C-type Natriuretic Peptide mRNA Expression Research Articles

C-type natriuretic peptide in childhood obesity

According to the World Health Organization obesity is the result of an energy imbalance between calories assumed and expended and over the past 30 years its incidence has dramatically increased. Recently, the problem of obesity has drastically increased also in childhood, assuming a social relevance. Childhood obesity, in fact, increases the possibility to be obese in adulthood, representing a risk for cardiovascular morbidity and mortality. Aim of this review was to carry out a revision of the literature on childhood obesity focusing on natriuretic peptides (NPs) and in particular on the role of C-type natriuretic peptide (CNP). In obesity NPs play a fundamental role in the regulation of body weight and energy metabolism. Data on plasma CNP levels in children are scarce. The review of the literature relating to the role of CNP in adolescents showed a progressive reduction in the CNP plasma levels in overweight/obese adolescents compared to normal-weight subjects, as previously observed in obese adults, as well as a different modulation in CNP mRNA expression. An independent association between CNP levels and obesity as well as a significant association with the endothelial dysfunction index was reported, indicating that the peptide could play a very important role as a marker of risk of developing obesity. The results of these studies indicate the importance of adopting healthy lifestyles to improve glucometabolic control as well as to provide the rationale for designing and developing new drugs to modulate the NPs system.

C-type natriuretic peptide plasma levels and whole blood mRNA expression show different trends in adolescents with different degree of endothelial dysfunction

C-type natriuretic peptide (CNP) is an endogenous adipogenesis regulator whose plasma levels in childhood are known, while no data are available on its expression. Our aim was to evaluate both CNP plasma levels and CNP system expression in whole blood obtained from normal-weight (N, n = 24) and obese (O, n = 16) adolescents (age:13.5 ± 0.4 years). Endothelial function was assessed measuring reactive hyperemia index (RHI). CNP plasma levels, evaluated with specific RIA, resulted significantly lower in O than in N (6.1 ± 0.8 vs.15.2 ± 1.3 pg/mL; p < 0.0001), while CNP/NPR-B/NPR-C mRNA, measured by Real-Time PCR, resulted similar in N (4.1 ± 1.7; 5.0 ± 1.6; 2.2 ± 0.9) and in O (4.3 ± 1.6; 3.5 ± 1.1; 2.3 ± 0.8). RHI was significantly lower in O than in N (1.4 ± 0.08 vs.2.1 ± 0.04, p < 0.0001). Dividing all subjects according to the RHI median value, irrespective of the presence or absence of obesity (Group 1 > 1.9, n = 23, Group 2 < 1.9, n = 17), CNP plasma concentrations resulted significantly (p = 0.014) higher in Group 1 (14.6 ± 1.6) than in Group 2 (7.5 ± 1.0), showing a significant correlation with RHI (p = 0.0026), while CNP mRNA expression was, surprisingly, higher in Group 2 (7.0 ± 2.3) than in Group 1 (1.8 ± 0.4; p = 0.02). NPR-B mRNA resulted similar in both Groups (4.3 ± 1.6; 4.7 ± 1.3) and NPR-C significantly higher in Group 2 (p = 0.02). Our data suggest different trends between CNP plasma levels and expression, assessed for the first time in whole blood, that could reflect changes occurring both at CNP transcriptional level in activated leukocytes due to inflammation, and at circulating levels, due to CNP paracrine/autocrine activities. This could represent an interesting area for new therapies able to modulate endothelial dysfunction.

C-type natriuretic peptide transcriptomic profiling increases in human leukocytes of patients with chronic heart failure as a function of clinical severity

