RESUMEN
The aim of this study was to evaluate the effects of C-type natriuretic peptide (CNP) treatment prior to in vitro maturation (IVM) on mitochondria biogenesis in bovine oocyte matured in vitro and explore the related causes. The results showed that treatment with CNP before IVM significantly improved mitochondrial content, elevated the expression of genes related to mitochondria biogenesis, and increased the protein levels of phosphorylation of cAMP-response element binding protein (p-CREB) in bovine oocytes following IVM. However, further studies revealed that treatment with CNP before IVM could not increased the protein levels of p-CREB in bovine oocytes when natriuretic peptide receptor 2 activities was inhibited using the relative specific inhibitor Gö6976. In addition, treatment with CNP before IVM could not improved mitochondrial content or elevated the expression of genes related to mitochondria biogenesis in bovine oocytes when CREB activities was abolished using the specific inhibitor 666-15. In summary, these results provide evidence that treatment of bovine oocytes with CNP before IVM promotes mitochondrial biogenesis in vitro, possibly by activating CREB.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Mitocondrias , Péptido Natriurético Tipo-C , Oocitos , Biogénesis de Organelos , Animales , Bovinos , Péptido Natriurético Tipo-C/farmacología , Péptido Natriurético Tipo-C/metabolismo , Oocitos/metabolismo , Oocitos/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/métodos , Fosforilación/efectos de los fármacosRESUMEN
BACKGROUND: Preeclampsia is a serious condition of pregnancy, complicated by aberrant maternal vascular dysfunction. CNP (C-type natriuretic peptide) contributes to vascular homeostasis, acting through NPR-B (natriuretic peptide receptor-B) and NPR-C (natriuretic peptide receptor-C). CNP mitigates vascular dysfunction of arteries in nonpregnant cohorts; this study investigates whether CNP can dilate maternal arteries in ex vivo preeclampsia models. METHODS: Human omental arteries were dissected from fat biopsies collected during cesarean section. CNP, NPR-B, and NPR-C mRNA expression was assessed in arteries collected from pregnancies complicated by preeclampsia (n=6) and normotensive controls (n=11). Using wire myography, we investigated the effects of CNP on dilation of arteries from normotensive pregnancies. Arteries were preconstricted with either serum from patients with preeclampsia (n=6) or recombinant ET-1 (endothelin-1; vasoconstrictor elevated in preeclampsia; n=6) to model vasoconstriction associated with preeclampsia. Preconstricted arteries were treated with recombinant CNP (0.001-100 µmol/L) or vehicle and vascular relaxation assessed. In further studies, arteries were preincubated with NPR-B (5 µmol/L) and NPR-C (10 µmol/L) antagonists before serum-induced constriction (n=4-5) to explore mechanistic signaling. RESULTS: CNP, NPR-B, and NPR-C mRNAs were not differentially expressed in omental arteries from preeclamptic pregnancies. CNP potently stimulated maternal artery vasorelaxation in our model of preeclampsia (using preeclamptic serum). Its vasodilatory actions were driven through the activation of NPR-B predominantly; antagonism of this receptor alone dampened CNP vasorelaxation. Interestingly, CNP did not reduce ET-1-driven omental artery constriction. CONCLUSIONS: Collectively, these data suggest that enhancing CNP signaling through NPR-B offers a potential therapeutic strategy to reduce systemic vascular constriction in preeclampsia.
