Paracrine and endocrine pathways of natriuretic peptides assessed by ligand-receptor mapping in the Japanese eel brain.

The natriuretic peptide (NP) family consists of cardiac NPs (ANP, BNP, and VNP) and brain NPs (CNPs) in teleosts. In addition to CNP1-4, a paralogue of CNP4 (named CNP4b) was recently discovered in basal teleosts including Japanese eel. Mammals have lost most Cnps during the evolution, but teleost cnps were conserved and diversified, suggesting that CNPs are important hormones for maintaining brain functions in teleost. The present study evaluated the potency of each Japanese eel CNP to their NP receptors (NPR-A, NPR-B, NPR-C, and NPR-D) overexpressed in CHO cells. A comprehensive brain map of cnps- and nprs-expressing neurons in Japanese eel was constructed by integrating the localization results obtained by in situ hybridization. The result showed that CHO cells expressing NPR-A and NPR-B induced strong cGMP productions after stimulation by cardiac and brain NPs, respectively. Regarding brain distribution of cnps, cnp1 is engaged in the ventral telencephalic area and periventricular area including the parvocellular preoptic nucleus (Pp), anterior/posterior tuberal nuclei, and periventricular gray zone of the optic tectum. cnp3 is found in the habenular nucleus and prolactin cells in the pituitary. cnp4 is expressed in the ventral telencephalic area, while cnp4b is expressed in the motoneurons in the medullary area. Such CNP isoform-specific localizations suggest that function of each CNP has diverged in the eel brain. Furthermore, the Pp lacking the blood-brain barrier expressed both npra and nprb, suggesting that endocrine and paracrine NPs interplay for regulating the Pp functions in Japanese eels.

Promising horizons in achondroplasia along with the development of new drugs.

Achondroplasia (ACH) is a representative skeletal disorder characterized by rhizomelic shortened limbs and short stature. ACH is classified as belonging to the fibroblast growth factor receptor 3 (FGFR3) group. The downstream signal transduction of FGFR3 consists of STAT1 and RAS/RAF/MEK/ERK pathways. The mutant FGFR3 found in ACH is continuously phosphorylated and activates downstream signals, resulting in abnormal proliferation and differentiation of chondrocytes in the growth plate and cranial base synchondrosis. A patient registry has been developed and has contributed to revealing the natural history of ACH patients. Concerning the short stature, the adult height of ACH patients ranges between 126.7-135.2 cm for men and 119.9-125.5 cm for women in many countries. Along with severe short stature, foramen magnum stenosis and spinal canal stenosis are major complications: the former leads to sleep apnea, breathing disorders, myelopathy, hydrocephalus, and sudden death, and the latter causes pain in the extremities, numbness, muscle weakness, movement disorders, intermittent claudication, and bladder-rectal disorders. Growth hormone treatment is available for ACH only in Japan. However, the effect of the treatment on adult height is not satisfactory. Recently, the neutral endopeptidase-resistant CNP analogue vosoritide has been approved as a new drug for ACH. Additionally in development are a tyrosine kinase inhibitor, a soluble FGFR3, an antibody against FGFR3, meclizine, and the FGF2-aptamer. New drugs will bring a brighter future for patients with ACH.

Auto/Paracrine C-Type Natriuretic Peptide/Cyclic GMP Signaling Prevents Endothelial Dysfunction.

Endothelial dysfunction is cause and consequence of cardiovascular diseases. The endothelial hormone C-type natriuretic peptide (CNP) regulates vascular tone and the vascular barrier. Its cGMP-synthesizing guanylyl cyclase-B (GC-B) receptor is expressed in endothelial cells themselves. To characterize the role of endothelial CNP/cGMP signaling, we studied mice with endothelial-selective GC-B deletion. Endothelial EC GC-B KO mice had thicker, stiffer aortae and isolated systolic hypertension. This was associated with increased proinflammatory E-selectin and VCAM-1 expression and impaired nitric oxide bioavailability. Atherosclerosis susceptibility was evaluated in such KO and control littermates on Ldlr (low-density lipoprotein receptor)-deficient background fed a Western diet for 10 weeks. Notably, the plaque areas and heights within the aortic roots were markedly increased in the double EC GC-B/Ldlr KO mice. This was accompanied by enhanced macrophage infiltration and greater necrotic cores, indicating unstable plaques. Finally, we found that EC GC-B KO mice had diminished vascular regeneration after critical hind-limb ischemia. Remarkably, all these genotype-dependent changes were only observed in female and not in male mice. Auto/paracrine endothelial CNP/GC-B/cGMP signaling protects from arterial stiffness, systolic hypertension, and atherosclerosis and improves reparative angiogenesis. Interestingly, our data indicate a sex disparity in the connection of diminished CNP/GC-B activity to endothelial dysfunction.

