C-type Natriuretic Peptide Release Research Articles

THU156 Significantly Improved Annual Height Velocity With Once-Weekly TransCon CNP In Children With Achondroplasia: The ACcomplisH Phase 2, Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Trial

THU156 Significantly Improved Annual Height Velocity With Once-Weekly TransCon CNP In Children With Achondroplasia: The ACcomplisH Phase 2, Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Trial

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-β1 (TGF-β1)-induced myofibrogenic differentiation. It is also shown that treatment with NPCNP attenuated contraction of collagen gels by cardiac myofibroblasts stimulated with TGF-β1. 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.

OP.3D.05] HYPERTENSION AND VASCULAR INJURY

Objective: C -type natriuretic peptide (CNP) and nitric oxide (NO) have effects on the endothelium and the vascular smooth muscle and are considered to be two major factors involved in the regulation of blood pressure and cardiovascular homeostasis. Growing evidence shows that the expression and release of a number of inflammatory cytokines are increased in hypertension and are also present at sites of vascular injury. In addition, pro-inflammatory cytokines such as tumoral necrosis factor alpha (TNF-α) and interleukin 1 (IL-1) stimulate the release of C-type natriuretic peptide (CNP), showing a potential role of CNP as a modulator of the inflammatory process. The aim of the present study was to evaluate the effects of CNP chronic treatment on vascular NO system, oxidative stress and inflammation in spontaneously hypertensive rats (SHR). Design and method: 12-week-old male Wistar and SHR were infused with CNP (0.75 μg/hr) or saline (S) (osmotic pumps, 14 days). Systolic blood pressure (SBP, mmHg) was recorded by tail-cuff method. After treatment, animals were decapitated and the thoracic aorta artery was removed to determine: protein expression of endothelial NO synthase (eNOS, % relative to actin density) and its phosphorylation in Ser1177 (% relative to eNOS density) by Western blot, superoxide anion production (O2.-, AU/mg dry weight) using lucigenine, content of thiobarbituric acid reactive species (TBARS, nmol/mg protein), IL-6 and TNF-α (% of staining area) by immunohistochemistry, and IL-1β (pg/mg protein) by ELISA. Statistics: 2 way ANOVA, Bonferroni test ad hoc or t test. n = 6 rats/group except IL-1β: n = 3 rats/group. Results: Results are expressed as mean ± SEM.Conclusions: Conclusions: Our results show that CNP chronic treatment attenuates the expression of pro-inflammatory markers and oxidative stress in vascular tissue of hypertensive rats. Also, CNP enhances NO system and induces a drop in blood pressure. These beneficial effects could be related to an overall improvement of target organ damage in this model of hypertension.

C-type natriuretic peptide chronic administration attenuates cardiac fibrosis and inflammation in spontaneously hypertensive rats

Background Growing evidence shows that the expression and release of a number of inflammatory cytokines are increased in hypertension [1,2] and are also present at sites of cardiovascular fibrosis. In fact, many of these inflammatory mediators are responsible for the activation of collagen producing fibroblasts [3]. In addition, proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-a )a nd interleukin-1 (IL-1) have shown to stimulate the release of C-type natriuretic peptide (CNP), indicating a potential role of CNP as a modulator of the inflammatory process [4]. The aim of the present study was to evaluate the effects of chronic CNP treatment on cardiac fibrosis and inflammation in spontaneously hypertensive rats (SHR). Experimental design and methods 12-week-old male SHR and Wistar rats were infused through osmotic pumps with CNP (0,75 µg/hr) or saline (S) for 14 days. Systolic blood pressure (SBP, mmHg) was recorded by tail-cuff method. At the end of the treatment, the left ventricle (LV) was extracted to determine left ventricular mass index (LVMI, g LV/mm tibia length), the myocyte area (µm 2 , Haematoxylin and Eosin-stain), fibrosis (% collagen, Picrosirius Red stain) and inflammation parameters (IL-6 and TNF-a ,% stain/mm 2 by immunohistochemistry, IHC and IL-6, TNF-a and IL-1b ,p g/mg protein by ELISA). Results Conclusions Characteristic high SBP values in SHR are accompanied by hypertrophy, fibrosis and a higher presence of inflammatory cytokines. Our results show that chronic treatment with CNP attenuates the expression of proinflammatory markers and the early signs of fibrosis in cardiac tissue of hypertensive rats. These effects, combined with the drop in blood pressure we observed, indicate that CNP could possibly have an important pathophysiological and therapeutical role in preventing or even reversing cardiac fibrosis and inflammation accompanying left ventricular remodelling in arterial hypertension.

Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1β in cartilage explants.

C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1β (IL-1β). SEM showed uniform, 2 to 3 μm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1β and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1β in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

IntroductionThe present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.MethodsNatriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF4-23. Nitric oxide (NO) production, prostaglandin E2 (PGE2) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data.ResultsThe present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE2 release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF4-23 had the opposite effect and reduced NO and PGE2 release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE2 release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE2 release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression.ConclusionsMechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.

Mismeasure of C-Type Natriuretic Peptide

It has been difficult to establish an endocrine role for C-type natriuretic peptide (CNP). Release of CNP from the heart remains to be convincingly demonstrated (1)(2)(3)(4)(5), and data concerning CNP in cardiac disease have been conflicting, possibly because of specificity problems in measurement of CNP. All natriuretic peptides (NPs) display strong homology, and antibodies raised against one NP may consequently bind other NPs. With the relatively high plasma concentrations of A- and B-type NPs (ANP and BNP), a CNP assay with even a small degree of ANP or BNP cross-reactivity may produce falsely high CNP concentrations. This is especially relevant in heart failure with augmented cardiac ANP and BNP expression. We examined the selectivity of a widely used CNP RIA. To evaluate cross-reactivity in the CNP assay, we measured serial dilutions of human CNP-22 (4.5–582 pmol/L), ANP-28 (5–100 000 pmol/L), and BNP-32 (4–81 000 pmol/L) against the calibrators of the CNP assay. We obtained human …

Natriuretic peptide receptor-C regulates coronary blood flow and prevents myocardial ischemia/reperfusion injury: novel cardioprotective role for endothelium-derived C-type natriuretic peptide.

Ischemia/reperfusion (I/R) injury complicates myocardial infarction and stroke by exacerbating tissue damage and increasing risk of mortality. We have recently identified C-type natriuretic peptide (CNP) as an endothelium-derived hyperpolarizing factor in the mesenteric resistance vasculature and described a novel signaling pathway involving activation of natriuretic peptide receptor C (NPR-C), which plays a pivotal role in the regulation of local blood flow. We tested the hypothesis that CNP/NPR-C signaling is a novel regulatory pathway governing coronary blood flow and protecting against I/R injury. CNP and (Cys18)-atrial natriuretic factor (4-23) amide (cANF(4-23)) elicited dose-dependent decreases in coronary perfusion pressure (CPP) that were blocked by Ba(2+) and ouabain in the isolated Langendorff rat heart. The endothelium-dependent vasodilator acetylcholine elicited the release of CNP from the coronary endothelium. CNP and cANF(4-23) reduced infarct size after 25 minutes of global ischemia and 120 minutes of reperfusion, maintaining CPP and left ventricular pressure at preischemic values. The vasorelaxant and protective activity of CNP and cANF(4-23) were enhanced in the absence of endothelium-derived nitric oxide. Endothelium-derived CNP is involved in the regulation of the coronary circulation, and NPR-C activation underlies the vasorelaxant activity of this peptide. Moreover, this newly defined pathway represents a protective mechanism against I/R injury and a novel target for therapeutic intervention in ischemic cardiovascular disorders.

Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor.

