RESUMO
Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI.
Title: Les phosphodiestérases des nucléotides cycliques - Cibles thérapeutiques dans l'hypertrophie et l'insuffisance cardiaques. Abstract: Les phosphodiestérases des nucléotides cycliques (PDE) modulent la régulation neuro-hormonale de la fonction cardiaque en dégradant l'AMPc et le GMPc. Dans les cardiomyocytes, de multiples isoformes de PDE, aux propriétés enzymatiques et aux localisations subcellulaires différentes, régulent localement les niveaux de nucléotides cycliques et les fonctions cellulaires associées. Cette organisation est fortement perturbée au cours de l'hypertrophie et de l'insuffisance cardiaque à fraction d'éjection réduite (IC), ce qui peut contribuer à la progression de la maladie. Sur le plan clinique, l'inhibition des PDE a été considérée comme une approche prometteuse pour compenser la désensibilisation aux catécholamines qui accompagne l'IC. Bien que des inhibiteurs de la PDE3, tels que la milrinone ou l'énoximone, puissent être utilisés cliniquement pour améliorer la fonction systolique et soulager les symptômes de l'IC aiguë, leur utilisation chronique s'est avérée préjudiciable. D'autres PDE, telles que les PDE1, PDE2, PDE4, PDE5, PDE9 et PDE10, sont apparues comme de nouvelles cibles potentielles pour le traitement de l'IC, chacune ayant un rôle unique dans les voies de signalisation locales des nucléotides cycliques. Dans cette revue, nous décrivons la signalisation de l'AMPc et du GMPc dans les cardiomyocytes et présentons les différentes familles de PDE exprimées dans le cÅur ainsi que leurs modifications dans l'hypertrophie cardiaque pathologique et dans l'IC. Nous évaluons également les résultats issus de modèles précliniques ainsi que les données cliniques indiquant l'utilisation d'inhibiteurs ou d'activateurs de PDE spécifiques qui pourraient avoir un potentiel thérapeutique dans l'IC.
Assuntos
Cardiomegalia , Insuficiência Cardíaca , Inibidores de Fosfodiesterase , Humanos , Cardiomegalia/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Animais , Inibidores de Fosfodiesterase/uso terapêutico , Inibidores de Fosfodiesterase/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Terapia de Alvo Molecular/métodos , GMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/fisiologiaRESUMO
Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.
Assuntos
Linfócitos do Interstício Tumoral/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiotaxia/fisiologia , Feminino , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias , Diester Fosfórico Hidrolases/fisiologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/fisiologia , Microambiente TumoralRESUMO
PURPOSE: The objective of the current study was to evaluate the effects of the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. METHODS: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. RESULTS: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. CONCLUSIONS: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX-LPA signaling axis.
Assuntos
Lisofosfolipídeos/farmacologia , Diester Fosfórico Hidrolases/farmacologia , Malha Trabecular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Diester Fosfórico Hidrolases/fisiologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/fisiologia , Malha Trabecular/fisiologiaRESUMO
The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus. Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis.
Assuntos
Padronização Corporal/genética , Rim/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Transdução de Sinais , Proteínas de Xenopus/fisiologia , Xenopus laevis/genética , Animais , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Redes Reguladoras de Genes , Rim/embriologia , Diester Fosfórico Hidrolases/genética , Proteínas de Xenopus/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
AIM: Diabetic cardiomyopathy (DCM) accomodates a spectrum of cardiac abnormalities. This study aims to investigate whether DCM is associated with changes in cyclic adenosine 3'-5' monophosphate (cAMP) signaling, particularly cyclic nucleotide phosphodiesterases (PDEs). MAIN METHODS: Type 1 diabetes (T1D) was induced in rats by streptozotocin (STZ, 65 mg/kg) injection. Myocardial remodeling, structure and function were evaluated by histology and echocardiography, respectively. We delineated the sequential changes affecting cAMP signaling and characterized the expression pattern of the predominant cardiac PDE isoforms (PDE 1-5) and ß-adrenergic (ß-AR) receptors at 4, 8 and 12 weeks following diabetes induction, by real-time quantitative PCR and Western blot. cAMP levels were measured by immunoassays. KEY FINDINGS: T1D-induced DCM was associated with cardiac remodeling, steatosis and fibrosis. Upregulation of ß1-AR receptor transcripts was noted in diabetic hearts at 4 weeks along with an increase in cAMP levels and an upregulation in the ejection fraction and fraction shortening. However, ß2-AR receptors expression remained unchanged regardless of the disease stage. Moreover, we noted an early and specific upregulation of cardiac PDE1A, PDE2A, PDE4B, PDE4D and PDE5A expression at week 4, followed by increases in