Asunto(s)
Péptido Natriurético Tipo-C , Preeclampsia , Receptores del Factor Natriurético Atrial , Vasodilatación , Femenino , Preeclampsia/fisiopatología , Preeclampsia/metabolismo , Preeclampsia/tratamiento farmacológico , Humanos , Embarazo , Péptido Natriurético Tipo-C/farmacología , Adulto , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Epiplón/irrigación sanguínea , Vasoconstricción/efectos de los fármacos , Arterias/efectos de los fármacos , Arterias/metabolismo , Arterias/fisiopatologíaRESUMEN
C-type natriuretic peptide (CNP) plays a crucial role in enhancing endochondral bone growth and holds promise as a therapeutic agent for impaired skeletal growth. To overcome CNP's short half-life, we explored the potential of dampening its clearance system. Neprilysin (NEP) is an endopeptidase responsible for catalyzing the degradation of CNP. Thus, we investigated the effects of NEP inhibition on skeletal growth by administering sacubitril, a NEP inhibitor, to C57BL/6 mice. Remarkably, we observed a dose-dependent skeletal overgrowth phenotype in mice treated with sacubitril. Histological analysis of the growth plate revealed a thickening of the hypertrophic and proliferative zones, mirroring the changes induced by CNP administration. The promotion of skeletal growth observed in wild-type mice treated with sacubitril was nullified by the knockout of cartilage-specific natriuretic peptide receptor B (NPR-B). Notably, sacubitril promoted skeletal growth in mice only at 3 to 4 weeks of age, a period when endogenous CNP and NEP expression was higher in the lumbar vertebrae. Additionally, sacubitril facilitated endochondral bone growth in organ culture experiments using tibial explants from fetal mice. These findings suggest that NEP inhibition significantly promotes skeletal growth via the CNP/NPR-B pathway, warranting further investigations for potential applications in people with short stature.
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Compuestos de Bifenilo , Desarrollo Óseo , Ratones Endogámicos C57BL , Péptido Natriurético Tipo-C , Neprilisina , Animales , Neprilisina/metabolismo , Neprilisina/antagonistas & inhibidores , Neprilisina/genética , Péptido Natriurético Tipo-C/farmacología , Péptido Natriurético Tipo-C/metabolismo , Desarrollo Óseo/efectos de los fármacos , Ratones , Compuestos de Bifenilo/farmacología , Ratones Noqueados , Aminobutiratos/farmacología , Transducción de Señal/efectos de los fármacos , Masculino , Valsartán/farmacología , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/metabolismo , Combinación de Medicamentos , Tetrazoles/farmacologíaRESUMEN
Studies have shown that Sacubitril/valsartan (Sac/Val) can reduce myocardial inflammation in myocarditis mice, in addition to its the recommended treatment of heart failure. However, the underlying mechanisms of Sac/Val in myocarditis remain unclear. C-type natriuretic peptide (CNP), one of the targeting natriuretic peptides of Sac/Val, was recently reported to exert cardio-protective and anti-inflammatory effects in cardiovascular systems. Here, we focused on circulating levels of CNP in patients with acute myocarditis (AMC) and whether Sac/Val modulates inflammation by targeting CNP in experimental autoimmune myocarditis (EAM) mice as well as LPS-induced RAW 264.7 cells and bone marrow derived macrophages (BMDMs) models. Circulating CNP levels were higher in AMC patients compared to healthy controls, and these levels positively correlated with the elevated inflammatory cytokines IL-6 and monocyte count. In EAM mice, Sac/Val alleviated myocardial inflammation while augmenting circulating CNP levels rather than BNP and ANP, accompanied by reduction in intracardial M1 macrophage infiltration and expression of inflammatory cytokines IL-1ß, TNF-α, and IL-6. Furthermore, Sac/Val inhibited CNP degradation and directly blunted M1 macrophage polarization in LPS-induced RAW 264.7 cells and BMDMs. Mechanistically, the effects might be mediated by the NPR-C/cAMP/JNK/c-Jun signaling pathway apart from NPR-B/cGMP/NF-κB pathway. In conclusion, Sac/Val exerts a protective effect in myocarditis by increasing CNP concentration and inhibiting M1 macrophages polarization.
