-17-associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse RORγt+ Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity.
Asunto(s)
Artritis Reumatoide/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 2/inmunología , Linfocitos T Reguladores/inmunología , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Polyethylene glycol (PEG)-modified carbon nanotubes have been successfully employed for intra-articular delivery in mice without systemic or local toxicity. However, the fate of the delivery system itself remains to be understood. In this study 2 kDa PEG-modified single-walled carbon nanotubes (PNTs) are synthesized, and trafficking and degradation following intra-articular injection into the knee-joint of healthy mice are studied. Using confocal Raman microspectroscopy, PNTs can be imaged in the knee-joint and are found to either egress from the synovial cavity or undergo biodegradation over a period of 3 weeks. Raman analysis discloses that PNTs are oxidatively degraded mainly in the chondrocyte-rich cartilage and meniscus regions while PNTs can also be detected in the synovial membrane regions, where macrophages can be found. Furthermore, using murine chondrocyte (ATDC-5) and macrophage (RAW264.7) cell lines, biodegradation of PNTs in activated, nitric oxide (NO)-producing chondrocytes, which is blocked upon pharmacological inhibition of inducible nitric oxide synthase (iNOS), can be shown. Biodegradation of PNTs in macrophages is also noted, but after a longer period of incubation. Finally, cell-free degradation of PNTs upon incubation with the peroxynitrite-generating compound, SIN-1 is demonstrated. The present study paves the way for the use of PNTs as delivery systems in the treatment of diseases of the joint.
Asunto(s)
Condrocitos/metabolismo , Articulación de la Rodilla/metabolismo , Nanotubos de Carbono/química , Óxido Nítrico/metabolismo , Polietilenglicoles/química , Animales , Condrocitos/patología , Femenino , Inyecciones Intraarticulares , Articulación de la Rodilla/patología , Ratones , Células RAW 264.7RESUMEN
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFß signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFß signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC-ERK-SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFß-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Tirosina Fosfatasas/metabolismo , Esclerodermia Sistémica/enzimología , Factor de Crecimiento Transformador beta/fisiología , Animales , Línea Celular , Dermis/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Proteína smad3/metabolismoRESUMEN
PURPOSE OF REVIEW: The pathogenesis of systemic sclerosis depends on a complex interplay between autoimmunity, vasculopathy, and fibrosis. Reversible phosphorylation on tyrosine residues, in response to growth factors and other stimuli, critically regulates each one of these three key pathogenic processes. Protein tyrosine kinases, the enzymes that catalyze addition of phosphate to tyrosine residues, are known players in systemic sclerosis, and tyrosine kinase inhibitors are undergoing clinical trials for treatment of this disease. Until recently, the role of tyrosine phosphatases-the enzymes that counteract the action of tyrosine kinases by removing phosphate from tyrosine residues-in systemic sclerosis has remained largely unknown. Here, we review the function of tyrosine phosphatases in pathways relevant to the pathogenesis of systemic sclerosis and their potential promise as therapeutic targets to halt progression of this debilitating rheumatic disease. RECENT FINDINGS: Protein tyrosine phosphatases are emerging as important regulators of a multitude of signaling pathways and undergoing validation as molecular targets for cancer and other common diseases. Recent advances in drug discovery are paving the ways to develop new classes of tyrosine phosphatase modulators to treat human diseases. Although so far only few reports have focused on tyrosine phosphatases in systemic sclerosis, these enzymes play a role in multiple pathways relevant to disease pathogenesis. Further studies in this field are warranted to explore the potential of tyrosine phosphatases as drug targets for systemic sclerosis.
Asunto(s)
Terapia Molecular Dirigida/métodos , Proteínas Tirosina Fosfatasas/fisiología , Esclerodermia Sistémica/enzimología , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/uso terapéutico , Fibrosis , Sustancias de Crecimiento/fisiología , Humanos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Receptores de Interleucina/inmunología , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/inmunología , Transducción de Señal/inmunología , Investigación Biomédica Traslacional/métodosRESUMEN
The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1ß stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.