PDE3A levels in diabetic hearts at week 8. However, DCM was not associated with changes in PDE4A gene expression irrespective of the disease stage. SIGNIFICANCE: We show for the first time differential and time-specific regulations in cardiac PDEs, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Diester Fosfórico Hidrolases/metabolismo , Animais , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Cardiomiopatias Diabéticas/metabolismo , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Estreptozocina/farmacologiaRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for 3'-end DNA lesions, predominantly stalled DNA-topoisomerase 1 (Top1) cleavage complexes. Tdp1 is a promising target for anticancer therapy based on DNA damage caused by Top1 poisoning. Earlier, we have reported about usnic acid enamine derivatives that are Tdp1 inhibitors sensitizing tumor cells to the action of Top1 poison (Zakharenko in J Nat Prod 79:2961-2967, 2016). In the present work, we showed a sensitizing effect of an enamine derivative of usnic acid (when administered intragastrically) on Lewis lung carcinoma in mice in combination with topotecan (TPT, Top1 poison used in the clinic). In the presence of the usnic acid derivative, both the volume of the primary tumor and the number of metastases significantly diminished. The absence of acute toxicity of this compound was demonstrated, as was the importance of the method of its administration for the manifestation of the sensitizing properties.
Assuntos
Benzofuranos/farmacologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/fisiologia , Topotecan/uso terapêutico , Animais , Carcinoma Pulmonar de Lewis/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Metástase Neoplásica , Transplante de NeoplasiasRESUMO
BACKGROUND: Elevated transforming growth factor (TGF)-ß2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-ß2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators. METHODS: The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-ß levels were measured using the Bio-Plex Pro TGF-ß Assay. RNA scope was used to assess the expression of ATX and TGF-ß2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-ß2 regulates ATX expressions. RESULTS: TGF-ß2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-ß2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-ß2 with concentration-dependent manner. In counter, ATX also induced TGF-ß1, TGF-ß2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-ß and ATX inhibition. CONCLUSIONS: Trans-signaling of TGF-ß2 regulates ATX expressions and thereby induced upregulations of TGF-ßs or fibrosis of hTM. TGF-ß2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.
Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Glaucoma de Ângulo Aberto/classificação , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to identify rmcA and morA, two genes which encode bis-(3',5')-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the ΔrmcA and ΔmorA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms.IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process.
Assuntos
Biofilmes , GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/fisiologia , Pseudomonas aeruginosa/fisiologia , GMP Cíclico/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a "self-trapping" mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2.
Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I/fisiologia , Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Diester Fosfórico Hidrolases/fisiologia , RNA/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Substituição de Aminoácidos , Clivagem do DNA , Técnicas de Inativação de Genes , Células HCT116 , Células HEK293 , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteólise , Estruturas R-Loop , Clivagem do RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , UbiquitinaçãoRESUMO
Glucose metabolism is the initiator of a large number of molecular secretory processes in ß cells. Cyclic nucleotides as a second messenger are the main physiological regulators of these processes and are functionally divided into compartments in pancreatic cells. Their intracellular concentration is limited by hydrolysis led by one or more phosphodiesterase (PDE) isoenzymes. Literature data confirmed multiple expressions of PDEs subtypes, but the specific roles of each in pancreatic ß-cell function, particularly in humans, are still unclear. Isoforms present in the pancreas are also found in various tissues of the body. Normoglycemia and its strict control are supported by the appropriate release of insulin from the pancreas and the action of insulin in peripheral tissues, including processes related to homeostasis, the regulation of which is based on the PDE- cyclic AMP (cAMP) signaling pathway. The challenge in developing a therapeutic solution based on GSIS (glucose-stimulated insulin secretion) enhancers targeted at PDEs is the selective inhibition of their activity only within ß cells. Undeniably, PDEs inhibitors have therapeutic potential, but some of them are burdened with certain adverse effects. Therefore, the chance to use knowledge in this field for diabetes treatment has been postulated for a long time.