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Aminobutiratos , Compuestos de Bifenilo , Combinación de Medicamentos , Macrófagos , Miocarditis , Péptido Natriurético Tipo-C , Valsartán , Animales , Ratones , Miocarditis/tratamiento farmacológico , Miocarditis/metabolismo , Miocarditis/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Aminobutiratos/farmacología , Valsartán/farmacología , Células RAW 264.7 , Masculino , Humanos , Compuestos de Bifenilo/farmacología , Péptido Natriurético Tipo-C/farmacología , Tetrazoles/farmacología , Enfermedad Aguda , Modelos Animales de Enfermedad , Femenino , Citocinas/metabolismo , Citocinas/sangre , Ratones Endogámicos C57BL , Antiinflamatorios/farmacología , Polaridad Celular/efectos de los fármacosRESUMEN
Background: Fertility plays a great role in animal reproduction since high-quality semen improves sheep industry reproduction. The current worldwide data revealed the close relation of C-type natriuretic peptide (CNP) to the reproductive function of rams. Aims: Evaluation of the effect of CNP on cooled sperms using traditional and molecular assays. Methods: Totally, of 20 testicular samples were collected, processed to obtain the semen samples, and divided into two parts; one was treated with a suitable dose of CNP, and the other served as a control. Sperm samples of both groups were cooled for 3 days and tested at 0, 24, 48, and 72 hours. Results: The findings revealed that the suitable dose of CNP-treated sperms was 0.01 × 10-13. Values of individual motility, live sperms, and sperm concentration were reduced significantly in CNP-24h, CNP-48h, and CNP-72h when compared to control; however, abnormal sperms were increased in both control and CNP groups at 24, 48, and 72 hours when compared to values of 0 hour. Concerning turbidmetric analysis, a significant reduction in values of lag time was observed in CNP when compared to control at all times of cooling intervals. In both CNP and control groups, motility index was decreased at 24, 48, and 72 hours when compared to 0 hour. For velocity, significant increases were shown in CNP compared with control at all cooling intervals. However, values of both groups were increased significantly at 24, 48, and 72 hours times when compared to 0 hour. Fraction of rapidly moving sperm of CNP was elevated at 0 hour and decreased at 24, 48, and 72 hours when compared to control. Expression of the hNPR-B gene was reduced gradually in sperms of CNP and control groups at times of cooling intervals. Conclusion: To the best of our knowledge, this first Iraqi study targets the effect of CNP on epididymal sperms of rams. However, changes that occur after excessive CNP exposure remain unclear, and the toxicological profile of CNP requires furthermore supplements.
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Péptido Natriurético Tipo-C , Semen , Masculino , Ovinos , Animales , Péptido Natriurético Tipo-C/genética , Péptido Natriurético Tipo-C/metabolismo , Péptido Natriurético Tipo-C/farmacología , Irak , Semen/metabolismo , Espermatozoides/metabolismo , Análisis de Semen/veterinaria , Oveja DomésticaRESUMEN
Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.
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Tiburones , Animales , Tiburones/metabolismo , Glándula de Sal/metabolismo , Cloruros/metabolismo , Cloruros/farmacología , Cazón/metabolismo , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/farmacología , Péptido Natriurético Tipo-C/metabolismo , Péptido Natriurético Tipo-C/farmacología , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Sodio/metabolismo , Sodio/farmacología , Potasio/metabolismo , Potasio/farmacologíaRESUMEN
BACKGROUND: ß-AR (ß-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca2+ homeostasis via cAMP-dependent mechanisms; however, enhanced ß-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca2+ homeostasis, and atrial arrhythmogenesis is incompletely understood. METHODS: Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B+/-) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca2+ imaging, and in mouse and human atrial tissues using molecular biology. RESULTS: Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B+/- mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B+/- mice leading to larger increases in atrial cAMP in the presence of the ß-AR agonist isoproterenol. NPR-B+/- mice displayed larger increases in action potential duration and L-type Ca2+ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca2+ release events and delayed afterdepolarizations in NPR-B+/- atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B+/- atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca2+ current through PDE2 in mouse and human atrial myocytes. CONCLUSIONS: NPR-B protects against AF by preventing enhanced atrial responses to ß-adrenergic receptor agonists.
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Fibrilación Atrial , Humanos , Ratones , Animales , Fibrilación Atrial/prevención & control , Fibrilación Atrial/metabolismo , Isoproterenol/farmacología , Péptido Natriurético Tipo-C/farmacología , Atrios Cardíacos , Miocitos Cardíacos/metabolismoRESUMEN
C-natriuretic peptide (CNP) is the central regulator of oocyte meiosis progression, thus coordinating synchronization of oocyte nuclear-cytoplasmic maturation. However, whether CNP can independently regulate cytoplasmic maturation has been long overlooked. Mitochondrial DNA (mtDNA) accumulation is the hallmark event of cytoplasmic maturation, but the mechanism underlying oocyte mtDNA replication remains largely elusive. Herein, we report that CNP can directly stimulate oocyte mtDNA replication at GV stage, and deficiency of follicular CNP may contribute largely to lower mtDNA copy number in in vitro matured oocytes. The mechanistic study showed that cAMP-PKA-CREB1 signaling cascade underlies the regulatory role of CNP in stimulating mtDNA replication and upregulating related genes. Of interest, we also report that CNP-NPR2 signaling is inhibited in aging follicles, and this inhibition is implicated in lower mtDNA copy number in oocytes from aging females. Together, our study provides the first direct functional link between follicular CNP and oocyte mtDNA replication, and identifies its involvement in aging-associated mtDNA loss in oocytes. These findings, not only update the current knowledge of the functions of CNP in coordinating oocyte maturation but also present a promising strategy for improving in vitro fertilization outcomes of aging females.