The aim of this study was to evaluate the transcriptomic profiling of C-type natriuretic peptide (CNP) and of its specific receptor, NPR-B in human leukocytes of heart failure (HF) patients as a function of clinical severity, assessing the possible changes with respect to healthy subjects (C). mRNA expression was evaluated by Real-Time PCR and total RNA was extracted from leukocytes of C (n=8) and of HF patients (NYHA I–II, n=7; NYHA III–IV, n=13) with PAXgene Blood RNA Kit. Significantly higher levels of CNP mRNA expression were found in HF patients as a function of clinical severity (C=0.23±0.058, NYHA I–II=0.47±0.18, NYHA III–IV=2.58±0.71, p=0.005 C vs NYHA III–IV, p=0.017 NYHA I–II vs NYHA III–IV) and NPR-B transcript levels resulted down-regulated in HF patients with higher NYHA class (C=2.2±0.61, NYHA I–II=2.76±0.46, NYHA III–IV=0.29±0.13, p=0.001 C vs NYHA III–IV, p<0.0001 NYHA I–II vs NYHA III–IV). A significant negative correlation between CNP and NPR-B mRNA expression (r=0.5, p=0.03) was also observed. These results suggest a co-regulation of NPR-B and CNP expression supporting the relevance of this receptor in human disease characterized by a marked inflammatory/immune component and suggesting the possibility of manipulating inflammation via pharmacological agents selective for this receptor.

Transforming growth factor-β1regulation of C-type natriuretic peptide expression in human vascular smooth muscle cells: dependence on TSC22D1

C-type natriuretic peptide (CNP) possesses nitric oxide-like signaling mechanisms and actions in the vasculature, including the inhibition of fibrosis and vascular remodeling through counterregulation of transforming growth factor-β (TGF-β) signaling. The leucine zipper protein transforming growth factor stimulated clone 22 domain 1 (TSC22D1), cloned via its presumed binding to a GC-rich element in the CNP promoter, was the first protein to be described as a CNP transcription factor, but the lack of supporting evidence since its discovery and its lack of a classical DNA-binding site have left in question its role in the regulation of CNP by TGF-β and other factors. To define a specific role for TSC22D1 in CNP transcription, we have examined the effects of the profibrotic growth factors TGF-β1 and PDGF-BB on CNP mRNA expression in cultured human vascular smooth muscle cells (SMC) in which TSC22D1 expression was suppressed with small interfering RNA. Results showed that TGF-β and PDGF-BB significantly increased CNP expression in all three SMC types. Twenty-four-hour TGF-β-induced elevations in CNP were strongly correlated with changes in TSC22D1 mRNA levels, and both genes exhibited their greatest response to TGF-β1 in coronary artery SMC. Furthermore, siRNA suppression of TSC22D1 expression in coronary artery and aortic SMC by ∼90% resulted in 45-65% reductions of both PDGF- and TGF-β-stimulated CNP expression, respectively. These results support a postulated role of TSC22D1 as an enhancer of CNP transcription and suggest that TGF-β-induced upregulation of CNP expression in SMC may be mediated in part by increased transcription of TSC22D1.

Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure

High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF ( n = 8) and without (control, n = 6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65 ± 0.11 vs. 0.35 ± 0.04, p = 0.02), but not in the OS (BNP/GAPDH: 0.36 ± 0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089 ± 0.036, PS 0.289 ± 0.23, OS 0.54 ± 0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69 ± 0.13; PS = 1.56 ± 0.19; OS = 1.70 ± 0.42 pg/mg protein; p = 0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C. leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis scyllia and Scyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0 ± 7.9 fmol ml −1 in FW to 144.9 ± 19.5 fmol ml −1 in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed.

Presence of C-type natriuretic peptide (CNP) in guinea pig caecum: role and mechanisms of CNP in circular smooth muscle relaxation.

The distribution and role of C-type natriuretic peptide (CNP) in the gastrointestinal tract are still unclear. This study was designed to investigate the distribution of CNP in guinea pig caecum and the inhibitory mechanisms of CNP in caecal circular smooth muscle cells. CNP immunoreactivity was recognized in smooth muscle cells, myenteric and submucosal neurons of the caecum by immunohistochemistry. CNP mRNA expression was demonstrated in both freshly dispersed and cultured smooth muscle cells by reverse-transcription polymerase chain reaction. CNP inhibited 1 nmol L(-1) cholecystokinin octapeptide (CCK-8)-induced smooth muscle cell contraction in a dose-dependent manner, with an IC(50) value of 0.24 nmol L(-1), and significantly stimulated the production of intracellular cyclic guanosine monophosphate. Furthermore, inhibitors of both soluble and particulate guanylate cyclase (GC) partially but significantly inhibited CNP-induced relaxation. This is the first report demonstrating that CNP localizes in gastrointestinal smooth muscle cells and the enteric nervous system. These results suggest that CNP acts locally through neural and autocrine pathways to modulate colonic motility via both particulate and soluble GC systems. These two pathways appear to be through natriuretic peptide receptor (NPR)-B, which has particulate GC domain, and NPR-C, which activates soluble GC, judging from previous findings that NPR-A is not expressed in these cells.

