RESUMO
BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].
Assuntos
AMP Cíclico , Miócitos Cardíacos , Camundongos , Animais , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Peptídeos/metabolismoRESUMO
Abdominal aortic aneurysm (AAA) is characterized by aorta dilation due to wall degeneration, which mostly occurs in elderly males. Vascular aging is implicated in degenerative vascular pathologies, including AAA. Cyclic nucleotide phosphodiesterases, by hydrolyzing cyclic nucleotides, play critical roles in regulating vascular structure remodeling and function. Cyclic nucleotide phosphodiesterase 1C (PDE1C) expression is induced in dedifferentiated and aging vascular smooth muscle cells (SMCs), while little is known about the role of PDE1C in aneurysm. We observed that PDE1C was not expressed in normal aorta but highly induced in SMC-like cells in human and murine AAA. In mouse AAA models induced by Angiotensin II or periaortic elastase, PDE1C deficiency significantly decreased AAA incidence, aortic dilation, and elastin degradation, which supported a causative role of PDE1C in AAA development in vivo. Pharmacological inhibition of PDE1C also significantly suppressed preestablished AAA. We showed that PDE1C depletion antagonized SMC senescence in vitro and/or in vivo, as assessed by multiple senescence biomarkers, including senescence-associated ß-galactosidase activity, γ-H2AX foci number, and p21 protein level. Interestingly, the role of PDE1C in SMC senescence in vitro and in vivo was dependent on Sirtuin 1 (SIRT1). Mechanistic studies further showed that cAMP derived from PDE1C inhibition stimulated SIRT1 activation, likely through a direct interaction between cAMP and SIRT1, which leads to subsequent up-regulation of SIRT1 expression. Our findings provide evidence that PDE1C elevation links SMC senescence to AAA development in both experimental animal models and human AAA, suggesting therapeutical significance of PDE1C as a potential target against aortic aneurysms.
Assuntos
Aneurisma da Aorta Abdominal/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Angiotensina II/toxicidade , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Biomarcadores , Senescência Celular , AMP Cíclico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/genética , Inibidor de Quinase Dependente de Ciclina p21 , Histonas , Masculino , Camundongos , Camundongos Knockout para ApoE , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Regulação para Cima , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Heart failure is a leading cause of death worldwide. Cyclic nucleotide phosphodiesterases (PDEs), through degradation of cyclic nucleotides, play critical roles in cardiovascular biology and disease. Our preliminary screening studies have revealed PDE10A upregulation in the diseased heart. However, the roles of PDE10A in cardiovascular biology and disease are largely uncharacterized. The current study is aimed to investigate the regulation and function of PDE10A in cardiac cells and in the progression of cardiac remodeling and dysfunction. METHODS: We used isolated adult mouse cardiac myocytes and fibroblasts, as well as preclinical mouse models of hypertrophy and heart failure. The PDE10A selective inhibitor TP-10, and global PDE10A knock out mice were used. RESULTS: We found that PDE10A expression remains relatively low in normal and exercised heart tissues. However, PDE10A is significantly upregulated in mouse and human failing hearts. In vitro, PDE10A deficiency or inhibiting PDE10A with selective inhibitor TP-10, attenuated cardiac myocyte pathological hypertrophy induced by Angiotensin II, phenylephrine, and isoproterenol, but did not affect cardiac myocyte physiological hypertrophy induced by IGF-1 (insulin-like growth factor 1). TP-10 also reduced TGF-ß (transforming growth factor-ß)-stimulated cardiac fibroblast activation, proliferation, migration and extracellular matrix synthesis. TP-10 treatment elevated both cAMP and cGMP levels in cardiac myocytes and cardiac fibroblasts, consistent with PDE10A as a cAMP/cGMP dual-specific PDE. In vivo, global PDE10A deficiency significantly attenuated myocardial hypertrophy, cardiac fibrosis, and dysfunction induced by chronic pressure overload via transverse aorta constriction or chronic neurohormonal stimulation via Angiotensin II infusion. Importantly, we demonstrated that the pharmacological effect of TP-10 is specifically through PDE10A inhibition. In addition, TP-10 is able to reverse pre-established cardiac hypertrophy and dysfunction. RNA-Sequencing and bioinformatics analysis further identified a PDE10A-regualted transcriptome involved in cardiac hypertrophy, fibrosis, and cardiomyopathy. CONCLUSIONS: Taken together, our study elucidates a novel role for PDE10A in the regulation of pathological cardiac remodeling and development of heart failure. Given that PDE10A has been proven to be a safe drug target, PDE10A inhibition may represent a novel therapeutic strategy for preventing and treating cardiac diseases associated with cardiac remodeling.