Assuntos
Diabetes Mellitus/etiologia , Diester Fosfórico Hidrolases/fisiologia , Animais , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina/fisiologia , Isoenzimas/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Patients with chronic hepatitis C virus (HCV) develop hepatocellular carcinoma (HCC) regardless of achieving a sustained viral response (SVR). Because advanced liver fibrosis is a powerful risk factor for HCC, we analyzed the association between autotaxin (ATX), a liver fibrosis marker, and post-SVR HCC development within 3 years after antiviral treatment. We included 670 patients with HCV who received direct-acting antivirals, achieved SVR and were followed up for at least 6 months (270 of them were followed up for 3 years or more). We measured serum ATX levels before treatment and 12/24 weeks after treatment. The diagnosis of HCC was based on imaging modalities, such as dynamic computed tomography and dynamic magnetic resonance imaging and/or liver biopsy. The present study revealed that high levels of serum ATX predicted post-SVR HCC development (area under the receiver operating characteristic: 0.70-0.76). However, Wisteria floribunda agglutinin positive Mac-2 binding protein (M2BPGi), another liver fibrosis marker, was a more useful predictive marker especially post-treatment according to a multivariate analysis. Patients with a high rate of ATX reduction before and after antiviral treatment did not develop HCC regardless of high pretreatment ATX levels. In conclusion, post-treatment M2BPGi level and the combination of pretreatment ATX levels and rate of ATX reduction were useful predictive markers for post-SVR HCC development in patients with chronic HCV infection.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Hepatite C Crônica/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Idoso , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/genética , Antivirais/uso terapêutico , Biomarcadores/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Feminino , Hepacivirus/patogenicidade , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Humanos , Cirrose Hepática/complicações , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/sangue , Diester Fosfórico Hidrolases/fisiologia , Fatores de RiscoRESUMO
Autologous T cells engineered with T receptor genes (TCR) are being studied to treat cancers. We have recently identified a panel of mouse TCRs specific for the HLA-A0201/alpha fetoprotein epitope (AFP158) complex and have shown that human T cells engineered with these TCR genes (TCR-Ts) can eradicate hepatocellular carcinoma (HCC) xenografts in NSG mice. However, due to TCR's promiscuity, their off-target cross-reactivity must be studied prior to conducting clinical trials. In this study, we conducted in vitro X-scan assay and in silico analysis to determine the off-target cross-reactivity of 3 AFP158-specific TCR-Ts. We found that the 3 AFP158-specific TCR-Ts could be cross-activated by ENPP1436 peptide and that the TCR3-Ts could also be activated by another off-target peptide, RCL1215. However, compared to AFP158, it requires 250 times more ENPP1436 and 10,000 times more RCL1215 peptides to achieve the same level of activation. The EC50 of ENPP1436 peptide for activating TCR-Ts is approximately 17-33 times higher than AFP158. Importantly, the ENPP1+ tumor cells did not activate TCR1-Ts and TCR2-Ts, and only weakly activated TCR3-Ts. The IFNγ produced by TCR3-Ts after ENPP1+ cell stimulation was >22x lower than that after HepG2 cells. And, all TCR-Ts did not kill ENPP1 + tumor cells. Furthermore, ectopic over-expression of ENPP1 protein in HLA-A2+ tumor cells did not activate TCR-Ts. In silico analysis showed that the ENPP1436 peptide affinity for HLA-A0201 was ranked 40 times lower than AFP158 and the chance of ENPP1436 peptide being processed and presented by HLA-A0201 was 100 times less likely than AFP158. In contrast, the two off-targets (Titin and MAGE-A3) that did cause severe toxicity in previous trials have the same or higher MHC-binding affinity and the same or higher chance of being processed and presented. In conclusion, our data shows that TCR-Ts can be activated by off-target ENPP1436 peptide. But, compared to target AFP158, it requires at least 250 times more ENPP1436 to achieve the same level of activation. Importantly, ENPP1436 peptide in human cells is not processed and presented to a sufficient level to activate the AFP158-specific TCR-Ts. Thus, these TCR-Ts, especially the TCR1-Ts and TCR2-Ts, will unlikely cause significant off-target toxicity.
Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , alfa-Fetoproteínas/imunologia , Animais , Células Cultivadas , Reações Cruzadas , Antígeno HLA-A2/imunologia , Humanos , Ativação Linfocitária , Camundongos , Diester Fosfórico Hidrolases/fisiologia , Pirofosfatases/fisiologiaRESUMO
Carnosol is a natural compound with pharmacological action due to its anti-cancer properties. However, the precise mechanism for its anti-carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double-strand breaks (DSBs). We also found that cells lacking tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1-DNA cleavage complex (TOP1cc) and TOP2-DNA cleavage complex (TOP2cc). When comparing the accumulation of γ-H2AX foci and the number of chromosomal aberrations (CAs) with wild-type (WT) cells, the susceptivity of the TDP1-/- and TDP2-/- cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti-cancer effect of carnosol.
