RESUMO
Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood-retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.
Assuntos
Lipocalinas/farmacologia , Terapia de Alvo Molecular , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Complexo Antígeno-Anticorpo/metabolismo , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Barreira Hematorretiniana/patologia , Permeabilidade Capilar , Proliferação de Células/efeitos dos fármacos , Feminino , Meia-Vida , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipocalinas/farmacocinética , Lipocalinas/uso terapêutico , Camundongos Nus , Camundongos Transgênicos , Mitógenos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Polietilenoglicóis/química , Engenharia de Proteínas , Coelhos , Receptores de IgG/metabolismo , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Trombose/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Our overview covers several key areas related to recent results obtained for collagen type VI and endotrophin (ETP): i) An introduction to the history of ETP, including how it was identified, how it is released and its function and potential receptors. ii) An introduction to the collagen family, with a focus on what differentiates collagen type VI from an evolutionary standpoint. iii) An overview of collagen type VI, the six individual chains (COL6A1, A2, A3, A4, A5 and A6), their differences and similarities, as well as their expression profiles and function. iv) A detailed analysis of COL6A3, including the cleaved product endotrophin, and what separates it from the other five collagen 6 molecules, including its suggested function based on insights gained from knockout and gain of function mouse models. v) An introduction to the history of ETP, including how it was identified, how it is released and its function and potential receptors. vi) The pathology of ETP. What leads to its presence and release and what are the consequences thereof? vii) Functional implications of circulating ETP. Here we review the data with the functional roles of ETP in mind. viii) We propose that ETP is a mediator for fibrotic (or fibro-inflammatory? ) disorders. Based on what we know about ETP, we have to consider it as a target for the treatment of fibrotic (or fibro-inflammatory) disorders. What segment(s) of the patient population would most dramatically respond to an ETP-targeted intervention? How can we find the population that would profit most from an intervention? We aim to present a broad overview over the ETP field at large, providing an assessment of where the future research efforts need to be placed to tap into the vast potential of ETP, both as a marker and as a target in different diseases.
RESUMO
BACKGROUND: Immune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role of discoidin domain-containing receptor 1 (DDR1) in promoting IE in breast cancer and validated its critical role in IE using neutralizing rabbit monoclonal antibodies (mAbs) in multiple mouse tumor models. METHODS: To develop a DDR1-targeting mAb as a potential cancer therapeutic, we humanized mAb9 with a complementarity-determining region grafting strategy. The humanized antibody named PRTH-101 is currently being tested in a Phase 1 clinical trial. We determined the binding epitope of PRTH-101 from the crystal structure of the complex between DDR1 extracellular domain (ECD) and the PRTH-101 Fab fragment with 3.15 Å resolution. We revealed the underlying mechanisms of action of PRTH-101 using both cell culture assays and in vivo study in a mouse tumor model. RESULTS: PRTH-101 has subnanomolar affinity to DDR1 and potent antitumor efficacy similar to the parental rabbit mAb after humanization. Structural information illustrated that PRTH-101 interacts with the discoidin (DS)-like domain, but not the collagen-binding DS domain of DDR1. Mechanistically, we showed that PRTH-101 inhibited DDR1 phosphorylation, decreased collagen-mediated cell attachment, and significantly blocked DDR1 shedding from the cell surface. Treatment of tumor-bearing mice with PRTH-101 in vivo disrupted collagen fiber alignment (a physical barrier) in the tumor extracellular matrix (ECM) and enhanced CD8+ T cell infiltration in tumors. CONCLUSIONS: This study not only paves a pathway for the development of PRTH-101 as a cancer therapeutic, but also sheds light on a new therapeutic strategy to modulate collagen alignment in the tumor ECM for enhancing antitumor immunity.
Assuntos
Anticorpos Monoclonais , Receptor com Domínio Discoidina 1 , Neoplasias , Animais , Camundongos , Colágeno/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Matriz Extracelular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Microambiente Tumoral , Anticorpos Monoclonais/farmacologiaRESUMO
Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.