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

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.

Opposite regulation of brain and C-type natriuretic peptides in the streptozotocin-diabetic cardiopathy.

C-type natriuretic peptide (CNP), a recent addition to the family of natriuretic peptides including atrial and brain natriuretic peptide (ANP, BNP), is believed to be an endothelium-derived vasodilator and to have an antimitotic effect. ANP and BNP concentrations are increased in conditions such as congestive heart failure, but cardiac CNP concentrations have not been investigated in this connection. Diabetes mellitus also involves myocardial dysfunctions without coronary artery disease or systemic hypertension. We therefore investigated the cardiac expression of CNP mRNA compared with that of BNP mRNA in streptozotocin (STZ)-diabetic rats. STZ- diabetic male Wistar rats (n=6) were studied in comparison with controls (n=6). The animals were characterised by their mean arterial blood pressure and plasma glucose concentrations. After extraction of total cardiac RNA, a specific cDNA probe of BNP was used for northern blot analysis, whereas myocardial CNP expression was analysed by an RNase-protection assay. Twelve weeks after diabetes was induced, the rats were normotensive (96.4+/-2.0 compared with 95.1+/-1.9 mmHg) and hyperglycaemic (615+/-61 compared with 165+/-21 mg/dl; P<0.001). Left ventricular pressure was significantly impaired (76.8+/-6.4 compared with 51.2+/-3.6 mmHg). STZ-diabetic rats had a 3.2-fold increase in cardiac BNP expression compared with controls. In contrast, cardiac CNP mRNA concentrations were decreased 2.6-fold. CNP seems to be downregulated like other peptides with antimitotic and vasodilator activities (nitric oxide, prostacyclin, kinins). This may contribute to cardiac dysfunction in diabetes mellitus and suggests that stimulation of CNP expression could provide cardiac protection in such cases.

Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin.

Excess oxidative stress is one of the major metabolic abnormalities on vascular walls in hypertension and atherosclerosis. In order to further elucidate the endothelial function under oxidative stress, the effect of hydrogen peroxide (H2O2) on expression of two novel endothelium-derived vasorelaxing peptides, C-type natriuretic peptide (CNP) and adrenomedullin (AM) from bovine carotid artery endothelial cells (BCAECs) was examined. BCAECs were treated with H2O2 (0.1-1.0 mmol/ l) and/or an antioxidant, N-acetylcysteine (NAC) (5-10 mmol/l), and incubated for 48 h. The concentrations of CNP and AM were measured with the specific radioimmuno assays that we originally developed. CNP and AM mRNA expressions were also examined by reverse transcription-polymerase chain reaction (RT-PCR). Treatment of BCAECs with 0.5 and 1 mmol/l H2O2 induced 9-and 10-fold increases of CNP concentration in the media. Addition of 10 mmol/l NAC significantly suppressed the effect of H2O2 by 52%. RT-PCR analysis showed that CNP mRNA expression in BCAECs was also rapidly augmented within 1 h with H2O2 (1 mmol/l) treatment, and reached a peak at 3 h to show a 10-fold increase. AM secretion from BCAECs also increased to two-fold with exposure to 0.5 mmol/l H2O2, accompanied with the augmented level of AM mRNA. NAC 10 mmol/l completely suppressed the effect of H2O2 on AM secretion. In this study, it has been demonstrated that H2O2 augments endothelial secretion of the two endothelium-derived relaxing peptides, CNP and AM. Our findings suggest the increased secretion of CNP and AM from endothelium under oxidative stress may function to compensate the impaired nitric oxide-dependent vasorelaxation in hypertension and atherosclerosis.