Assuntos
Abietanos/toxicidade , Antineoplásicos/toxicidade , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Diester Fosfórico Hidrolases/genéticaRESUMO
Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP â phosphate) was greater than eNPP activity (ATP â pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate â phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Fosfatase Alcalina/fisiologia , Doenças da Aorta/prevenção & controle , Apirase/antagonistas & inibidores , Calcinose/prevenção & controle , Difosfatos/metabolismo , Levamisol/uso terapêutico , Progéria/tratamento farmacológico , Pirofosfatases/antagonistas & inibidores , Trifosfato de Adenosina/uso terapêutico , Fosfatase Alcalina/antagonistas & inibidores , Animais , Antígenos CD/fisiologia , Doenças da Aorta/enzimologia , Apirase/deficiência , Apirase/fisiologia , Calcinose/enzimologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Lamina Tipo A/genética , Longevidade/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Miócitos de Músculo Liso/metabolismo , Diester Fosfórico Hidrolases/deficiência , Diester Fosfórico Hidrolases/fisiologia , Progéria/genética , Progéria/metabolismo , Progéria/patologia , Pirofosfatases/deficiência , Pirofosfatases/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The accumulation of fibroblasts is a critical step in the development of fibrosis, and lysophosphatidic acid (LPA) promotes fibrosis by regulating multiple fibroblast functions. Autotaxin (ATX) is a key LPA-producing enzyme, and we hypothesized that ATX contributes to the development of renal interstitial fibrosis through LPA-mediated effects on fibroblast functions. In a mouse model of renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO), the levels of renal ATX protein and activity increased with the progression of fibrosis in ligated kidneys, despite concurrent reductions in renal ATX mRNA. UUO enhanced vascular permeability in the renal interstitium, and ATX protein localized to areas of vascular leak, suggesting that vascular leak allowed ATX to enter the renal interstitium. In vitro studies showed that ATX induces the migration and proliferation of renal fibroblasts and enhances the vascular permeability of endothelial monolayers. Finally, pharmacological inhibition of ATX partially attenuated renal interstitial fibrosis. These results suggest that during the development of renal fibrosis, ATX accumulates in the renal interstitium and drives fibroblast accumulation and promotes renal interstitial vascular leak, thereby partially contributing to the pathogenesis of renal interstitial fibrosis. Taken together, ATX inhibition may have the potential to be a novel therapeutic strategy to combat renal interstitial fibrosis.
Assuntos
Permeabilidade Capilar/fisiologia , Fibroblastos/fisiologia , Rim/patologia , Diester Fosfórico Hidrolases/fisiologia , Animais , Progressão da Doença , Feminino , Fibrose , Rim/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Diester Fosfórico Hidrolases/metabolismoRESUMO
The dual-specific cAMP/cGMP phosphodiesterase PDE10A is exclusively localised to regions of the brain and specific cell types that control crucial brain circuits and behaviours. The downside to this expression pattern is that PDE10A is also positioned to be a key player in pathology when its function is perturbed. The last decade of research has seen a clear role emerge for PDE10A inhibition in modifying behaviours in animal models of psychosis and Huntington's disease. Unfortunately, this has not translated to the human diseases as expected. More recently, a series of families with hyperkinetic movement disorders have been identified with mutations altering the PDE10A protein sequence. As these mutations have been analysed and characterised in other model systems, we are beginning to learn more about PDE10A function and perhaps catch a glimpse into how PDE10A activity could be modified for therapeutic benefit.
Assuntos
Corpo Estriado/metabolismo , Doença de Huntington/metabolismo , Hipercinese/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Animais , Corpo Estriado/patologia , Modelos Animais de Doenças , Humanos , Camundongos , RatosRESUMO
Serotonin (5-hydroxy-tryptamine, 5-HT) exerted concentration-dependent positive inotropic effects or positive chronotropic effects in transgenic (TG) mice which overexpress the human 5-HT4a receptor in the heart but not in littermate wild-type (WT) mice. These positive inotropic effects and positive chronotropic effects are thought to be mediated by cyclic adenosine 3',5'-monophosphate (cAMP) in TG cardiomyocytes. To determine whether these effects are antagonized by endogenous phosphodiesterases (PDEs), the inotropic and chronotropic effects of 5-HT were tested in the additional presence of the PDE inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) (1 µM, a PDE2 inhibitor) or cilostamide (1 µM, a PDE3 inhibitor), rolipram (0.1 µM and 1 µM, a PDE4 inhibitor), and their combinations. For comparison, 3-isobutyl-1-methylxanthine (IBMX), an unspecific PDE inhibitor, was investigated. The use of 10 µM IBMX, the combination of rolipram (1 µM) and EHNA (1 µM), and the combination of rolipram (0.1 µM) and cilostamide (1 µM) each increased the potency of 5-HT to elevate the force of contraction in TG mice, but not the potency of 5-HT to increase the beating rate in TG mice. This indicates that PDE4 and PDE2 regulate the inotropic but not the chronotropic effects of 5-HT in TG mice. In contrast, cilostamide (1 µM) alone, EHNA (1 µM) alone, or in combination decreased the potency of 5-HT to increase force of contraction in TG mice. In summary, our present data suggest that the positive chronotropic effect of 5-HT in TG mice does not involve PDE activities, whereas the positive inotropic effect of 5-HT and the basal force in TG mice are diminished by endogenous activity of PDE4. Phosphorylation of PDE4, when PDE2 or PDE3 is inhibited, might enhance the activity of PDE4.