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Cardiomegalia/enzimologia , Fibroblastos/enzimologia , Miócitos Cardíacos/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Remodelação Ventricular , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Modelos Animais de Doenças , Fibroblastos/patologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Diester Fosfórico Hidrolases/genética , TranscriptomaRESUMO
BACKGROUND: cAMP plays a critical role in regulating cardiomyocyte survival. Various cAMP signaling pathways behave distinctly or in opposition. We have previously reported that activation of cAMP hydrolysis by cyclic nucleotide phosphodiesterase 1C (PDE1C) promotes cardiomyocytes death/apoptosis, yet the underlying molecular mechanism remains unknown. In this study, we aimed to identify the specific cAMP signaling pathway modulated by PDE1C and determine the mechanism by which Ca2+/calmodulin-stimulated PDE1C is activated. METHODS: To study cardiomyocyte death/apoptosis, we used both isolated mouse adult cardiomyocytes in vitro and doxorubicin-induced cardiotoxicity in vivo. We used a variety of pharmacological activators and inhibitors as well as genetically engineered molecular tools to manipulate the expression and activity of proteins of interest. RESULTS: We found that the protective effect of PDE1C inhibition/deficiency on Ang II or doxorubicin-induced cardiomyocyte death/apoptosis is dependent on cAMP-generating adenosine A2 receptors (A2Rs), suggesting that PDE1C's cAMP-hydrolyzing activity selectively modulates A2R-cAMP signaling in cardiomyocytes. In addition, we found that the effects of PDE1C activation on Ang II-mediated cAMP reduction and cardiomyocyte death are dependent on transient receptor potential-canonical (TRPC) channels, in particular TRPC3. We also observed synergistic protective effects on cardiomyocyte survival from the combination of A2R stimulation together with PDE1 or TRPC inhibition. Coimmunostaining and coimmunoprecipitation studies showed that PDE1C is localized in proximity with A2R and TRPC3 in the plasma membrane and perhaps T tubules. It is important to note that we found that doxorubicin-induced cardiac toxicity and dysfunction in mice are attenuated by the PDE1 inhibitor IC86340 or in PDE1C knockout mice, and this protective effect is significantly diminished by A2R antagonism. CONCLUSIONS: We have characterized a novel multiprotein complex comprised of A2R, PDE1C, and TRPC3, in which PDE1C is activated by TRPC3-derived Ca2+, thereby antagonizing A2R-cAMP signaling and promoting cardiomyocyte death/apoptosis. Targeting these molecules individually or in combination may represent a compelling therapeutic strategy for potentiating cardiomyocyte survival.
Assuntos
AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Receptores A2 de Adenosina/metabolismo , Canais de Cátion TRPC/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Angiotensina II/toxicidade , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Doxorrubicina/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores A2 de Adenosina/química , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genéticaRESUMO
Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-ß-stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance.
