Efficiency of C-type natriuretic peptide on improvement of Iraqi local ram's epididymal sperms.

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.

The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

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.

"Meiosis arrester" C-natriuretic peptide directly stimulates oocyte mtDNA accumulation and is implicated in aging-associated oocyte mtDNA loss.

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.

Natriuretic Peptide Receptor B Protects Against Atrial Fibrillation by Controlling Atrial cAMP Via Phosphodiesterase 2.

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.

A Redesigned Method for CNP-Synchronized In Vitro Maturation Inhibits Oxidative Stress and Apoptosis in Cumulus-Oocyte Complexes and Improves the Developmental Potential of Porcine Oocytes.

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.

C-type natriuretic peptide improves maternally aged oocytes quality by inhibiting excessive PINK1/Parkin-mediated mitophagy.

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.

Effects of cyclic adenosine monophosphate modulating agents during oocyte pre-maturation and the role of melatonin on in vitro maturation of bovine cumulus-oocyte complexes.

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.

Bioinspired Oligo-Urethane Nanoparticles for Delivering Exogenous C-Type Natriuretic Peptide: Synthetic Biomaterial Nanocarrier Complexes and Their Interactions with Cardiac Myofibroblasts.

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.

Natriuretic peptides potentiate cardiac hypertrophic response to noradrenaline in rats.

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.

Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice.

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.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

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.

CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2.

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.

Circulatory C-type natriuretic peptide reduces mucopolysaccharidosis-associated craniofacial hypoplasia in vivo.

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.

C-Type Natriuretic Peptide (CNP) Could Improve Sperm Motility and Reproductive Function of Asthenozoospermia.

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.

The loading of C-type natriuretic peptides improved hemocompatibility and vascular regeneration of electrospun poly(ε-caprolactone) grafts.

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.

A Series of Substituted Bis-Aminotriazines Are Activators of the Natriuretic Peptide Receptor C.

C-type natriuretic peptide (CNP) is involved in the regulation of vascular homeostasis, which is at least partly mediated through agonism of natriuretic peptide receptor C (NPR-C), and loss of this signaling has been associated with vascular dysfunction. As such, NPR-C is a novel therapeutic target to treat cardiovascular diseases. A series of novel small molecules have been designed and synthesized, and their structure-activity relationships were evaluated by a surface plasmon resonance binding assay. The biological activity of hit compounds was confirmed through organ bath assays measuring vascular relaxation and inhibition of cAMP production, which was shown to be linked to its NPR-C activity. Lead compound 1 was identified as a potent agonist (EC50 ∼ 1 µM) with promising in vivo pharmacokinetic properties.

C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth.

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide evidence from experiments using mutant mice, indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

C-type Natriuretic Peptide-induced PKA Activation Promotes Endochondral Bone Formation in Hypertrophic Chondrocytes.

Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.

Assessment of cGMP level in medium during in vitro growth period of murine preantral follicles with and without supplementation of C-type natriuretic peptide.

To enhance the developmental competency of murine ovarian follicles cultured in vitro, C-type natriuretic peptide (CNP) was supplemented in the culture system. Although the mechanism is not fully elucidated, it was reported that the effect of CNP supplementation was mediated by increased cyclic guanosine monophosphate (cGMP). In the present study, cGMP levels in media for murine preantral follicle culture were compared both between a control group without CNP supplementation and an experimental group with CNP supplementation and between days in each group. In addition, follicle growth patterns and oocyte maturity were assessed and compared between the two groups. Results demonstrated that along with in vitro culture, cGMP levels increased (P < 0.05) both in the control group and the experimental group, whereas cGMP levels were not significantly different between the two groups on the same day of in vitro culture (P > 0.05). The oocyte's maturity was superior in the experimental group compared with the control group (P < 0.05). As ovarian follicles grew three-dimensionally in the experimental group but were flattened in the control group, CNP might improve oocyte maturity through maintaining the three-dimensional architecture of the ovarian follicle because of increased transzonal projections (TZP) and functional gap junctions between oocyte and surrounding granulosa cells.

Natriuretic peptide receptor B maintains heart rate and sinoatrial node function via cyclic GMP-mediated signalling.

AIMS: Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP), have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signalling in regulating HR and SAN function. METHODS AND RESULTS: We have used NPR-B deficient mice (NPR-B+/-) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anaesthetized mice, high-resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/- mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/- mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signalling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/- mice. NPR-B+/- mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function. CONCLUSIONS: NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signalling mechanism.