Assuntos
Artrite/metabolismo , Artrite/patologia , Leucotrieno B4/fisiologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Animais , Artrite/genética , Artrite/imunologia , Células Cultivadas , Predisposição Genética para Doença , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Leucotrieno B4/biossíntese , Leucotrieno B4/deficiência , Leucotrieno B4/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologiaRESUMO
Neuropathic pain produced by damage to or dysfunction of the nervous system is a common and severely disabling state that affects millions of people worldwide. Recent evidence indicates that activated microglia are key cellular intermediaries in the pathogenesis of neuropathic pain and that ATP serves as the mediator. However, the in vivo mechanism underlying the retention of activated microglia in the injured region has not yet been completely elucidated. Prostaglandin E(2) (PGE(2)) is the principal proinflammatory prostanoid and plays versatile roles by acting via four PGE receptor subtypes, EP1-EP4. In the present study, we investigated the role of PGE(2) in spinal microglial activation in relation to neuropathic pain by using genetic and pharmacological methods. Mice deficient in microsomal prostaglandin E synthase-1 impaired the activation of microglia and the NMDA-nitric oxide (NO) cascade in spinal neurons in the dorsal horn and did not exhibit mechanical allodynia after peripheral nerve injury. The intrathecal injection of indomethacin, a nonsteroidal anti-inflammatory drug, ONO-8713, a selective EP1 antagonist, or 7-nitroindole, a neuronal NO synthase inhibitor, attenuated mechanical allodynia and the increase in activated microglia observed in the established neuropathic-pain state. We further demonstrated that ATP-induced microglial migration was blocked in vitro by PGE(2) via EP2 and by S-nitrosoglutathione, an NO donor. Taken together, the present study suggests that PGE(2) participated in the maintenance of neuropathic pain in vivo not only by activating spinal neurons, but also by retaining microglia in the central terminals of primary afferent fibers via EP2 subtype and via EP1-mediated NO production.
Assuntos
Movimento Celular/fisiologia , Dinoprostona/metabolismo , Microglia/fisiologia , Neuralgia/metabolismo , Neuralgia/patologia , Medula Espinal/patologia , Trifosfato de Adenosina/farmacologia , Animais , Movimento Celular/genética , Córtex Cerebral/citologia , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Indazóis/farmacologia , Indazóis/uso terapêutico , Oxirredutases Intramoleculares/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Microglia/efeitos dos fármacos , Neuralgia/complicações , Neuralgia/tratamento farmacológico , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Prostaglandina-E Sintases , S-Nitrosoglutationa/farmacologia , Medula Espinal/efeitos dos fármacos , Nervos Espinhais/lesõesRESUMO
Prostaglandin E(2) (PGE(2)) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE(2) from the cyclooxygenase metabolite PGH(2) have been described. Here, we examine the contribution of one of these enzymes to PGE(2) production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE(2) levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE(2) synthase.
Assuntos
Dinoprostona/biossíntese , Oxirredutases Intramoleculares/fisiologia , Animais , Northern Blotting , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Interferon gama/farmacologia , Oxirredutases Intramoleculares/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Prostaglandina-E SintasesRESUMO
We studied the febrile response in mice deficient in microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal isomerase expressed in cytokine-sensitive brain endothelial cells. These animals showed no fever and no central prostaglandin (PG) E2 synthesis after peripheral injection of bacterial-wall lipopolysaccharide, but their pyretic capacity in response to centrally administered PGE2 was intact. Our findings identify mPGES-1 as the central switch during immune-induced pyresis and as a target for the treatment of fever and other PGE2-dependent acute phase reactions elicited by the brain.
Assuntos
Febre/imunologia , Oxirredutases Intramoleculares/fisiologia , Microssomos/enzimologia , Animais , Temperatura Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Dinoprostona/líquido cefalorraquidiano , Dinoprostona/farmacologia , Modelos Animais de Doenças , Febre/induzido quimicamente , Febre/fisiopatologia , Regulação Enzimológica da Expressão Gênica , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Prostaglandina-E Sintases , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
BACKGROUND AND PURPOSE: Anaemia of chronic disease (ACD) has been linked to iron-restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin-derived, small protein-based anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. EXPERIMENTAL APPROACH: Anticalin- and hepcidin-specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS-080-PEG30 administration in vivo. KEY RESULTS: PRS-080 had a hepcidin-binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS-080-PEG30), a t1/2 of 43 h in cynomolgus monkeys. Dose-dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS-080-PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment-free interval, all iron parameters returned to pre-dose values. CONCLUSIONS AND IMPLICATIONS: In conclusion, we developed a dose-dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron-restricted erythropoiesis that can address the root cause of ACD. PRS-080-PEG30 is currently in early clinical development.