RESUMO
In order to define the role of oxalic acid (OA) in the invasion of Botrytis cinerea in tomato plants, the OA induction of resistance related to oxalate oxidase (O×O) and germin was examined. In greenhouse experiments, OA at 3 mmol/L significantly induced resistance in tomato plants against B. cinerea strains B05.10 and T4, reducing lesion size of 37.55% and 24.91% by compared with distilled water control, respectively, while 20 mmol/L OA increasing by 36.14% and 41.48%. OA contents were 98 and 46 µg/mL when tomato plants were infected by B. cinerea strains B05.10 and T4, respectively. To define the molecular-genetic mechanisms, we compared the gene expression under four different conditions: 3 mmol/L OA-treated plants, 20 mmol/L OA-treated plants, B. cinerea strain B05.10-infected plants (B05.10 Inf plants) and B. cinerea strain T4-infected plants (T4 Inf plants). In 3 mmol/L OA-treated plants, the expressions of O×O and Germin peaked at 48 h after spraying, with approximate threefold and 18-fold increase compared with the control expression, respectively. In T4 Inf plants, the expression (mRNA accumulation) of O×O and Germin reached the highest levels at 24 h after inoculation, with 3- and 13-times that immediately after inoculation, respectively. In total, these findings suggest that elevated levels of OA correlated with increased fungal invasion and lower OA induced resistance in tomato plants by increasing expressions of O×O and Germin.
Assuntos
Botrytis/fisiologia , Ácido Oxálico/imunologia , Doenças das Plantas/microbiologia , Solanum lycopersicum/imunologia , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Oxirredutases/genética , Oxirredutases/imunologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologiaRESUMO
PURPOSE: Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). METHODS: Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. RESULTS: We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFß-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). CONCLUSIONS: Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.
Assuntos
Cardiomegalia/prevenção & controle , Cardiomiopatias/prevenção & controle , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Alcaloides de Vinca/farmacologia , Angiotensina II , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/enzimologia , Cardiomiopatias/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (ß2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by ß2-AR but not ß1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a ß2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the ß2-AR/PI3K/AKT cascade.
Assuntos
Alcaloides/farmacologia , Cardiotônicos/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Alcaloides/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cardiotônicos/uso terapêutico , Caspase 3/metabolismo , Células Cultivadas , Peróxido de Hidrogênio , Masculino , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tetra-Hidroisoquinolinas/uso terapêuticoRESUMO
Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.
Assuntos
Miosinas Cardíacas/fisiologia , Glicoproteínas de Membrana/genética , Miocárdio/metabolismo , Cadeias Leves de Miosina/fisiologia , NADPH Oxidases/genética , Animais , Miosinas Cardíacas/análise , Núcleo Celular/química , Células Cultivadas , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Cadeias Leves de Miosina/análise , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , NADPH Oxidase 2 , Estresse Oxidativo , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Hsp70 (heat shock protein 70) plays critical roles in cancer cell proliferation and apoptosis. Recently, accumulating evidences have demonstrated the cancer promoting effects of Hsp70 in bladder cancer. The development of novel therapeutic approaches targeting Hsp70 thus received great attention from researchers. In this study, we demonstrated that silibinin, a natural polyphenolic flavonoid isolated from the milk thistle, targeted Hsp70 by inhibiting its transcription in bladder cancer cells. We also demonstrated that knockdown of endogenous Hsp70 enhanced silibinin-induced apoptosis, while overexpression of exogenous Hsp70 could partially reverse the effects of silibinin-induced cell apoptosis. Furthermore, we found that silibinin could activate HSF1/Hsp70-regulated mitochondrial apoptotic pathway. Mechanically, silibinin inhibited the interaction between Apaf-1 and Hsp70, thus increasing the recruitment of pro caspase-9. Results from in vivo study demonstrated that silibinin suppressed the growth of bladder cancer xenografts, which was accompanied with the activation of caspase-3 and downregulation of HSF1 and Hsp70. Taken together, our data indicates that silibinin induces mitochondrial apoptosis via inhibiting HSF1/Hsp70 pathway and also suggests the therapeutic potential of silibinin in the treatment of bladder cancer.
