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.

"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.

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.

C-type natriuretic peptide induces inotropic and lusitropic effects in human 3D-engineered cardiac tissue: Implications for the regulation of cardiac function in humans.

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.

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.

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.

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.

C-type natriuretic peptide improves growth retardation in a mouse model of cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome, a genetic disorder caused by germline mutations in BRAF, KRAS, MAP2K1 and MAP2K2, is characterized by growth retardation, heart defects, dysmorphic facial appearance and dermatologic abnormalities. We have previously reported that knock-in mice expressing the CFC syndrome-associated mutation, Braf Q241R, showed growth retardation because of gastrointestinal dysfunction. However, other factors associated with growth retardation, including chondrogenesis and endocrinological profile, have not been examined. Here, we show that 3- and 4-week-old BrafQ241R/+ mice have decreased body weight and length, as well as reduced growth plate width in the proximal tibiae. Furthermore, proliferative and hypertrophic chondrocyte zones of the growth plate were reduced in BrafQ241R/+ mice compared with Braf+/+ mice. Immunohistological analysis revealed that extracellular signal-regulated kinase (ERK) activation was enhanced in hypertrophic chondrocytes in BrafQ241R/+ mice. In accordance with growth retardation and reduced growth plate width, decreased serum levels of insulin-like growth factor 1 (IGF-1) and IGF binding protein 3 (IGFBP-3) were observed in BrafQ241R/+ mice at 3 and 4 weeks of age. Treatment with C-type natriuretic peptide (CNP), a stimulator of endochondral bone growth and a potent inhibitor of the FGFR3-RAF1-MEK/ERK signaling, increased body and tail lengths in Braf+/+ and BrafQ241R/+ mice. In conclusion, ERK activation in chondrocytes and low serum IGF-1/IGFBP-3 levels could be associated with the growth retardation observed in BrafQ241R/+ mice. Our data also suggest that CNP is a potential therapeutic target in CFC syndrome.

Inhibition of fibrotic changes in infrapatellar fat pad alleviates persistent pain and articular cartilage degeneration in monoiodoacetic acid-induced rat arthritis model.

OBJECTIVE: We have reported that fibrotic changes in infrapatellar fat pad (IFP) after acute joint inflammation are closely associated with persistent pain in rats. In this study, to examine the effects of anti-fibrotic treatment on persistent pain, we used C-type natriuretic peptides (CNP) at the recovery phase after acute joint inflammation. DESIGN: Thirty-two male Wistar rats were used in this study. Monoiodoacetic acid (MIA) was injected intra-articularly to induce IFP fibrosis and persistent pain. CNP was injected after acute inflammatory phase in the same knee joint. Time-course pain-avoidance behavior tests and histological analyses were performed to examine the effects of CNP. RESULTS: Histological evaluations indicated that intra-articular injection of CNP inhibited fibrotic changes in IFP after acute inflammation. Incapacitance tests indicated that MIA injection into rat knee joint quickly decreased the percent weight on ipsilateral limb. In the vehicle group, the decrease was maintained up to day 28, suggesting that pain persistence occurred after acute inflammation (Day 0/Day 28, Est Dif -8.15, CI -10.78∼-5.53, Linear mixed-effect model). In contrast, the pain was alleviated in the CNP group after day 14 (Day0/Day 14, -0.51, -2.62-1.59). In addition, we observed significant improvement in the degree of articular cartilage degeneration at day 14 in the CNP group (OARSI score: vehicle 16.14 ± 4.37 vs CNP 6.87 ± 3.44, P < 0.01; Wilcoxon rank sum test). CONCLUSION: Fibrotic changes in IFP may play important roles in both persistent pain and articular cartilage degeneration.

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide).

OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.

C-Type Natriuretic Peptide Ameliorates Vascular Injury and Improves Neurological Outcomes in Neonatal Hypoxic-Ischemic Brain Injury in Mice.

C-type natriuretic peptide (CNP) is an important vascular regulator that is present in the brain. Our previous study demonstrated the innate neuroprotectant role of CNP in the neonatal brain after hypoxic-ischemic (HI) insults. In this study, we further explored the role of CNP in cerebrovascular pathology using both in vivo and in vitro models. In a neonatal mouse HI brain injury model, we found that intracerebroventricular administration of recombinant CNP dose-dependently reduces brain infarct size. CNP significantly decreases brain edema and immunoglobulin G (IgG) extravasation into the brain tissue, suggesting a vasculoprotective effect of CNP. Moreover, in primary brain microvascular endothelial cells (BMECs), CNP dose-dependently protects BMEC survival and monolayer integrity against oxygen-glucose deprivation (OGD). The vasculoprotective effect of CNP is mediated by its innate receptors NPR2 and NPR3, in that inhibition of either NPR2 or NPR3 counteracts the protective effect of CNP on IgG leakage after HI insult and BMEC survival under OGD. Of importance, CNP significantly ameliorates brain atrophy and improves neurological deficits after HI insults. Altogether, the present study indicates that recombinant CNP exerts vascular protection in neonatal HI brain injury via its innate receptors, suggesting a potential therapeutic target for the treatment of neonatal HI brain injury.

Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in Mice.

RATIONALE: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo. OBJECTIVE: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease. METHODS AND RESULTS: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein-coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2+/- and Npr2+/-;Ldlr-/- mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2+/- fibroblasts was insensitive to CNP treatment, whereas aged Npr2+/- and Npr2+/-;Ldlr-/- mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2+/- and Npr2+/-;Ldlr-/- mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2+/- with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2+/- valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2+/-;Ldlr-/- mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2+/- mice with bicuspid aortic valves than those with tricuspid valves. CONCLUSIONS: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

Mechanism of CNP-mediated DG-PKC and IP4 signaling pathway in diabetic rats with gastric motility disorder.

In the precedent research conducted by the same team, it concluded that the activities in C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP)/ß-type phospholipase C (PLCß) pathways of rat antral smooth muscle were changed due to diabetes, which was the key pathogenetic mechanism for diabetic gastric dysmotility. As the follow-on step, this study was designed to probe into the downstream signaling pathway of CNP/PLCß. The results showed that level of α-type protein kinase C (PKCα),cell membrane to cytoplasm ratio of PKCα, cell membrane to cytoplasmic ratio of ßI-type protein kinase C (PKCßI) and level of Phosphor-PKCα (P-PKCα) were significantly reduced in diabetes rat antral smooth muscle samples. The content of tetraphosphate inositol (IP4) in gastric antral smooth muscle of diabetic rats reduced, and the content of diacyl-glycerol (DG) was unchanged. CNP significantly decreased the content of IP4 and DG, this effect was more obvious in diabetic rats. Subsequent to the addition of protein kinase A (PKA) blocker N-[2- (p-Bromocin-namylamino)ethyl]-5 -isoquinolinesulfonamide dihydrochloride (H-89) before CNP treatment, the inhibitory effect of CNP was reduced; subsequent to the addition of protein kinase G (PKG) blocker KT5823 before CNP treatment, the inhibitory effect of CNP was also reduced. With the addition of the combination of H-89 and KT5823 before CNP treatment, the inhibition by CNP could be offset. These results were concluded that CNP inhibited the activity of PKC family in rat smooth muscle and reduced the levels of IP4 and DG through the PKG/PKA-PLCß pathways, causing inhibited muscular contractions, which may be a key pathogenetic factor for diabetic gastroparesis.

Endothelial C-Type Natriuretic Peptide Acts on Pericytes to Regulate Microcirculatory Flow and Blood Pressure.

BACKGROUND: Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone, but the target cells remain controversial. The cGMP-producing guanylyl cyclase-B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (SMCs). However, whereas endothelial cell-specific CNP knockout mice are hypertensive, mice with deletion of GC-B in vascular SMCs have unaltered blood pressure. METHODS: We analyzed whether the vasodilating response to CNP changes along the vascular tree, ie, whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B ( Npr2) gene were interbred with Tie2-Cre or PDGF-Rß-Cre ERT2 lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMCs and capillary pericytes. Intravital microscopy, invasive and noninvasive hemodynamics, fluorescence energy transfer studies of pericyte cAMP levels in situ, and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. RESULTS: Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases toward small-diameter arterioles and capillaries. CNP consistently did not prevent endothelin-1-induced acute constrictions of proximal arterioles, but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, ie, in pericytes. It is notable that the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion, but abolished in mice lacking GC-B in microcirculatory SMCs and pericytes. CNP, via GC-B/cGMP signaling, modulates 2 signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator-activated phosphoprotein, and it inhibits phosphodiesterase 3A, thereby enhancing pericyte cAMP levels. These pathways ultimately prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMCs and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. CONCLUSIONS: Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMCs and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure.

Chronic treatment with C-type natriuretic peptide impacts differently in the aorta of normotensive and hypertensive rats.