Assuntos
Hepcidinas/antagonistas & inibidores , Hepcidinas/sangue , Ferro/sangue , Animais , Feminino , Macaca fascicularis , Masculino , Modelos BiológicosRESUMO
Rheumatoid arthritis (RA) is a chronic inflammatory disorder leading to bone and cartilage destruction. A substantial body of evidence suggests that prostaglandin E2 (PGE2) contributes to the pathogenesis of RA, and nonsteroidal anti-inflammatory drugs, inhibitors of the synthesis of PGE2 and other prostanoids, continue to be used in the treatment of this disease. To begin to understand the mechanism by which prostaglandins modulate the pathophysiology of this disease, we examined mice lacking each of the four known PGE2 (EP) receptors after generation of collagen antibody-induced arthritis, an animal model of RA. Homozygous deletion of the EP1, EP2, or EP3 receptors did not affect the development of arthritis, whereas EP4 receptor-deficient mice showed decreased incidence and severity of disease. These animals also showed reduced inflammation as assessed by circulating IL-6 and serum amyloid A levels. Joint histopathology of EP4(-/-) animals revealed reduced bone destruction, proteoglycan loss, and type II collagen breakdown in cartilage compared with EP4(+/+) mice. Furthermore, liver and macrophages isolated from EP4(-/-) animals produced significantly less IL-1 beta and IL-6 than control samples. Thus, PGE2 contributes to disease progression at least in part by binding to the EP4 receptor. Antagonists of this receptor might therefore provide novel agents for the treatment of RA.
Assuntos
Artrite Reumatoide/metabolismo , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E/fisiologia , Animais , Artrite/metabolismo , Progressão da Doença , Deleção de Genes , Homozigoto , Inflamação/metabolismo , Interleucina-1/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Amiloide A Sérica/metabolismo , Fatores de TempoRESUMO
The P2X7 nucleotide receptor is an ATP-gated ion channel expressed widely in cells of hematopoietic origin. Our purpose was to explore the involvement of the P2X7 receptor in bone development and remodeling by characterizing the phenotype of mice genetically modified to disrupt the P2X7 receptor [knockout (KO)]. Femoral length did not differ between KO and wild-type (WT) littermates at 2 or 9 months of age, indicating that the P2X7 receptor does not regulate longitudinal bone growth. However, KO mice displayed significant reduction in total and cortical bone content and periosteal circumference in femurs, and reduced periosteal bone formation and increased trabecular bone resorption in tibias. Patch clamp recording confirmed expression of functional P2X7 receptors in osteoclasts from WT but not KO mice. Osteoclasts were present in vivo and formed in cultures of bone marrow from KO mice, indicating that this receptor is not essential for fusion of osteoclast precursors. Functional P2X7 receptors were also found in osteoblasts from WT but not KO mice, suggesting a direct role in bone formation. P2X7 receptor KO mice demonstrate a unique skeletal phenotype that involves deficient periosteal bone formation together with excessive trabecular bone resorption. Thus, the P2X7 receptor represents a novel therapeutic target for the management of skeletal disorders such as osteoporosis.
Assuntos
Desenvolvimento Ósseo/genética , Reabsorção Óssea/genética , Receptores Purinérgicos P2/fisiologia , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Feminino , Fêmur/anatomia & histologia , Fêmur/crescimento & desenvolvimento , Fêmur/patologia , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Técnicas de Patch-Clamp , Receptores Purinérgicos P2X7 , Tomografia/métodosRESUMO
The metabolism of arachidonic acid through the cyclooxygenase pathway is a highly regulated cellular process that results in the formation of PGH2. This unstable intermediate can be enzymatically metabolized to PGE2 by the actions of a microsomal 17 kDa PGE synthase (mPGES1). Treatment of A549 cells with IL-1beta for 24 h resulted in a twofold increase in mPGES1 mRNA, protein expression, and PGES specific activity. To understand the relationship between expression of mPGES1 and PGE2 formation, IL-1beta treated cells were incubated with increasing concentrations of antisense oligonucleotides (ASO) and their effects compared to cells treated with reverse sense oligonucleotides (RSO) designed against the ATG translation initiation codon of mPGES1. Incubation with ASO resulted in a 44% reduction in mRNA expression level as compared to RSO-treated cells. Microsomal preparations isolated from ASO- and RSO-treated cells were analyzed for their ability to convert PGH2 to PGE2 in the presence 2.5 mM reduced glutathione. An approximate 50% reduction (ASO: 1.8 nmol/min/mg, RSO: 3.7 nmol/min/mg) in PGES activity, protein expression by immunodetection, and extracellular PGE2 release was detected in these samples. As a control in these studies, the protein levels of COX2 and secreted IL-8 were quantified; no change in these levels was observed. These results demonstrate the direct association between mPGES1 expression, its enzymatic activity, and total PGE2 production following an inflammatory stimulus.