RESUMO
Metal ions are vital components in many proteins for the inference and engineering of protein function, with coordination complexity linked to structural (4-residue predominate), catalytic (3-residue predominate), or regulatory (2-residue predominate) roles. Computational tools for modeling metal ions in protein structures, especially for transient, reversible, and concentration-dependent regulatory sites, remain immature. We present PinMyMetal (PMM), a sophisticated hybrid machine learning system for predicting zinc ion localization and environment in macromolecular structures. Compared to other predictors, PMM excels in predicting regulatory sites (median deviation of 0.34 Å), demonstrating superior accuracy in locating catalytic sites (median deviation of 0.27 Å) and structural sites (median deviation of 0.14 Å). PMM assigns a certainty score to each predicted site based on local structural and physicochemical features independent of homolog presence. Interactive validation through our server, CheckMyMetal, expands PMM's scope, enabling it to pinpoint and validates diverse functional zinc sites from different structure sources (predicted structures, cryo-EM and crystallography). This facilitates residue-wise assessment and robust metal binding site design. The lightweight PMM system demands minimal computing resources and is available at https://PMM.biocloud.top. While currently trained on zinc, the PMM workflow can easily adapt to other metals through expanded training data.
RESUMO
Influenza virus infection poses a continual menace to public health. Here, we developed soluble trimeric HA ectodomain vaccines by establishing interprotomer disulfide bonds in the stem region, which effectively preserve the native antigenicity of stem epitopes. The stable trimeric H1 ectodomain proteins exhibited higher thermal stabilities in comparison with unmodified HAs and showed strong binding activities towards a panel of anti-stem cross-reactive antibodies that recognize either interprotomer or intraprotomer epitopes. Negative stain transmission electron microscopy (TEM) analysis revealed the stable trimer architecture of the interprotomer disulfide-stapled WA11#5, NC99#2, and FLD#1 proteins as well as the irregular aggregation of unmodified HA molecules. Immunizations of mice with those trimeric HA ectodomain vaccines formulated with incomplete Freund's adjuvant elicited significantly more potent cross-neutralizing antibody responses and offered broader immuno-protection against lethal infections with heterologous influenza strains compared to unmodified HA proteins. Additionally, the findings of our study indicate that elevated levels of HA stem-specific antibody responses correlate with strengthened cross-protections. Our design strategy has proven effective in trimerizing HA ectodomains derived from both influenza A and B viruses, thereby providing a valuable reference for designing future influenza HA immunogens.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Dissulfetos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vacinas contra Influenza , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae , Animais , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Anticorpos Antivirais/imunologia , Camundongos , Dissulfetos/química , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Anticorpos Neutralizantes/imunologia , Feminino , Proteção Cruzada/imunologia , Reações Cruzadas , Humanos , Influenza Humana/prevenção & controle , Influenza Humana/imunologia , Influenza Humana/virologia , Epitopos/imunologia , Epitopos/genética , Epitopos/química , Multimerização Proteica , Vírus da Influenza B/imunologia , Vírus da Influenza B/genética , Vírus da Influenza B/químicaRESUMO
Vascular peroxidase 1 (VPO1) can utilize reactive oxygen species (ROS) generated from NADPH oxidase (NOX) to catalyze peroxidative reactions. This study was performed to identify a novel pathway of NOX/VPO1 in mediating the oxidative injury following myocardial ischemia reperfusion (IR). In a rat model of myocardial IR, the infarct size, serum creatine kinase (CK) activity, apoptosis, NOX activity, NOX2 and VPO1 expression were measured. In a cell (rat heart-derived H9c2 cells) model of hypoxia/reoxygenation (HR), the apoptosis, NOX activity, NOX2 and VPO1 expression, and H(2)O(2) and HOCl levels were examined. In vivo, IR caused 54.8 ± 1.7 % infarct size in myocardium accompanied by elevated activities of CK, caspase-3 and NOX, up-regulated VPO1 expression and high numbers of myocardial apoptotic cells; these effects were attenuated by pretreatment with the inhibitor of NOX. In vitro, inhibition of NOX or silencing of NOX2 or VPO1 expression significantly suppressed HR-induced cellular apoptosis concomitantly with decreased HOCl production. Inhibition of NOX or silencing of NOX2 led to a decrease in H(2)O(2) production accompanied by a decrease in VPO1 expression and HOCl production. However, silencing of VPO1 expression did not affect NOX2 expression and H(2)O(2) production. H(2)O(2)-induced VPO1 expression was partially reversed by JNK or p38 MAPK inhibitor. Our results demonstrate a novel pathway of NOX2/VPO1 in myocardium, where VPO1 coordinates with NOX2 and amplifies the role of NOX-derived ROS in oxidative injury following IR.