The aim of this study was to determine whether exogenous administration of C-type natriuretic peptide (CNP) induces functional and morphological vascular changes in spontaneously hypertensive rats (SHR) compared with normotensive rats. Male 12-week-old normotensive Wistar and SHR were administered with saline (NaCl 0.9%) or CNP (0.75 µg/h/100 g) for 14 days (subcutaneous micro-osmotic pumps). Systolic blood pressure (SBP) was measured in awake animals and renal parameters were evaluated. After decapitation, the aorta was removed, and vascular morphology, profibrotic markers, and vascular reactivity were measured. In addition, nitric oxide (NO) system and oxidative stress were evaluated. After 14-days of treatment, CNP effectively reduced SBP in SHR without changes in renal function. CNP attenuated vascular remodeling in hypertensive rats, diminishing both profibrotic and pro-inflammatory cytokines. Also, CNP activated the vascular NO system and exerted an antioxidant effect in aortic tissue of both groups, diminishing superoxide production and thiobarbituric acid-reactive substances, and increasing glutathione content. These results show that chronic treatment with CNP attenuates the vascular damage development in a model of essential hypertension, inducing changes in fibrotic, inflammatory, oxidative, and NO pathways that could contribute to beneficial long-term effects on vascular morphology, extracellular matrix composition, and function. The knowledge of these effects of CNP could lead to improved therapeutic strategies to not only control BP but also reduce vascular damage, primarily responsible for the risk of cardiovascular events.

TransCon CNP, a Sustained-Release C-Type Natriuretic Peptide Prodrug, a Potentially Safe and Efficacious New Therapeutic Modality for the Treatment of Comorbidities Associated with Fibroblast Growth Factor Receptor 3-Related Skeletal Dysplasias.

TransCon CNP is a C-type natriuretic peptide (CNP-38) conjugated via a cleavable linker to a polyethylene glycol carrier molecule, designed to provide sustained systemic CNP levels upon weekly subcutaneous administration. TransCon CNP is in clinical development for the treatment of comorbidities associated with achondroplasia. In both mice and cynomolgus monkeys, sustained exposure to CNP via TransCon CNP was more efficacious in stimulating bone growth than intermittent CNP exposure. TransCon CNP was well tolerated with no adverse cardiovascular effects observed at exposure levels exceeding the expected clinical therapeutic exposure. At equivalent dose levels, reductions in blood pressure and/or an increase in heart rate were seen following single subcutaneous injections of the unconjugated CNP-38 molecule or a daily CNP-39 molecule (same amino acid sequence as Vosoritide, USAN:INN). The half-life of the daily CNP-39 molecule in cynomolgus monkey was estimated to be 20 minutes, compared with 90 hours for CNP-38, released from TransCon CNP. C max for the CNP-39 molecule (20 µg/kg) was approximately 100-fold higher, compared with the peak CNP level associated with administration of 100 µg/kg CNP as TransCon CNP. Furthermore, CNP exposure for the daily CNP-39 molecule was only evident for up to 2 hours postdose (lower limit of quantification 37 pmol/l), whereas TransCon CNP gave rise to systemic exposure to CNP-38 for at least 7 days postdose. The prolonged CNP exposure and associated hemodynamically safe peak serum concentrations associated with TransCon CNP administration are suggested to improve efficacy, compared with short-lived CNP molecules, due to better therapeutic drug coverage and decreased risk of hypotension. SIGNIFICANCE STATEMENT: The hormone C-type natriuretic peptide (CNP) is in clinical development for the treatment of comorbidities associated with achondroplasia, the most common form of human dwarfism. The TransCon Technology was used to design TransCon CNP, a prodrug that slowly releases active CNP in the body over several days. Preclinical data show great promise for TransCon CNP to be an effective and well-tolerated drug that provides sustained levels of CNP in a convenient once-weekly dose, while avoiding high systemic CNP bolus concentrations that can induce cardiovascular side effects.

C-type natriuretic peptide enhances mouse preantral follicle growth.

Compared to ovarian antral follicle development, the mechanism underlying preantral follicle growth has not been well documented. Although C-type natriuretic peptide (CNP) involvement in preantral folliculogenesis has been explored, its detailed role has not been fully defined. Here, we used mouse preantral follicles and granulosa cells (GCs) as a model for investigating the dynamic expression of CNP and natriuretic peptide receptor 2 (NPR2) during preantral folliculogenesis, the regulatory role of oocyte-derived growth factors (ODGFs) in natriuretic peptide type C (Nppc) and Npr2 expression, and the effect of CNP on preantral GC viability. Both mRNA and protein levels of Nppc and Npr2 were gradually activated during preantral folliculogenesis. CNP supplementation in culture medium significantly promoted the growth of in vitro-cultured preantral follicles and enhanced the viability of cultured GCs in a follicle-stimulating hormone (FSH)-independent manner. Using adult and prepubertal mice as an in vivo model, CNP pre-treatment via intraperitoneal injection before conventional superovulation also had a beneficial effect on promoting the ovulation rate. Furthermore, ODGFs enhanced Nppc and Npr2 expression in the in vitro-cultured preantral follicles and GCs. Mechanistic study demonstrated that the regulation of WNT signaling and estrogen synthesis may be implicated in the promoting role of CNP in preantral folliculogenesis. This study not only proves that CNP is a critical regulator of preantral follicle growth, but also provides new insight in understanding the crosstalk between oocytes and somatic cells during early folliculogenesis.