Assuntos
Dinoprostona/biossíntese , Interleucina-1/antagonistas & inibidores , Oxirredutases Intramoleculares/genética , Oligonucleotídeos Antissenso/genética , Prostaglandina-Endoperóxido Sintases/genética , Animais , Sequência de Bases , Dinoprostona/genética , Expressão Gênica , Camundongos , Microssomos/enzimologia , Dados de Sequência Molecular , Prostaglandina-E Sintases , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: To report the nonrandomized first-in-human phase I trial of PRS-050, a novel, rationally engineered Anticalin based on human tear lipocalin that targets and antagonizes vascular endothelial growth factor A (VEGF-A). METHODS: Patients with advanced solid tumors received PRS-050 at 0.1 mg/kg to 10 mg/kg by IV in successive dosing cohorts according to the 3+3 escalation scheme. The primary end point was safety. RESULTS: Twenty-six patients were enrolled; 25 were evaluable. Two patients experienced dose-limiting toxicity, comprising grade (G) 3 hypertension and G3 pyrexia, respectively. The maximum tolerated dose was not reached. Most commonly reported treatment-emergent adverse events (AEs) included chills (52%; G3, 4%), fatigue (52%; G3, 4%), hypertension (44%; G3, 16%), and nausea (40%, all G1/2). No anti-PRS-050 antibodies following multiple administration of the drug were detected. PRS-050 showed dose-proportional pharmacokinetics (PK), with a terminal half-life of approximately 6 days. Free VEGF-A was detectable at baseline in 9/25 patients, becoming rapidly undetectable after PRS-050 infusion for up to 3 weeks. VEGF-A/PRS-050 complex was detectable for up to 3 weeks at all dose levels, including in patients without detectable baseline-free VEGF-A. We also detected a significant reduction in circulating matrix metalloproteinase 2, suggesting this end point could be a pharmacodynamic (PD) marker of the drug's activity. CONCLUSIONS: PRS-050, a novel Anticalin with high affinity for VEGF-A, was well-tolerated when administered at the highest dose tested, 10 mg/kg. Based on target engagement and PK/PD data, the recommended phase II dose is 5 mg/kg every 2 weeks administered as a 120-minute infusion. TRIAL REGISTRATION: ClinicalTrials.gov NCT01141257 http://clinicaltrials.gov/ct2/show/NCT01141257.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacocinética , Lipocalina 1 , Lipocalinas/administração & dosagem , Lipocalinas/farmacocinética , Neoplasias/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adulto , Idoso , Inibidores da Angiogênese/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Humanos , Lipocalinas/efeitos adversos , Metaloproteinase 2 da Matriz/sangue , Pessoa de Meia-Idade , Neoplasias/sangue , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
Approval of an anti-CD20 chimeric monoclonal antibody, rituximab, has revolutionized cancer treatment and also validated CD20 targeting for providing benefit and improvement of overall response rate in B cell malignancies. Although many patients have benefited from the treatment of rituximab, there are still significant numbers of patients who are refractory or develop resistance to the treatment. Here we discuss pre-clinically well-defined potential mechanisms of action for rituximab and review the ways next generation anti-CD20 monoclonal antibodies can potentially exploit them to further enhance the treatment of B cell malignancies. Although the relative importance of each of these mechanism remains to be established in the clinic, well-designed clinical trials will help to define the efficacy and understanding of which effector activity of modified next generation anti-CD20 mAb will be important in the treatment of B-cell malignancies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/efeitos dos fármacos , Imunoterapia , Linfoma de Células B/tratamento farmacológico , Oncologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Apoptose/imunologia , Linfócitos B/imunologia , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos , Humanos , Linfoma de Células B/imunologia , Rituximab , Transdução de Sinais/imunologiaRESUMO
Prostaglandin (PG)E(2) has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. Although microsomal PGE synthase (mPGES)1 is the only genetically verified PGE synthase, results of previous studies examining the consequences of mPGES1 deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1(-/-) mice on 2 distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1(-/-) mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1(-/-) animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1(-/-) and WT mice developed mild hypertension of similar magnitude, whereas 129-mPGES1(-/-) mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1(-/-) animals. In WT mice of both strains, the increase in urinary excretion of PGE(2) with angiotensin II was attenuated in mPGES1(-/-) animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1(-/-) mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.