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ADN Mitocondrial , Técnicas de Maduración In Vitro de los Oocitos , Femenino , Humanos , ADN Mitocondrial/genética , Péptido Natriurético Tipo-C/genética , Péptido Natriurético Tipo-C/farmacología , Oocitos/fisiología , Meiosis , Péptidos Natriuréticos/genética , VasodilatadoresRESUMEN
In vitro embryo production depends on high-quality oocytes. Compared with in vivo matured oocytes, in vitro oocytes undergo precocious meiotic resumption, thus compromising oocyte quality. C-type natriuretic peptide (CNP) is a follicular factor maintaining meiotic arrest. Thus, CNP-pretreatment has been widely used to improve the in vitro maturation (IVM) of oocytes in many species. However, the efficacy of this strategy has remained unsatisfactory in porcine oocytes. Here, by determining the functional concentration and dynamics of CNP in inhibiting spontaneous meiotic resumption, we improved the current IVM system of porcine oocytes. Our results indicate that although the beneficial effect of the CNP pre-IVM strategy is common among species, the detailed method may be largely divergent among them and needs to be redesigned specifically for each one. Focusing on the overlooked role of cumulus cells surrounding the oocytes, we also explore the mechanisms relevant to their beneficial effect. In addition to oocytes per se, the enhanced anti-apoptotic and anti-oxidative gene expression in cumulus cells may contribute considerably to improved oocyte quality. These findings not only emphasize the importance of screening the technical parameters of the CNP pre-IVM strategy for specific species, but also highlight the critical supporting role of cumulus cells in this promising strategy.
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Técnicas de Maduración In Vitro de los Oocitos , Péptido Natriurético Tipo-C , Animales , Porcinos , Péptido Natriurético Tipo-C/farmacología , Péptido Natriurético Tipo-C/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/métodos , Meiosis , Oocitos/metabolismo , Estrés Oxidativo , ApoptosisRESUMEN
The overall oocyte quality declines with aging, and this effect is strongly associated with a higher reactive oxygen species (ROS) level and the resultant oxidative damage. C-type natriuretic peptide (CNP) is a well-characterized physiological meiotic inhibitor that has been successfully used to improve immature oocyte quality during in vitro maturation. However, the underlying roles of CNP in maternally aged oocytes have not been reported. Here, we found that the age-related reduction in the serum CNP concentration was highly correlated with decreased oocyte quality. Treatment with exogenous CNP promoted follicle growth and ovulation in aged mice and enhanced meiotic competency and fertilization ability. Interestingly, the cytoplasmic maturation of aged oocytes was thoroughly improved by CNP treatment, as assessed by spindle/chromosome morphology and redistribution of organelles (mitochondria, the endoplasmic reticulum, cortical granules, and the Golgi apparatus). CNP treatment also ameliorated DNA damage and apoptosis caused by ROS accumulation in aged oocytes. Importantly, oocyte RNA-seq revealed that the beneficial effect of CNP on aged oocytes was mediated by restoration of mitochondrial oxidative phosphorylation, eliminating excessive mitophagy. CNP reversed the defective phenotypes in aged oocytes by alleviating oxidative damage and suppressing excessive PINK1/Parkin-mediated mitophagy. Mechanistically, CNP functioned as a cAMP/PKA pathway modulator to decrease PINK1 stability and inhibit Parkin recruitment. In summary, our results demonstrated that CNP supplementation constitutes an alternative therapeutic approach for advanced maternal age-related oocyte deterioration and may improve the overall success rates of clinically assisted reproduction in older women.