Assuntos
Hemeproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Infarto do Miocárdio/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , NADPH Oxidases/metabolismo , Estresse Oxidativo , Peroxidases/metabolismo , Transdução de Sinais , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular , Creatina Quinase/sangue , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Hemeproteínas/genética , Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Estresse Oxidativo/efeitos dos fármacos , Peroxidases/genética , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Myeloperoxidase (MPO) is involved in myocardial ischemia-reperfusion (IR) injury and vascular peroxidase (VPO) is a newly identified isoform of MPO. This study was conducted to explore whether VPO is involved in IR-induced cardiac dysfunction and apoptosis. In a rat Langendorff model of myocardial IR, the cardiac function parameters (left ventricular pressure and the maximum derivatives of left ventricular pressure and coronary flow), creatine kinase (CK) activity, apoptosis, VPO1 activity were measured. In a cell (rat-heart-derived H9c2 cells) model of hypoxia-reoxygenation (HR), apoptosis, VPO activity, and VPO1 mRNA expression were examined. In isolated heart, IR caused a marked decrease in cardiac function and a significant increase in apoptosis, CK, and VPO activity. These effects were attenuated by pharmacologic inhibition of VPO. In vitro, pharmacologic inhibition of VPO activity or silencing of VPO1 expression significantly suppressed HR-induced cellular apoptosis. Our results suggest that increased VPO activity contributes to IR-induced cardiac dysfunction and inhibition of VPO activity may have the potential clinical value in protecting the myocardium against IR injury.
Assuntos
Apoptose/fisiologia , Cardiopatias/enzimologia , Coração/fisiopatologia , Hemeproteínas/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/enzimologia , Peroxidases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Catalase/metabolismo , Células Cultivadas , Creatina Quinase/antagonistas & inibidores , Creatina Quinase/metabolismo , Coração/efeitos dos fármacos , Cardiopatias/genética , Cardiopatias/patologia , Hemeproteínas/genética , Hemeproteínas/metabolismo , Masculino , Reperfusão Miocárdica/métodos , Miocárdio/enzimologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Peroxidase/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Floroglucinol/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-DawleyRESUMO
Capsiate is a non-pungent analogue of capsaicin from CH-19 Sweet peppers. Capsaicin is reported to trigger calcitonin gene-related peptide (CGRP) release through activation of transient receptor potential vanilloid subfamily member 1 (TRPV1) and produces beneficial effects on gastric mucosa. This study aimed to investigate whether capsiate is able to produce beneficial effects on gastric mucosa and whether the protective effects of capsipate occur through a mechanism involving the activation of TRPV1 and CGRP release. A rat model of gastric mucosal injury was established by the oral administration of acidified ethanol. Gastric tissues were collected for analysis of the gastric ulcer index, cellular apoptosis, activities of caspase-3, catalase and superoxide dismutase (SOD), and levels of CGRP, TNF-α, and malondialdehyde (MDA). Our results show that the acute administration of ethanol significantly increased the gastric ulcer index concomitantly with an increase in cellular apoptosis, caspase-3 activity, and TNF-α and MDA levels, as well as a decrease in the activities of catalase and SOD. Pretreatment with 1 mg/kg capsiate attenuated ethanol-induced gastric mucosal injury and cellular apoptosis accompanied by an increase in CGRP level, catalase, and SOD activities, and a decrease in caspase-3 activity, and TNF-α and MDA levels. The effects of capsiate were inhibited by capsazepine, an antagonist of TRPV1. These results suggest that capsiate is able to produce beneficial effects on ethanol-induced gastric mucosal injury. These effects are related to the stimulation of CGRP release through the activation of TRPV1.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/análogos & derivados , Capsicum/química , Mucosa Gástrica/efeitos dos fármacos , Fitoterapia , Úlcera Gástrica/tratamento farmacológico , Canais de Cátion TRPV/metabolismo , Animais , Apoptose/efeitos dos fármacos , Capsaicina/farmacologia , Capsaicina/uso terapêutico , Caspase 3/metabolismo , Modelos Animais de Doenças , Etanol , Mucosa Gástrica/metabolismo , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Masculino , Malondialdeído/sangue , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/sangueRESUMO
AIMS: Intimal hyperplasia is a common feature of vascular remodelling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo. METHODS AND RESULTS: RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodelling of cultured human saphenous vein segments. CONCLUSIONS: Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signalling and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease.