Assuntos
Coração/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/efeitos dos fármacos , Receptores 5-HT4 de Serotonina/genética , Antagonistas do Receptor 5-HT4 de Serotonina/farmacologia , Animais , Cardiotônicos/farmacologia , Eletrocardiografia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Diester Fosfórico Hidrolases/fisiologia , Serotonina/farmacologiaRESUMO
AIMS: 24-Hydroxycholesterol (24HC) is the main brain cholesterol metabolite, which level in the circulation is significantly changed under physiological and pathological conditions. Here, we have studied the effect of 24HC on the inotropic responses to ß-adrenoceptor (AR) stimulation. MAIN METHODS: Electrical stimulation-evoked contractions were recorded in isolated atria from mice. Fluorescent dyes, Fluo-4 and DAF-FM, were used for estimation of Ca2+ transient and NO production, respectively. KEY FINDINGS: We revealed that 24HC in the submicromolar range attenuated ß-AR-induced positive inotropy in isolated atria. This was accompanied by a decrease in Ca2+ transient and unchanged nitric oxide (NO) production. However, ß1-AR-induced positive inotropy and enhancement of Ca2+ transient were increased by 24HC due to suppression of NO production. Only ß2-AR-dependent inotropy and enhancement of Ca2+ transient were decreased by 24HC in a NO-independent manner. Inhibition of phosphodiesterase (PDE) suppressed effect of 24HC on ß2-AR-dependent contractility as well as on non-subtype specific ß-AR activation. Moreover, 24HC counteracted positive inopropic action of PDE inhibitors, IBMX and rolipam. Thus, 24HC modulates the effects of ß1- and ß2-AR stimulation via different mechanisms linked with change in activity of NO synthase or PDE, respectively. Under conditions of non-selective activation of ß-ARs, the depressant effect of 24HC related with ß2-AR-dependent signaling dominates. SIGNIFICANCE: We suggest that 24HC could serve as a modulator of atrial ß-AR signaling, contributing to regulation of contractility.
Assuntos
Colesterol 24-Hidroxilase/metabolismo , Hidroxicolesteróis/metabolismo , Óxido Nítrico/metabolismo , Antagonistas Adrenérgicos beta/metabolismo , Animais , Encéfalo/metabolismo , Colesterol/metabolismo , Colesterol 24-Hidroxilase/fisiologia , Átrios do Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/metabolismo , Oxisteróis/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Phosphodiesterase enzymes (PDEs) are responsible for the adjustment of cyclic nucleotide levels. Alterations in PDE expressions in different tissues cause conflicts between functional and clinical effects of PDE inhibitors. Therefore, the aim of this study was to investigate the gene and protein expressions and the functional role of PDEs in atrium and ventricle of rat heart. The expressions of PDEs were examined in cardiac intact tissues and enzymatically isolated cells. The effects of PDE1-5 inhibitors (vinpocetine, EHNA, milrinone, rolipram, sildenafil, and IBMX) on basal and isoprenaline-stimulated contractions and sinus rate were recorded in the isolated spontaneously beating right atrium and electrically stimulated left papillary muscles. The mRNA and protein levels of PDEs were significantly different in atrial and ventricular intact tissues and isolated myocytes. Atrial contractions were increased with vinpocetine while suppressed by EHNA, milrinone, rolipram, sildenafil and IBMX. Milrinone, sildenafil and IBMX increased the heart rate whereas vinpocetine caused negative chronotropy. Papillary muscle contractions have been increased only with the vinpocetine and IBMX. Both the expression and the action of PDE-1-5 show atrial and ventricular differences. Therefore, these differences should be taken into account in the experimental or therapeutic approaches of the heart.