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Técnicas de Maduración In Vitro de los Oocitos , Péptido Natriurético Tipo-C , Animales , Femenino , Ratones , Células del Cúmulo/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/métodos , Meiosis , Mitofagia , Péptido Natriurético Tipo-C/farmacología , Péptido Natriurético Tipo-C/genética , Péptido Natriurético Tipo-C/metabolismo , Oocitos/metabolismo , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10-6, 10-7 or 10-8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10-8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10-7 or 10-8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10-6 or 10-8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10-8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10-8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.
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Melatonina , Animales , Bovinos , Femenino , Melatonina/farmacología , Melatonina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Péptido Natriurético Tipo-C/farmacología , Colforsina/farmacología , Colforsina/metabolismo , Oocitos/fisiología , AMP Cíclico/metabolismo , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Células del CúmuloRESUMEN
The role of C-type natriuretic peptide (CNP) in the regulation of cardiac function in humans remains to be established as previous investigations have been confined to animal model systems. Here, we used well-characterized engineered cardiac tissues (ECTs) generated from human stem cell-derived cardiomyocytes and fibroblasts to study the acute effects of CNP on contractility. Application of CNP elicited a positive inotropic response as evidenced by increases in maximum twitch amplitude, maximum contraction slope and maximum calcium amplitude. This inotropic response was accompanied by a positive lusitropic response as demonstrated by reductions in time from peak contraction to 90% of relaxation and time from peak calcium transient to 90% of decay that paralleled increases in maximum contraction decay slope and maximum calcium decay slope. To establish translatability, CNP-induced changes in contractility were also assessed in rat ex vivo (isolated heart) and in vivo models. Here, the effects on force kinetics observed in ECTs mirrored those observed in both the ex vivo and in vivo model systems, whereas the increase in maximal force generation with CNP application was only detected in ECTs. In conclusion, CNP induces a positive inotropic and lusitropic response in ECTs, thus supporting an important role for CNP in the regulation of human cardiac function. The high degree of translatability between ECTs, ex vivo and in vivo models further supports a regulatory role for CNP and expands the current understanding of the translational value of human ECTs. NEW FINDINGS: What is the central question of this study? What are the acute responses to C-type natriuretic peptide (CNP) in human-engineered cardiac tissues (ECTs) on cardiac function and how well do they translate to matched concentrations in animal ex vivo and in vivo models? What is the main finding and its importance? Acute stimulation of ECTs with CNP induced positive lusitropic and inotropic effects on cardiac contractility, which closely reflected the changes observed in rat ex vivo and in vivo cardiac models. These findings support an important role for CNP in the regulation of human cardiac function and highlight the translational value of ECTs.
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Péptido Natriurético Tipo-C , Animales , Humanos , Ratas , Calcio , Contracción Miocárdica/fisiología , Miocitos Cardíacos , Péptido Natriurético Tipo-C/farmacologíaRESUMEN
In a healthy heart, cells naturally secrete C-type natriuretic peptide (CNP), a cytokine that protects against myofibroblast differentiation of cardiac fibroblasts and extracellular matrix deposition leading to fibrosis. CNP availability during myocardial remodeling is important to prevent cardiac fibrosis, but CNP is limited after an injury because of the loss of cardiomyocytes and the activation of cardiac fibroblasts to myofibroblasts. We hypothesized that the sustained release of exogenous CNP from oligo-urethane nanoparticles (NPs) would reduce differentiation of human cardiac fibroblasts toward a myofibrogenic phenotype. Our work used a modified form of a degradable polar hydrophobic ionic (D-PHI) oligo-urethane, which has shown the ability to self-assemble into NPs for the delivery of peptide and oligonucleotide biomolecules. The CNP-loaded NPs (NPCNP) were characterized for a diameter of 129 ± 1.4 nm and a ζ potential of -46 ± 7.8 mV. Treatment of cardiac fibroblasts with NPCNP increased cyclic guanosine-monophosphate (cGMP) synthesis, confirming that exogenous CNP delivered via oligo-urethane NPs is bioactive and can induce downstream signaling that has been implicated in antagonizing transforming growth factor-ß1 (TGF-ß1)-induced myofibrogenic differentiation. It is also shown that treatment with NPCNP attenuated contraction of collagen gels by cardiac myofibroblasts stimulated with TGF-ß1. Coating with heparin on the NPCNP (HEP-NPCNP) exemplified an approach to extend the release of CNP from the NPs. Both HEP-NPCNP and NPCNP show minimal cell toxicity, studied up to 0.25 × 1010 NPs/mL in culture media. These findings support further investigation of CNP delivery via NPs as a future therapy for suppressing cardiac fibrosis.