Neprilysin Inhibition Promotes Skeletal Growth via the CNP/NPR-B Pathway.

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.

Vosoritide treatment for children with hypochondroplasia: a phase 2 trial.

Background: Hypochondroplasia is a rare autosomal dominant skeletal dysplasia due to activating variants in FGFR3. It presents with disproportionate short stature with a wide range of clinical severity. There are currently no approved medications to treat short stature in children with hypochondroplasia. Vosoritide is a C-type natriuretic peptide analog that was recently approved for improving growth in children with achondroplasia. We aimed to evaluate the safety and efficacy of vosoritide in children with hypochondroplasia. Methods: We conducted a single-arm, phase 2, open-label trial at a single centre in the USA and enrolled 26 children with hypochondroplasia. The trial consists of a 6-month observation period to establish a baseline annualized growth velocity followed by a 12-month intervention period during which vosoritide is administered daily via subcutaneous injection at a dose of 15 µg/kg/day. The trial's co-primary endpoints included the incidence of adverse events and the change from baseline in age-sex standardized annualized growth velocity and height standardized deviation score (SDS) after 12 months of treatment. This trial is registered with ClinicalTrials.gov (NCT04219007). Findings: Twenty-four participants with a mean age of 5.86 years received vosoritide therapy. The first participant was enrolled on August 4, 2020, and the final participant completed the 18-month trial on September 8, 2023. Vosoritide was well tolerated with no treatment-related serious adverse events. Injection site reactions occurred in 83.3% of participants. No participants discontinued therapy due to an adverse event. Annualized growth velocity increased by 2.26 standard deviations (SD) and height SDS increased by 0.36 SD during the treatment period versus the observation period. Hypochondroplasia specific height SDS increased by 0.38 SD. There was a 1.81 cm/year increase in absolute annualized growth velocity. Interpretation: Vosoritide was safe and effective in increasing growth velocity in children with hypochondroplasia. Efficacy was similar to what has been reported in children with achondroplasia. Funding: This study was supported by an investigator-initiated grant from BioMarin Pharmaceutical.

Sacubitril/valsartan attenuates atrial conduction disturbance and electrophysiological heterogeneity with ameliorating fibrosis in mice.

Background: Sacubitril/valsartan (SacVal) has been shown to improve the prognosis of heart failure; however, whether SacVal reduces the occurrence of atrial fibrillation (AF) in heart failure has not yet been elucidated. In this study, we aimed to determine whether SacVal is effective in reducing the occurrence of AF in heart failure and identify the underlying mechanism of its electrophysiological effect in mice. Methods: Adult male mice underwent transverse aortic constriction, followed by SacVal, valsartan, or vehicle treatment for two weeks. Electrophysiological study (EPS) and optical mapping were performed to assess the susceptibility to AF and the atrial conduction properties, and fibrosis was investigated using heart tissue and isolated cardiac fibroblasts (CFs). Results: EPS analysis revealed that AF was significantly less inducible in SacVal-treated mice than in vehicle-treated mice. Optical mapping of the atrium showed that SacVal-treated and valsartan-treated mice restored the prolonged action potential duration (APD); however, only SacVal-treated mice showed the restoration of decreased conduction velocity (CV) compared to vehicle-treated mice. In addition, the electrophysiological distribution analysis demonstrated that heterogeneous electrophysiological properties were rate-dependent and increased heterogeneity was closely related to the susceptibility to AF. SacVal attenuated the increased heterogeneity of CV at short pacing cycle length in atria, whereas Val could not. Histological and molecular evaluation showed that SacVal exerted the anti-fibrotic effect on the atria. An in vitro study of CFs treated with natriuretic peptides and LBQ657, the metabolite and active form of sacubitril, revealed that C-type natriuretic peptide (CNP) combined with LBQ657 had an additional anti-fibrotic effect on CFs. Conclusions: Our results demonstrated that SacVal can improve the conduction disturbance and heterogeneity through the attenuation of fibrosis in murine atria and reduce the susceptibility of AF in heart failure with pressure overload, which might be attributed to the enhanced function of CNP.

Two Cases of Cardiovascular Adverse Events Following Subcutaneous Vosoritide Injection in Early Infancy.