RESUMEN
OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.
Asunto(s)
Artritis Experimental/genética , Artritis Reumatoide/genética , Fibroblastos/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , Familia-src Quinasas/metabolismo , Animales , Articulación del Tobillo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Western Blotting , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Interleucina-1/farmacología , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/genética , Factor de Crecimiento Derivado de Plaquetas/farmacología , Reacción en Cadena de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Membrana Sinovial/citología , Factor de Necrosis Tumoral alfa/farmacología , Familia-src Quinasas/efectos de los fármacosRESUMEN
OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.
Asunto(s)
Artritis Reumatoide/patología , Fibroblastos/patología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/fisiología , Membrana Sinovial/patología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Artritis Reumatoide/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Fibroblastos/trasplante , Regulación Enzimológica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones Desnudos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , ARN Mensajero/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/trasplante , Regulación hacia ArribaRESUMEN
Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.
Asunto(s)
Antirreumáticos/farmacología , Artritis Reumatoide/prevención & control , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Heparina/análogos & derivados , Proteoglicanos/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/antagonistas & inhibidores , Membrana Sinovial/efectos de los fármacos , Animales , Artritis Reumatoide/enzimología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Heparina/metabolismo , Humanos , Ratones Noqueados , Terapia Molecular Dirigida , Fosforilación , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Sindecano-4/genética , Sindecano-4/metabolismo , Membrana Sinovial/enzimología , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Factores de Tiempo , TransfecciónRESUMEN
Osteoarthritis (OA) is a common and debilitating degenerative disease of articular joints for which no disease-modifying medical therapy is currently available. Inefficient delivery of pharmacologic agents into cartilage-resident chondrocytes after systemic administration has been a limitation to the development of anti-OA medications. Direct intra-articular injection enables delivery of high concentrations of agents in close proximity to chondrocytes; however, the efficacy of this approach is limited by the fast clearance of small molecules and biomacromolecules after injection into the synovial cavity. Coupling of pharmacologic agents with drug delivery systems able to enhance their residence time and cartilage penetration can enhance the effectiveness of intra-articularly injected anti-OA medications. Herein we describe an efficient intra-articular delivery nanosystem based on single-walled carbon nanotubes (SWCNTs) modified with polyethylene glycol (PEG) chains (PEG-SWCNTs). We show that PEG-SWCNTs are capable to persist in the joint cavity for a prolonged time, enter the cartilage matrix, and deliver gene inhibitors into chondrocytes of both healthy and OA mice. PEG-SWCNT nanoparticles did not elicit systemic or local side effects. Our data suggest that PEG-SWCNTs represent a biocompatible and effective nanocarrier for intra-articular delivery of agents to chondrocytes.
Asunto(s)
Condrocitos/metabolismo , Portadores de Fármacos/química , Articulaciones/metabolismo , Nanotubos de Carbono/química , Polietilenglicoles/química , Animales , Secuencia de Bases , Línea Celular , Femenino , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Articulaciones/citología , Ratones , Ratones Transgénicos , Morfolinos/química , Morfolinos/genética , Morfolinos/metabolismo , Osteoartritis/tratamiento farmacológicoRESUMEN
Single-walled carbon nanotubes (SWCNTs) coated or functionalized with PEG chains of different molecular weight were assessed for their propensity to undergo biodegradation under in vitro conditions using recombinant myeloperoxidase (MPO) or ex vivo using freshly isolated primary human neutrophils. Our findings suggest that under natural conditions, a combined process of 'stripping' (i.e., defunctionalization) and biodegradation of PEG-SWCNTs might occur and that PEG-SWCNTs are a promising--and degradable--nanomedicine vector.