Assuntos
Músculo Liso Vascular , Lesões do Sistema Vascular , Animais , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Proliferação de Células , Células Cultivadas , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Ciclina D1/metabolismo , Células Endoteliais/metabolismo , Guanilato Ciclase/metabolismo , Guanilato Ciclase/farmacologia , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Diester Fosfórico Hidrolases/metabolismo , RNA Mensageiro/metabolismo , Remodelação Vascular , Lesões do Sistema Vascular/tratamento farmacológico , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/metabolismoRESUMO
1. It has been shown that phloroglucinol has anti-inflammatory and anti-oxidant properties. Both inflammatory cell infiltration and myeloperoxidase (MPO) activation play an important role in myocardial reperfusion injury. The aim of the present study was to explore the effect of phloroglucinol on myocardial reperfusion injury and the mechanisms involved. 2. Anaesthetized rats were pretreated with phloroglucinol (15 or 30 mg/kg, i.g.) or vehicle (5 mmol/L carboxymethyl cellulose sodium) 30 min prior to experimentation. The left main coronary artery was subjected to 1 h occlusion followed by 3 h reperfusion. Infarct size, the release of creatine kinase (CK), inflammatory cell infiltration, MPO activity and protein content, catalase in the blood and myocardium, and myocardial apoptosis were measured. 3. Following myocardial ischaemia and reperfusion in vehicle-treated rats, infarct size was 43.5 ± 3.7% (relative to the area at risk). Accompanying detrimental changes included elevated CK, enhanced inflammatory cell infiltration, high numbers of myocardial apoptotic cells, elevated caspase 3 activity, increased MPO activity and content in the plasma and myocardium and reduced catalase activity. These effects were attenuated by pretreatment with both doses of phloroglucinol (15 and 30 mg/kg, i.g.). 4. The results of the present study suggest that phloroglucinol protects the myocardium against ischaemia-reperfusion injury in rats and that its beneficial effects are related to inhibition of MPO activity and inflammatory cell infiltration.
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Traumatismo por Reperfusão Miocárdica/prevenção & controle , Infiltração de Neutrófilos/efeitos dos fármacos , Peroxidase/antagonistas & inibidores , Floroglucinol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cardiotônicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Inflamação/imunologia , Masculino , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Infiltração de Neutrófilos/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Allopurinol, a xanthine oxidase (XO) inhibitor, is reported to alleviate myocardial ischemia/reperfusion (I/R) injury by reducing the production of reactive oxygen species (ROS). As an XO-derived product, H2O2 can act as a substrate of vascular peroxidase 1 (VPO1) to induce the generation of hypochlorous acid (HOCl), a potent oxidant. This study aims to explore whether the XO/VPO1 pathway is involved in the anti-oxidative effects of allopurinol on the myocardial I/R injury. In a rat heart model of I/R, allopurinol alleviated I/R oxidative injury accompanied by decreased XO activity, XO-derived products (H2O2 and uric acid), and VPO1 expression (mRNA and protein). In a cardiac cell model of hypoxia/reoxygenation (H/R), allopurinol or XO siRNA reduced H/R injury concomitant with decreased XO activity, VPO1 expression as well as the XO and VPO1-derived products (H2O2, uric acid, and HOCl). Although knockdown of VPO1 could also exert a beneficial effect on H/R injury, it did not affect XO activity, XO expression, and XO-derived products. Based on these observations, we conclude that the novel pathway of XO/VPO1 is responsible for, at least partly, myocardial I/R-induced oxidative injury, and allopurinol exerted the cardioprotective effects on myocardial I/R injury via inhibiting the XO/VPO1 pathway.