Assuntos
Albuminúria/metabolismo , Hipertensão/enzimologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Análise de Variância , Angiotensina II/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Dinoprostona/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Hipertensão/induzido quimicamente , Oxirredutases Intramoleculares/deficiência , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Microssomos/metabolismo , Probabilidade , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , RNA Mensageiro/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1) whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2) the clinical significance of its expression levels in human breast, colorectal, lung and prostate cancers. We found that the expression of Notch-1 protein was overexpressed in primary colorectal adenocarcinoma and nonsmall cell lung carcinoma (NSCLC), but not in primary ductal breast carcinoma or prostate adenocarcinoma. Further analysis revealed that higher levels of Notch-1 protein expression were significantly associated with poorer differentiation of breast and prostate tumors. Strikingly, for NSCLC, the expression levels of Notch-1 protein were found to be inversely correlated with tumor differentiation and progression. For colorectal tumors, however, no correlation of Notch-1 protein expression was found with any tumor clinicopathological parameters, in spite of its overexpression in tumor cells. Our data demonstrated the complexity of Notch-1 protein expression in human solid tumors and further supported the notion that the roles of Notch-1 expression in tumorigenesis are highly context-dependent. The findings could provide the basis for development of distinct therapeutic strategies of Notch-1 mAbs for its applications in the treatment of suitable types of human cancers.
RESUMO
BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
Assuntos
Anticorpos Monoclonais/farmacologia , Mutação , Receptor Notch1/imunologia , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Animais , Especificidade de Anticorpos/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Ligação Competitiva , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-2 , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Prostaglandins (PGs) are found in high levels in the synovial fluid of patients with rheumatoid arthritis, and nonsteroidal blockade of these bioactive lipids plays a role in patient care. The aim of this study was to explore the relative contribution of cyclooxygenase (COX) isoforms and PG species in the autoantibody-driven K/BxN serum-transfer arthritis. METHODS: The prostanoid content of arthritic ankles was assessed in ankle homogenates, and the importance of this pathway was confirmed with pharmacologic blockade. The presence of COX isoforms was assessed by Western blotting and their functional contribution was compared using COX-1-/- and COX-2-/- mice as well as isoform-specific inhibitors. The relative importance of PGE2 and PGI2 (prostacyclin) was determined using mice deficient in microsomal PGE synthase 1 (mPGES-1) and in the receptors for PGI2. RESULTS: High levels of PGE2 and 6-keto-PGF1alpha (a stable metabolite of PGI2) were detected in arthritic joint tissues, correlating strongly with the intensity of synovitis. Pharmacologic inhibition of PG synthesis prevented arthritis and ameliorated active disease. While both COX isoforms were found in inflamed joint tissues, only COX-1 contributed substantially to clinical disease; COX-1-/- mice were fully resistant to disease, whereas COX-2-/- mice remained susceptible. These findings were confirmed by isoform-specific pharmacologic inhibition. Mice lacking mPGES-1 (and therefore PGE2) developed arthritis normally, whereas mice incapable of responding to PGI2 exhibited a significantly attenuated arthritis course, confirming a role of PGI2 in this arthritis model. CONCLUSION: These findings challenge previous paradigms of distinct "housekeeping" versus inflammatory functions of the COX isoforms and highlight the potential pathogenic contribution of prostanoids synthesized via COX-1, in particular PGI2, to inflammatory arthritis.