Achondroplasia, characterized by short stature and skeletal abnormalities, is caused by a gain-of-function variant in the fibroblast growth factor receptor 3 gene. Vosoritide, a C-type natriuretic peptide analog, is an emerging treatment for achondroplasia that functions by promoting endochondral ossification. Vosoritide was approved for the treatment of achondroplasia in Europe and the United States in 2021, and in Japan, the following year. However, vosoritide is associated with a risk of hypotension and vomiting after subcutaneous injection due to its vasodilating effect. Herein, we present two cases of cardiovascular adverse events in infants following vosoritide injection. Case 1 involved a one-month-old female infant with achondroplasia who received the first subcutaneous injection of vosoritide 30 minutes after her last formula intake. Following injection, she developed transient symptomatic hypotension accompanied by vomiting. Although established guidelines recommend that injections be administered after approximately 30 minutes (Europe/Japan) or within one hour (USA) following the last feeding, an extended interval of 1.5 to two hours was required to prevent hypotension-associated vomiting. Case 2 involved a three-month-old female infant with achondroplasia. The first subcutaneous vosoritide injection was administered four hours after the last formula intake, and she subsequently developed prolonged compensated shock with marked tachycardia requiring intervention, including repetitive bolus saline injection. These cases indicate the need to monitor patients for cardiovascular adverse events following subcutaneous injection of vosoritide in early infancy.

Vascular endothelial cell-specific overexpression of CNP did not improve liver fibrosis in HFFCD-induced NASH, but did improve renal lesions.

Mice with endothelial-cell-specific overexpression of C-type natriuretic peptide (E-CNP Tg mice) were shown to be protected against hepatic fibrosis and inflammation induced by high fat diet (HFD) feeding, with improved insulin sensitivity and attenuated weight gain. A recently developed high-fat, high-fructose, high-cholesterol diet (HFFCD) is considered to be a superior model to HFD, owing to the resemblance to human non-alcoholic steatohepatitis (NASH). In this study, we therefore aimed to reveal whether these previous findings with E-CNP Tg mice on HFD can be observed in a newly developed NASH model.　Patients with NASH have been suggested to be at higher risk of developing chronic kidney disease, so we also assessed the kidney histology of these mice. After 8 months of HFFCD feeding, the livers of E-CNP Tg mice and controls showed progressive fibrosis, which resembled the features of human NASH. However, no significant differences were observed in NAFLD activity scores between E-CNP Tg mice and controls, although there was a tendency for improvement in E-CNP Tg mice. The reduced levels of GCB, a receptor for CNP, may have weakened the action of CNP in the current model. In the kidneys, HFFCD showed glomerular hypertrophy and tubular atrophy in the cortical region, which were suppressed in E-CNP Tg mice. The present study did not prove the therapeutic effect of CNP on NASH in the HFFCD model, but provided evidence of its potential beneficial effects on NASH-associated renal damage.

Molecular therapy and nucleic acid adeno-associated virus-based gene therapy delivering combinations of two growth-associated genes to MPS IVA mice.

Mucopolysaccharidosis type IVA (MPS IVA) is caused by a deficiency of the galactosamine (N-acetyl)-6-sulfatase (GALNS) enzyme responsible for the degradation of specific glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, hypoplasia, tracheal obstruction) and several symptoms in other organs. To date, no treatment is effective for patients with bone abnormalities. To improve bone pathology, we propose a novel combination treatment with the adeno-associated virus (AAV) vectors expressing GALNS enzyme and a natriuretic peptide C (CNP; NPPC gene) as a growth-promoting agent for MPS IVA. In this study, an MPS IVA mouse model was treated with an AAV vector expressing GALNS combined with another AAV vector expressing NPPC gene, followed for 12 weeks. After the combination therapy, bone growth in mice was induced with increased enzyme activity in tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP-treated mice with increased CNP activity in plasma. Delivering combinations of CNP and GALNS gene therapies enhanced bone growth in MPS IVA mice more than in GALNS gene therapy alone. Enzyme expression therapy alone fails to reach the bone growth region; our results indicate that combining it with CNP offers a potential alternative.

C-type natriuretic peptide (CNP): The cardiovascular system and beyond.

C-type natriuretic peptide (CNP) represents the 'local' member of the natriuretic peptide family, functioning in an autocrine or paracrine capacity to modulate a hugely diverse portfolio of physiological processes. Whilst the best-characterised of these regulatory roles are in the cardiovascular system, akin to its predominantly endocrine siblings atrial (ANP) and brain (BNP) natriuretic peptides, CNP governs many additional, unrelated mechanisms including bone growth, gamete maturation, auditory processing, and neuronal integrity. Furthermore, there is currently great interest in mimicking the biological activity of CNP for therapeutic gain in many of these disparate organ systems. Herein, we provide an overview of the physiology, pathophysiology and pharmacology of CNP in both cardiovascular and non-cardiovascular settings.

C-Type Natriuretic Peptide Pre-Treatment Improves Maturation Rate of Goat Oocytes by Maintaining Transzonal Projections, Spindle Morphology, and Mitochondrial Function.