Asunto(s)
Implantes de Medicamentos/química , Nanocápsulas/química , Nanotubos de Carbono/química , Neutrófilos/enzimología , Peroxidasa/química , Polietilenglicoles/química , Células Cultivadas , Materiales Biocompatibles Revestidos/síntesis química , Diseño de Fármacos , Humanos , Peso Molecular , Nanocápsulas/ultraestructura , Nanotubos de Carbono/ultraestructura , Tamaño de la Partícula , Especificidad por SustratoRESUMEN
The recent advent of nanomedicine holds potential to revolutionize cancer therapy. This innovative discipline has paved the way for the emergence of a new class of drugs based on nanoengineered particles. These "nanodrugs" are designed to greatly enhance drug therapeutic indices. First-generation nanodrugs consisted of conventional anti-cancer drugs loaded into/onto nanoengineered particles (nanocarriers) devoid of targeting features (non-targeted nanodrugs). Non-targeted nanodrugs have provided the opportunity to carry large amounts of drugs, including poorly water-soluble and/or permeable drugs, to several types of tumors, improving the therapeutic index with respect to comparable free drugs. Although effective, the primary delivery mechanism of non-targeted nanodrugs was through passive tissue accumulation, due to pathophysiological differences between tumor-associated and healthy vessels, and through non-specific targeting of cell subsets, posing the danger of off-target binding and effects. Recently, the therapeutic indices of certain anti-cancer drugs were further improved by attaching targeting ligands to nanodrugs (targeted-nanodrugs). Targeted-nanodrugs selectively bind to cognate receptors expressed on target cells and enter cells more efficiently than non-targeted formulations. Although these advancements have been sufficiently beneficial to place targeted-nanodrugs into clinical development for use in cancer therapy, they also come at a price. The addition of ligands to drug-loaded nanocarriers often leads to additional synthesis steps and costs, and more complex biological performance relative to ligand-devoid nanodrugs. Here, we will discuss the benefits and challenges facing the addition of targeting features to nanodrugs for cancer therapy.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Terapia Molecular Dirigida/métodos , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Antineoplásicos/química , Diseño de Fármacos , HumanosRESUMEN
BACKGROUND: Single wall carbon nanotubes (SWCNTs) are considered promising nanoparticles for industrial and biomedical applications; however their potential toxicity in several biological systems, including the feto-placental unit, has been demonstrated. Functionalization of SWCNTs with polyethylene glycol chains (PEG-SWCNTs) dramatically reduces their toxicity, and for this reason PEG-SWCNTs are candidates for biomedical applications. However, no data are available on their safety for the developing embryo, in spite of the clinical and social relevance of this topic. The purpose of this study is therefore to investigate the safety of PEG-SWCNTs for their use as biomedical carriers in pregnancy. METHODS: For toxicological studies, amino-functionalized PEG-SWCNT were intravenously injected in CD1 pregnant mice at different doses (range 0.1-30 µg/mouse), in single or multiple administrations. For biodistribution studies, fluorescently labeled PEG-SWCNTs were obtained by acylation of terminal PEG amino groups with near infrared emitting fluorochromes (PEG-SWCNT-750) and injected at the dosage of 10 µg/mouse, at either day 5.5 (when the placenta is still developing) or day 14.5 of gestation (when the maturation of the placenta is complete). RESULTS: We found no adverse effects both on embryos and dams up to the dose of 10 µg/mouse. At the dose of 30 µg/mouse, occasional teratogenic effects, associated with placental damage, were detected both when administered as a single bolus (1 out of 10 dams; 1 malformed embryo) or as multiple doses (2 out of 10 dams; 5 malformed embryos). The difference in the prevalence of dams with malformed embryos between the 30 µg exposed group and controls approached the statistical significance (p = 0.06). Hepatic damage in dams was seen only in the multiple exposure group (4 out of 10; p = 0.04 when compared with the single exposure group or controls). PEG-SWCNT-750 reached the conceptus when administered early in pregnancy. At later stages, PEG-SWCNT-750 were detected in the placenta and the yolk sac, but not in the embryo. CONCLUSIONS: PEG-SWCNTs may cause occasional teratogenic effects in mice beyond a threshold dose. Such effect might depend on their ability to reach the feto-placenta unit. Although not automatically transferable to humans, these data should be considered if exposing women during pregnancy.