Assuntos
Artrite/enzimologia , Artrite/imunologia , Autoanticorpos/imunologia , Ciclo-Oxigenase 1/fisiologia , Ciclo-Oxigenase 2/fisiologia , Prostaglandinas/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The inhibition of prostaglandin (PG) synthesis is at the center of current anti-inflammatory therapies. Because cyclooxygenase-2 (COX-2) inhibitors and nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of multiple PGs, there is currently a strong focus on characterizing the role of the different PGs in the inflammation process and development of arthritis. Evidence to date suggests that both PGE(2) and PGI(2) act as mediators of pain and inflammation. Most of the data indicating a role for PGI(2) in this context have been generated in animal models of acute pain. Herein, we describe the role of PGI(2) in models of osteoarthritis (OA) and rheumatoid arthritis using a highly selective PGI(2) receptor (IP, Ptgir) antagonist and IP receptor-deficient mice. In the rat OA model using monoiodoacetate injection into the knee joint, the IP antagonist reduced pain with an efficacy approaching that of the NSAID diclofenac. In a chronic model of inflammatory arthritis, collagen-antibody induced arthritis model in mice, IP receptor-deficient mice displayed a 91% reduction in arthritis score. Interestingly, pretreatment with the IP [N-[4-(imidazolidin-2-ylideneamino)-benzyl]-4-methoxy-benzamide] antagonist in this model also caused a significant reduction of the symptoms, whereas administration of the compound after the initiation of arthritis had no detectable effect. Our data indicate that, in addition to its role in acute inflammation, PGI(2) is involved in the development of chronic inflammation. The results also suggest that the inhibition of PGI(2) synthesis by NSAIDs and COX-2 inhibitors, in addition to that of PGE(2), contributes to their efficacy in treating the signs of arthritis.
Assuntos
Artrite Experimental/complicações , Artrite Experimental/tratamento farmacológico , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Dor/tratamento farmacológico , Dor/etiologia , Prostaglandinas I/antagonistas & inibidores , Animais , Carragenina , Cromatografia Líquida de Alta Pressão , Doença Crônica , Colágeno/imunologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Edema/induzido quimicamente , Edema/patologia , Epoprostenol/análogos & derivados , Epoprostenol/metabolismo , Epoprostenol/farmacologia , Temperatura Alta , Iodoacetatos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/induzido quimicamente , Osteoartrite/patologia , Ovalbumina , Ratos , Ratos Sprague-Dawley , Receptores de Epoprostenol/genéticaRESUMO
Prostacyclin is one of a number of lipid mediators elaborated from the metabolism of arachidonic acid by the cyclooxygenase (COX) enzymes. This prostanoid is a potent inhibitor of platelet aggregation, and its production by endothelial cells and protective role in the vasculature are well established. In contrast, much less is known regarding the function of this prostanoid in other disease processes. We show here that COX-2-dependent production of prostacyclin plays an important role in the development of fibrotic lung disease, limiting both the development of fibrosis and the consequential alterations in lung mechanics. In stark contrast, loss of prostaglandin E(2) synthesis and signaling through the G(s)-coupled EP2 and EP4 receptors had no effect on the development of disease. These findings suggest that prostacyclin analogs will protect against bleomycin-induced pulmonary fibrosis in COX-2(-/-) mice. If such protection is observed, investigation of these agents as a novel therapeutic approach to pulmonary fibrosis in humans may be warranted.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Ciclo-Oxigenase 2/metabolismo , Epoprostenol/metabolismo , Fibrose Pulmonar , Animais , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Pulmão/citologia , Pulmão/patologia , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prostaglandinas/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismoRESUMO
Prostaglandin E2 (PGE2), similar to beta-adrenergic receptor agonists, can protect airways from bronchoconstriction and resulting increase in airway resistance induced by a number of agents, including cholinergic receptor agonists and antigen. We examined the impact of sustained alterations in PGE2 pathways on changes in airway resistance. Genetic methods were utilized to alter PGE2 metabolism and signal transduction in the murine lung. PGE2 levels were elevated by generating mice lacking 15-hydroxyprostaglandin (Hpgd-/-), the major catabolic enzyme of PGE2, and by generating a transgenic line in which mouse PGE2 synthase (Ptges) expression is driven by a human lung-specific promoter, hSP-C. Conversely, to determine the impact of loss of PGE2 on airway reactivity, we examined mice lacking this synthase (Ptges-/-) and receptors that mediate the actions of PGE2, particularly the PGE2 EP2 receptor (Ptger2). Diminished capacity to produce and respond to PGE2 did not alter the response of mice to cholinergic stimuli. In contrast, the responsiveness to cholinergic stimulation was dramatically altered in animals with elevated PGE2 levels. The Hpgd-/- and hSP-C-Ptges transgenic lines both showed attenuated airway responsiveness to methacholine as measured by lung resistance. Thus, whereas compromise of the Ptges/PGE2/Ptger2 pathway does not alter airway responsiveness, genetic modulation that elevates PGE2 levels in the lung attenuates airway responsiveness.