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Alopurinol , Xantina Oxidase , Animais , Peróxido de Hidrogênio , RatosRESUMO
RATIONALE: Higenamine (HG), is one of the main active components in many widely used Chinese herbs, and a common ingredient of health products in Europe and North America. Several groups, including our own, have previously shown the beneficial effects of HG against cardiomyocyte death during acute ischemic damage. However, the effect of HG on chronic cardiac remodeling, such as cardiac fibrosis, remains unknown. OBJECTIVE: Herein, we aim to investigate the role of HG in cardiac fibrosis in vivo as well as its cellular and molecular mechanisms. METHODS AND RESULTS: Chronic pressure overload with transverse aortic constriction (TAC) significantly increased cardiac hypertrophy, fibrosis, and cardiac dysfunction in mice, which were significantly attenuated by HG. Consistently, cardiac fibrosis induced by the chronic infusion of isoproterenol (ISO), was also significantly reduced by HG. Interestingly, our results showed that HG had no effect on adult mouse CM hypertrophy in vitro. However, HG suppressed the activation of cardiac fibroblasts (CFs) in vitro. Furthermore, TGF-ß1-induced expression of ACTA2, a marker of fibroblast activation, was significantly suppressed by HG. Concomitantly, HG inhibited TGF-ß1-induced phosphorylation of Smad2/3 in CFs. HG also reduced the expression of extracellular matrix molecules such as collagen I and collagen III. To our surprise, the inhibitory effect of HG on CFs activation was independent of the activation of the beta2 adrenergic receptor (ß2-AR) that is known to mediate the effect of HG on antagonizing CMs apoptosis. CONCLUSION: Our findings suggest that HG ameliorates pathological cardiac fibrosis and dysfunction at least partially by suppressing TGF-ß1/Smad signaling and CFs activation.
Assuntos
Alcaloides/farmacologia , Fibrinolíticos/farmacologia , Fibroblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/antagonistas & inibidores , Tetra-Hidroisoquinolinas/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Actinas/antagonistas & inibidores , Agonistas Adrenérgicos beta , Animais , Aorta/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cardiomegalia/induzido quimicamente , Cardiomegalia/prevenção & controle , Fibrose/prevenção & controle , Cardiopatias/induzido quimicamente , Cardiopatias/prevenção & controle , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Isoproterenol , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
A recent study showed that resveratrol, a polyphenol found in many plant species, exerts dual effects on gastric mucosal injury. By using the model of ethanol-induced gastric mucosal injury in the present study, we explored the effect of trans-3,5,4'-trimethoxystilbene (BTM-0512), a novel analog of resveratrol, on gastric mucosal injury and the possible underlying mechanisms. Gastric mucosal injury in the rat was induced by oral administration of acidified ethanol. The gastric tissues were collected for determination of the gastric ulcer index, asymmetric dimethylarginine (ADMA) and nitric oxide (NO) contents, the activity of dimethylarginine dimethylaminohydrolase (DDAH) and superoxide anion (O2(-)) or hydroxyl radical (OH*) formation. The results showed that acute administration of ethanol significantly increased the gastric ulcer index concomitantly with the decrease in DDAH activity and NO content as well as the increase in ADMA content, effects that were reversed by pretreatment with BTM-0512 (100 mg/kg) or L-arginine (300 mg/kg). Administration of BTM-0512 did not show a significant effect on O2(-) or OH. formation. The results suggest that BTM-0512 could protect the gastric mucosa against ethanol-induced injury, which is mainly related to an increase in DDAH activity and subsequent decrease in ADMA content.