C-type natriuretic peptide (CNP) is a peptide molecule naturally found in follicles and can be used to extend meiotic resumption and enhance the potential for oocytes to develop. However, the mechanism by which CNP improves goat oocyte quality remains unclear. In this study, cumulus-oocyte complexes (COCs) from goats were pre-treated with CNP prior to IVM, and the results showed that pre-treatment with CNP enhanced goat oocyte maturation. First, we discovered that CNP maintained communication between cumulus cells and oocytes by regulating the transzonal projections (TZPs). We then found that CNP treatment reduced abnormal spindle formation and increased the expression of genes associated with spindle assembly and the spindle assembly checkpoint. Moreover, further analysis showed that oocytes exhibited better antioxidant ability in the CNP treatment group, which mainly manifested in higher glutathione (GSH) and lower reactive oxygen species (ROS) concentrations. Enhanced mitochondrial activity was signified via the augmented expression of mitochondrial oxidative metabolism and fusion and fission-related genes, thus diminishing the apoptosis of the oocytes. Overall, these results provide novel insights into the potential mechanism by which CNP treatment before IVM can improve oocyte quality.

Respuesta cardiovascular a la administración crónica de péptido natriurético tipo C en ratas espontáneamente hipertensas / Cardiovascular Response to Chronic Administration of C-type Natriuretic Peptide in Spontaneously Hypertensive Rats

Introducción: El péptido natriurético tipo C (CNP) ha cobrado relevancia por sus efectos sobre la regulación de la función y la morfología del corazón y los vasos sanguíneos. Previamente demostramos in vitro que el CNP incrementa la actividad del sistema del óxido nítrico (NO) en ratas espontáneamente hipertensas (SHR). Objetivo: Estudiar el efecto del tratamiento crónico con CNP sobre la presión arterial sistólica (PAS), la función cardíaca y vascular y el sistema del NO en ratas espontáneamente hipertensas y normotensas. Material y métodos: Se emplearon ratas Wistar macho de 12 semanas de edad normotensas y espontáneamente hipertensas. Los animales recibieron infusión crónica de solución salina o CNP (0,75 mg/hora/rata) durante 14 días mediante la implantación de bombas osmóticas subcutáneas. Se midió la PAS y se realizaron un electrocardiograma y un ecocardiograma. Se extrajeron el ventrículo izquierdo y la arteria aorta torácica y se determinó la actividad, con L-[U14C]-arginina, de la óxido nítrico sintasa (NOS) y se realizaron estudios de reactividad vascular. Resultados: La administración crónica de CNP disminuyó la PAS en las SHR. Se observó menor volumen minuto en las SHR y el CNP incrementó dicho volumen, en tanto que no indujo cambios en las ratas normotensas. En las SHR se observó un desequilibrio en las respuestas vasodilatadora y vasoconstrictora en la arteria aorta y el tratamiento con CNP mejoró la función vascular respecto de las ratas normotensas. En ambos tejidos, la actividad de la NOS fue mayor en las SHR y se incrementó con la infusión durante 14 días de CNP. Sin embargo, dicho incremento fue menor en las SHR. Conclusión: El CNP induce cambios a nivel cardiovascular y en el sistema del NO que podrían resultar beneficiosos en este modelo de hipertensión arterial.

Background: C-type natriuretic peptide (CNP) plays an important role in the regulation of cardiovascular function and morphology. We have previously demonstrated that CNP increases nitric oxide (NO) system activity in vivo in spontaneously hypertensive rats (SHR). Objective: The goal of this study is to evaluate the effect of chronic CNP administration on systolic blood pressure (SBP), cardiovascular function and the NO system in spontaneously hypertensive and normotensive rats. Methods: Twelve-week-old normotensive male Wistar rats and SHR were used. They received chronic infusion of saline or CNP (0.75 mg/h/rat) for 14 days via subcutaneously implanted osmotic pumps. Systolic blood pressure was measured and an electrocardiogram and echocardiogram were performed. The left ventricle and the thoracic aorta were resected; nitric oxide synthase (NOS) activity was determined using L-[U14C]-arginine and vascular reactivity was assessed. Results: Chronic administration of CNP decreased SBP in SHR. Cardiac output was lower in SHR and increased with CNP; however, CNP had no effect in normotensive rats. Spontaneously hypertensive rats had unbalanced aortic vasodilation and vasoconstriction responses, and CNP improved the vascular function. Nitric oxide synthase activity was greater in SHR and increased with the 14-day CNP infusion, but this increase was lower than in normotensive rats. Conclusion: C-type natriuretic peptide induces cardiovascular and NO system changes which may be beneficial in this model of hypertension.