Asunto(s)
Nanotubos de Carbono/toxicidad , Polietilenglicoles/farmacocinética , Polietilenglicoles/toxicidad , Anomalías Inducidas por Medicamentos/etiología , Animales , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Edad Gestacional , Inyecciones Intravenosas , Mediciones Luminiscentes , Exposición Materna/efectos adversos , Ratones , Imagen Óptica , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Medición de Riesgo , Distribución Tisular , Imagen de Cuerpo EnteroRESUMEN
Recent evidence regarding the role of regulatory T cells (Treg) in tumor development has suggested that the manipulation of Treg function selectively in the tumor microenvironment would be a desirable immunotherapy approach. Targeting intratumor immune populations would reduce side effects on peripheral healthy cells and increase antitumor efficacy of immunotherapies. However, no current approaches are available which enable selective in vivo targeting of intratumor Treg or other immune cell subpopulations. Herein, we investigated the ability of ligands against Treg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into Treg residing in the tumor microenvironment. We focused our attention on the glucocorticoid-induced TNFR-related receptor (GITR), as it showed higher overexpression on intratumor vs peripheral (i.e., splenic) Treg compared to other reported Treg-specific markers (folate receptor 4, CD103, and CD39). Ex vivo investigations showed that the Treg targeting efficiency and selectivity of PEG-SWCNTs depended on incubation time, dose, number of ligands per nanotube, and targeted surface marker. In vivo investigations showed that PEG-SWCNTs armed with GITR ligands targeted Treg residing in a B16 melanoma more efficiently then intratumor non-Treg or splenic Treg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor Treg vs effector T cell (Teff) ratio, and active targeting of markers that are enriched in intratumor vs splenic Treg. We also found that PEG-SWCNTs loaded with GITR ligands were internalized by Treg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus ex vivo and in vivo. This is the first example of intratumor immune cell targeting and we hope it will pave the way to innovative immunotherapies against cancer.
Asunto(s)
Nanotubos de Carbono/química , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Polietilenglicoles/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Animales , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Endogámicos C57BL , Polietilenglicoles/química , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Investigation of the nanoparticle protein corona, the shell of plasma proteins formed around nanoparticles immediately after they enter the bloodstream, is a benchmark in the study of the applications of nanoparticles in all fields of medicine, from pharmacology to toxicology. We report the first investigation of the protein corona adsorbed onto single-walled carbon nanotubes modified with 2 kDa molecular weight polyethylene glycol chains [PEG(2k)-modified SWCNTs or PEG2-SWCNTs] by using a large-scale gel-based proteomics method on biological replicates. More than 240 plasma proteins were selected, and their differences were analyzed among PEG2-SWCNTs differing in surface charge and PEG conformation. The protein corona of PEG2-SWCNTs showed that coagulation proteins, immunoglobulins, apolipoproteins, and proteins of the complement system were among the proteins bound by PEG2-SWCNTs and that their recruitment was independent from the isoelectric point, molecular weight, total hydrophobicity, and number of polyaromatic residues of the proteins. Statistical analysis on protein relative abundance revealed that PEG conformation had a higher influence on the PEG2-SWCNTs' protein corona repertoire than nanotube surface charge. PEG conformation also affected the biological performance of PEG2-SWCNTs. A change in PEG conformation from mushroom to mushroom-brush transition affected the competitive adsorption of the major constituents of the protein corona of PEG2-SWCNTs and promoted shorter blood circulation time, faster renal excretion, and higher relative spleen versus liver uptake of PEG2-SWCNTs. Our data suggest that the protein corona, along with steric stabilization, may mediate the action of PEG conformation on the pharmacokinetic profile of PEG-modified SWCNTs.