Endothelial cells in most vascular beds release a factor that hyperpolarizes the underlying smooth muscle, produces vasodilatation, and plays a fundamental role in the regulation of local blood flow and systemic blood pressure. The identity of this endothelium-derived hyperpolarizing factor (EDHF), which is neither NO nor prostacyclin, remains obscure. Herein, we demonstrate that in mesenteric resistance arteries, release of C-type natriuretic peptide (CNP) accounts for the biological activity of EDHF. Both produce identical smooth muscle hyperpolarizations that are attenuated in the presence of high [K(+)], the G(i) G protein (G(i)) inhibitor pertussis toxin, the G protein-gated inwardly rectifying K(+) channel inhibitor tertiapin, and a combination of Ba(2+) (inwardly rectifying K(+) channel blocker) plus ouabain (Na(+)K(+)-ATPase inhibitor). Responses to EDHF and CNP are unaffected by the natriuretic peptide receptor (NPR)-AB antagonist HS-142-1, but mimicked by the selective NPR-C agonist, cANF(4-23). EDHF-dependent relaxation is concomitant with liberation of endothelial CNP; in the presence of the myoendothelial gap-junction inhibitor 18alpha-glycyrrhetinic acid or after endothelial denudation, CNP release and EDHF responses are profoundly suppressed. These data demonstrate that acetylcholine-evoked release of endothelial CNP activates NPR-C on vascular smooth muscle that via a G(i) coupling promotes Ba(2+)ouabain-sensitive hyperpolarization. Thus, we have revealed the identity of EDHF and established a pivotal role for endothelial-derived CNP in the regulation of vascular tone and blood flow.

CNP production in the kidney and effects of protein intake restriction in nephrotic syndrome.

C-type natriuretic peptide (CNP) possesses well-established cardiovascular properties. Although present in the mammalian kidney, CNP production in human kidney and its modulation in human renal disease remain less defined. We investigated the presence of CNP in normal human kidney and in patients with nephrotic syndrome (NS). We also addressed whether or not a low-protein diet (LPD) alters plasma CNP and urinary CNP excretion in NS. In situ hybridization studies demonstrated CNP mRNA expression in tubular cells and glomeruli of normal human kidneys. CNP immunoreactivity was positive in proximal, distal, and medullary collecting duct tubular cells in both controls and patients with NS. The ratios of plasma CNP and urinary CNP to creatinine were significantly higher in patients with NS compared with controls. Urinary CNP, but not plasma CNP, was significantly lowered in patients with NS after an LPD. Similarly, the ratios of urinary protein to creatinine and urinary albumin to creatinine, but not urinary guanosine 3',5'-cyclic monophosphate to creatinine, decreased significantly with an LPD. These data confirm and extend previous reports and demonstrate for the first time the presence of CNP in human kidney with NS. We also report increased plasma CNP concentration and urinary CNP excretion in NS patients and a significant reduction of CNP excretion with an LPD. Our findings demonstrate that CNP metabolism is altered in patients with NS and support the hypothesis that activation of renal CNP can be partially offset by an LPD. These results underscore that the beneficial effect of an LPD on protein excretion is paralleled by a substantial reduction in intrarenal CNP release.

Nocturnal accumulation of cyclic 3',5'-guanosine monophosphate (cGMP) in the chick pineal organ is dependent on activation of guanylyl cyclase-B.

The role of cGMP in the avian pineal is not well understood. Although the light-sensitive secretion of melatonin is a well-known output of the circadian oscillator, pharmacologically elevated cGMP levels do not result in altered melatonin secretory amplitude or phase. This suggests that pineal cGMP signalling does not couple the endogenous circadian oscillator to the expression of melatonin rhythms. Nonetheless, the free-running rhythm of cGMP signalling implies a link to the circadian oscillator in chick pinealocytes. As the circadian rhythm of cGMP levels in vitro is not altered by pharmacological inhibition of phosphodiesterase activity, we infer that the synthesis, rather than the degradation of cGMP, is under circadian control. In vitro experiments with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine as well as with an inhibitor of the NO-sensitive soluble guanylyl cyclase (sGC), showed that the NOS-sGC pathway does not play a major role in the circadian control of cGMP generation. In organ culture experiments, we demonstrated that C-type natriuretic peptide (CNP), but not atrial natriuretic peptide (ANP), elevated daytime levels of cGMP. As CNP acts on the membrane guanylyl cyclase isoform B (GC-B), which is expressed at very high levels in mammalian pineals, we investigated the effect of the membrane GC-specific inhibitor HS-142 on chick pineal cGMP levels. CNP-induced daytime cGMP levels were reduced by HS-142. More importantly, 'spontaneously' high nocturnal levels of cGMP in vitro were reduced to daytime levels by acute addition of HS-142. These data implicate endogenous nocturnal CNP release and subsequent activation of GC-B as the major input driving cGMP synthesis in the chick pineal organ.