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Miofibroblastos , Factor de Crecimiento Transformador beta1 , Humanos , Péptido Natriurético Tipo-C/farmacología , Uretano , FibrosisRESUMEN
Excessive activation of the sympathetic nervous system is involved in cardiovascular damage including cardiac hypertrophy. Natriuretic peptides are assumed to exert protective actions for the heart, alleviating hypertrophy and/or fibrosis of the myocardium. In contrast to this assumption, we show in the present study that both atrial and C-type natriuretic peptides (ANP and CNP) potentiate cardiac hypertrophic response to noradrenaline (NA) in rats. Nine-week-old male Wistar rats were continuously infused with subcutaneous 30 micro-g/h NA without or with persistent intravenous administration of either 1.0 micro-g/h ANP or CNP for 14 days. Blood pressure (BP) was recorded under an unrestrained condition by a radiotelemetry system. Cardiac hypertrophic response to NA was evaluated by heart weight/body weight (HW/BW) ratio and microscopic measurement of myocyte size of the left ventricle. Mean BP levels at the light and dark cycles rose by about 20 mmHg following NA infusion for 14 days, with slight increases in HW/BW ratio and ventricular myocyte size. Infusions of ANP and CNP had no significant effects on mean BP in NA-infused rats, while two natriuretic peptides potentiated cardiac hypertrophic response to NA. Cardiac hypertrophy induced by co-administration of NA and ANP was attenuated by treatment with prazosin or atenolol. In summary, both ANP and CNP potentiated cardiac hypertrophic effect of continuously infused NA in rats, suggesting a possible pro-hypertrophic action of natriuretic peptides on the heart.
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Factor Natriurético Atrial , Norepinefrina , Ratas , Animales , Masculino , Ratas Wistar , Norepinefrina/farmacología , Cardiomegalia/inducido químicamente , Cardiomegalia/tratamiento farmacológico , Presión Sanguínea , Péptido Natriurético Tipo-C/farmacología , Péptido Natriurético Encefálico/farmacologíaRESUMEN
Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-ß. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.
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Péptido Natriurético Tipo-C , Remodelación Ventricular , Ratones , Animales , Masculino , Femenino , Humanos , Péptido Natriurético Tipo-C/genética , Péptido Natriurético Tipo-C/farmacología , Vasodilatadores/farmacología , Fibrosis , Angiotensina II/farmacología , Fibroblastos/metabolismoRESUMEN
STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-ß family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.
Asunto(s)
Factor de Crecimiento Epidérmico , Proteómica , Animales , Niño , Humanos , Femenino , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Estudios Prospectivos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/metabolismo , Péptido Natriurético Tipo-C/farmacología , Hormona Folículo Estimulante/metabolismo , Inhibinas/metabolismo , Activinas/metabolismo , MamíferosRESUMEN
BACKGROUND: Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways. METHODS: To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes. RESULTS: In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in ICaL, INaL, and Ca2+ spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca2+ handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion. CONCLUSIONS: CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies.
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Miocitos Cardíacos , Hidrolasas Diéster Fosfóricas , Ratones , Animales , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal , Catecolaminas/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/etiología , Arritmias Cardíacas/prevención & control , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Antiarrítmicos/metabolismo , GMP Cíclico/metabolismo , Péptido Natriurético Tipo-C/farmacologíaRESUMEN
Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities.