Effects of TAK-639, a novel topical C-type natriuretic peptide analog, on intraocular pressure and aqueous humor dynamics in mice.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness, and individuals with ocular hypertension are at risk to develop POAG. Currently, the only modifiable risk factor for glaucoma progression is lowering of intraocular pressure (IOP). A novel mechanism for lowering IOP involves activation of the type B natriuretic peptide receptor (NPR-B), the naturally occurring agonist of which is C-type natriuretic peptide (CNP). Being a cyclic peptide of 22 amino acids, CNP does not readily penetrate the cornea and its ocular hypotensive effect requires intraocular injection. TAK-639 is a synthetic, cornea-permeable, 9-amino acid CNP analog has been studied for the treatment of ocular hypertension and POAG. We assessed TAK-639 in a receptor binding profile and the effects of TAK-639 on NPR-B-mediated cyclic GMP production in cultured transformed human trabecular meshwork (TM) cells (GTM-3). We also evaluated the effects of topical ocular administration of TAK-639 on mouse IOP and aqueous humor dynamics. Among 89 non-natriuretic peptide receptors, transporters, and channels evaluated, TAK-639 at 10 µM displaced ligand binding by more than 50% to only two receptors: the type 2 angiotensin receptor (IC50 = 8.2 µM) and the cholecystokinin A receptor (IC50 = 25.8 µM). In vitro, TAK-639 selectively activates NPR-B (EC50 = 61 ±â€¯11 nM; GTM-3 cells) relative to NPR-A (EC50 = 2179 ±â€¯670 nM; 293T cells). In vivo, TAK-639 lowered mouse IOP by three mechanisms: increase in aqueous humor outflow facility (C), reduction in the aqueous humor formation rate (Fin), and reduction in episcleral venous pressure (Pe). The maximum mean IOP decreases from baseline were -12.1%, -21.0%, and -36.1% for 0.1%, 0.3%, and 0.6% doses of TAK-639, respectively. Maximum IOP-lowering effect was seen at 2 h, and the duration of action was >6 h. With TAK-639 0.6%, at 2 h post-dose, aqueous outflow facility (C) increased by 155.8%, Fin decreased by 41.0%, the uveoscleral outflow rate (Fu) decreased by 52.6%, and Pe decreased by 31.5% (all p < 0.05). No ocular adverse effects were observed. TAK-639 is an efficacious IOP-lowering agent, with a unique combination of mechanisms of action on both aqueous formation and aqueous outflow facility. Further study of this mechanism of treatment may optimize pharmacologic outcomes and provide disease management in patients with POAG and ocular hypertension.

The Novel Inodilator ORM-3819 Relaxes Isolated Porcine Coronary Arteries: Role of Voltage-Gated Potassium Channel Activation.

Relaxation and changes in the transmembrane potential of vascular smooth muscle induced by ORM-3819, a novel inodilating compound, were investigated in isolated porcine coronary arteries. Isometric tone was studied on arterial rings precontracted by KCl (30 mM), and resting membrane potential was investigated by a conventional microelectrode technique. ORM-3819 in the concentration range 0.38-230.6 µM evoked concentration-dependent relaxation with a maximum value of 58.1% and an effective concentration of the relaxing substance that caused 50% of maximum relaxation of 72.2 µM. The maximum hyperpolarization produced by ORM-3819 at a concentration of 120 µM (-2.6 ± 0.81 mV, N = 10) did not differ significantly from that induced by C-type natriuretic peptide (CNP), an endogenous hyperpolarizing mediator, at a concentration of 1.4 µM (-3.6 ± 0.38 mV, N = 17). The same effect elicited by the known inodilator levosimendan was less pronounced at a concentration of 3.7 µM: -1.82 ± 0.44 mV, N = 22 (P < 0.05 vs. CNP). The voltage-gated potassium channel inhibitor 4-aminopyridine, at a concentration of 5 mM, attenuated the relaxation induced by ORM-3819 at concentrations of 41.6 or 117.2 µM. These results suggest that ORM-3819 is a potent vasodilating agent able to relieve coronary artery vasospasm by causing hyperpolarization of vascular smooth muscle cells through processes involving activation of voltage-gated potassium channels.

A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

STUDY QUESTION: Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? SUMMARY ANSWER: A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. WHAT IS KNOWN ALREADY: Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. LIMITATIONS, REASONS FOR CAUTION: Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. WIDER IMPLICATIONS OF THE FINDINGS: IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. LARGE SCALE DATA: N/A. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.