Enhanced expression and release of C-type natriuretic peptide in freshwater eels.

C-type natriuretic peptide (CNP) is recognized as a paracrine factor acting locally in the brain and periphery. To assess the role of CNP in teleost fish, a cDNA encoding a CNP precursor was initially cloned from the eel brain. CNP message subsequently detected by ribonuclease protection assay, using the cDNA as probe, was most abundant in the brain followed by liver, gut, gills, and heart. Expression was generally higher in freshwater (FW) than in seawater (SW) eels, but not in the brain. Plasma CNP concentration measured by a newly developed homologous radioimmunoassay for eel CNP was higher in FW than in SW eels. The CNP concentration was also higher in the heart of FW eels but not in the brain. These results show that CNP is abundantly synthesized in peripheral tissues of FW eels and secreted constitutively into the circulation. Therefore, CNP is a circulating hormone as well as a paracrine factor in eels. Together with our previous demonstration that CNP-specific receptor expression is enhanced in FW eels, it appears that CNP is a hormone important for FW adaptation. Because atrial NP (ANP) promotes SW adaptation in eels, CNP and ANP, despite high sequence identity, appear to have opposite effects on environmental adaptation of the euryhaline fish.

Production of C-type natriuretic peptide in human aortic endothelial cells induced by activation of protein kinase C

The effects of activators and inhibitors of protein kinase C (PKC) on the production of C-type natriuretic peptide (CNP) by cultured human aortic endothelial cells were examined. The PKC activators phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetyl glycerol (OAG) stimulated CNP release; the maximal effects were apparent at 4 h, at which time release was 934 and 205% of the control value, respectively. The PKC inhibitors staurosporine and H-7 did not affect basal CNP release, but each abolished the increase in CNP release induced by PMA or OAG. PKC was activated and translocated from cytosolic to membrane fractions in endothelial cells exposed to PMA or OAG; the maximal effect was apparent at 1 h. PMA and OAG each increased the abundance of CNP mRNA, with the maximal effect at 2 h. These data suggest that activation of PKC by PMA or OAG results in an increase in the abundance of CNP mRNA and subsequent enhancement of CNP production and release in human aortic endothelial cells.

Lipoproteins regulate C-type natriuretic peptide secretion from cultured vascular endothelial cells.

We have shown that oxidized low-density lipoprotein (Ox-LDL) modulates various endothelial cell (EC) functions. C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family to be discovered, is secreted from peripheral vascular ECs and regulates body fluid homeostasis, vascular tone, and vascular growth. This study was designed to investigate the effects of lipoproteins on CNP secretion from cultured ECs. Treatment of bovine carotid ECs with OX-LDL and its extracted lipids resulted in a concentration-dependent suppression of the spontaneous and transforming growth factor-beta 1-stimulated secretion CNP. Native LDL, its extracted lipids, and acetylated LDL were inactive. OX-LDL depleted of its amphiphilic lipids, which was prepared by incubation with defatted albumin, lost its suppressive effect on CNP secretion. 7-Ketocholesterol, one of the amphiphilic lipids in OX-LDL that is transferable from OX-LDL to defatted albumin, suppressed CNP secretion by ECs, thus mimicking the effect of OX-LDL. Coincubation with high-density lipoprotein (HDL), which alone had no effect on CNP release, significantly prevented OX-LDL-induced inhibition of CNP secretion by ECs. Analysis by thin-layer chromatography demonstrated that oxysterols, including 7-ketocholesterol, in OX-LDL were transferred from OX-LDL to HDL during coincubation of these two lipoproteins. These results indicate that OX-LDL suppresses CNP secretion from ECs by 7-ketocholesterol or other transferable hydrophilic lipids in OX-LDL, and the suppressive effect of OX-LDL is reversed by HDL. Lipoproteins thus may regulate CNP secretion from the endothelium of atherosclerotic arteries.