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Mucopolisacaridosis VII , Péptido Natriurético Tipo-C , Humanos , Ratones , Animales , Péptido Natriurético Tipo-C/farmacología , Constricción Patológica/complicaciones , Mucopolisacaridosis VII/complicaciones , Mucopolisacaridosis VII/tratamiento farmacológico , Osteogénesis , Ratones TransgénicosRESUMEN
This study is to analyze the effect of C-type natriuretic peptide (CNP) on sperm motility of asthenozoospermia and explore the influence mechanism of CNP on the reproductive system and sperm motility. Our results showed that the concentration of CNP in asthenospermia patients' semen was lower than in normal people's. The motility of sperm could be improved markedly by CNP and 8-Br-cGMP, while the effect of CNP was inhibited by NPR-B antagonist and KT5823. In the asthenozoospermia mouse model induced by CTX, CNP injection could improve sperm motility in the epididymis, alleviate tissue damage in the testes and epididymis, and increase testosterone levels. The asthenospermia mouse model showed high activity of MDA and proinflammatory factors (TNF-α, IL-6), as well as low expression of antioxidants (SOD, GSH-Px, CAT) in the testis and epididymis, but this situation could be significantly ameliorated after being treated with CNP. Those studies indicated that the concentration of CNP in the semen of asthenospermia patients is lower than in normal people and could significantly promote sperm motility through the NPR-B/cGMP pathway. In the asthenospermia mouse model induced by CTX, CNP can alleviate the damage of cyclophosphamide to the reproductive system and sperm motility. The mechanism may involve increasing testosterone and reducing ROS and proinflammatory factors to damage the tissue and sperm.
Asunto(s)
Astenozoospermia , Animales , Antioxidantes/farmacología , Astenozoospermia/metabolismo , Ciclofosfamida/farmacología , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Péptido Natriurético Tipo-C/metabolismo , Péptido Natriurético Tipo-C/farmacología , Especies Reactivas de Oxígeno/metabolismo , Semen/metabolismo , Motilidad Espermática , Espermatozoides/metabolismo , Superóxido Dismutasa/metabolismo , Testosterona/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
As a result of thrombosis or intimal hyperplasia, synthetic artificial vascular grafts had a low success rate when they were used to replace small-diameter arteries (inner diameter < 6 mm). C-type natriuretic peptides (CNP) have anti-thrombotic effects, and can promote endothelial cell (EC) proliferation and inhibit vascular smooth muscle cell (SMC) over-growth. In this study, poly(ε-caprolactone) (PCL) vascular grafts loaded with CNP (PCL-CNP) were constructed by electrospinning. The PCL-CNP grafts were able to continuously release CNP at least 25 days in vitro. The results of scanning electron microscopy (SEM) and mechanical testing showed that the loading of CNP did not change the microstructure and mechanical properties of the PCL grafts. In vitro blood compatibility analysis displayed that PCL-CNP grafts could inhibit thrombin activity and reduce platelet adhesion and activation. In vitro cell experiments demonstrated that PCL-CNP grafts activated ERK1/2 and Akt signaling in human umbilical vein endothelial cells (HUVECs), as well as increased cyclin D1 expression, enhanced proliferation and migration, and increased vascular endothelial growth factor (VEGF) secretion and nitric oxide (NO) production. The rabbit arteriovenous (AV)-shunt ex vitro indicated that CNP loading significantly improved the antithrombogenicity of PCL grafts. The assessment of vascular grafts in rat abdominal aorta implantation model displayed that PCL-CNP grafts promoted the regeneration of ECs and contractile SMCs, modulated macrophage polarization toward M2 phenotype, and enhanced extracellular matrix remodeling. These findings confirmed for the first time that loading CNP is an effective approach to improve the hemocompatibility and vascular regeneration of synthetic vascular grafts. STATEMENT OF SIGNIFICANCE: Small-diameter (< 6 mm) vascular grafts (SDVGs) have not been made clinically available due to their prevalence of thrombosis, limited endothelial regeneration and intimal hyperplasia. The incorporation of bioactive molecules into SDVGs serves as an effective solution to improve hemocompatibility and endothelialization. In this study, for the first time, we loaded C-type natriuretic peptides (CNP) into PCL grafts by electrospunning and confirmed the effectiveness of loading CNP on improving the hemocompatibility and vascular regeneration of artificial vascular grafts. Regenerative advantages included enhancement of endothelialization, modulation of macrophage polarization toward M2 phenotypes, and improved contractile smooth muscle cell regeneration. Our investigation brings attention to CNP as a valuable bioactive molecule for modifying cardiovascular biomaterial.