C-type natriuretic peptide-like immunoreactivity in the rat inner ear

C-type natriuretic peptide (CNP) is a member of the atrial natriuretic peptide family (ANP family). The family also includes ANP and brain natriuretic peptide (BNP). These peptides regulate the homeostasis of body fluid and blood pressure as a neuropeptide in the central nervous system as well as a cardiac hormone in the periphery. We have recently reported the expression of CNP mRNA in the inner ear. To assess the possible physiological role of CNP in the inner ear, we investigated the localization of CNP peptide in the rat inner ear by immunohistochemistry at the light and electron microscopic level. CNP-like immunoreactivity was widely distributed in the secretory and the neuronal portion of the inner ear, i.e. the spiral ligament, the dark cell region of the utriculus, the epithelium of the endolymphatic sac, the spiral ganglion cells and the vestibular ganglion cells. The results suggest that CNP may play a role in the homeostasis of the perilymph and endolymph and may also influence nerve activities in the inner ear.

Expression of C-type natriuretic peptide during development of rat lung.

C-type natriuretic peptide (CNP), recently found to be secreted from vascular endothelial cells, is now viewed as a novel endothelium-derived relaxing peptide. However, the distribution and expression of CNP during cardiopulmonary development is unclear. To follow changes in the expression of CNP during lung development, we examined rat embryos and neonates using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay and immunohistochemistry for CNP protein. A substantial expression of CNP mRNA was first detected on postnatal day 2, and it thereafter remained fairly steady. The level of CNP protein also increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. CNP protein and mRNA were detected in the endothelium and smooth muscle cells of blood vessels and in bronchial airway and alveolar epithelia. Immunoreactivity for CNP protein in the endothelium of blood vessels increased to an intense level after the saccular stage. These results suggest that the changes in CNP levels may be related to the occurrence of pulmonary vasodilation after birth.

Shear stress induction of C-type natriuretic peptide (CNP) in endothelial cells is independent of NO autocrine signaling.

C-type natriuretic peptide (CNP) is secreted by endothelial cells and has vasodilatory and antiproliferative activity against smooth muscle cells. Using defined laminar shear stress exposures of cultured bovine aortic endothelial cells, we investigated the regulation of CNP gene by PhosphorImaging the ratio of CNP mRNA to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. A 6 h exposure to arterial shear stress of 25 dyn/cm2 caused a marked elevation (10.5 +/- 6.2-fold: n=10, p<0.001) of CNP/GAPDH mRNA ratio compared to stationary controls. Arterial shear stress was 2.6 times more potent than a venous level of shear stress of 4 dyn/cm2 in elevating the CNP/GAPDH mRNA ratio. After 6 h, CNP secretion by shear stressed BAEC was elevated over stationary controls by 3.1-fold (n=5, p<0.001) to a level of 34 +/- 7.5 pg/cm2 BAEC. Shear stress elevated CNP mRNA in the presence of L-NAME (400 microM) indicating that autocrine signaling through shear-induced NO production or guanylate cyclase activation was not involved. Similarly, the tyrosine kinase inhibitor genistein (10 microM), which can also block shear-induced NO production, had no effect on CNP mRNA induction by shear stress in BAEC. The intracellular calcium chelator BAPTA/AM (5 microM) attenuated the shear stress-induced CNP mRNA expression by 71%. Interestingly, dexamethasone (1 microM) potentiated by 2-fold the shear stress enhancement of CNP mRNA. Shear stress was a more potent inducer of CNP than either phorbol myristrate acetate or lipopolysaccharide. Hemodynamic shear stress may be an important physiological regulator of CNP expression with consequent effects on vasodilation and regulation of intimal hyperplasia.

Increased C-type natriuretic peptide mRNA expression in the kidney of diabetic rats.

To investigate the responsiveness of renal-synthesized C-type natriuretic peptide (CNP) to changes in water and electrolyte balance, we measured renal CNP mRNA levels, plasma CNP concentrations and urinary CNP excretion rates in streptozotocin-induced diabetic rats eating a normal (0.26% NaCl) or low (0.04% NaCl) salt diet. Using reverse transcription-PCR followed by Southern blot analysis, we found that renal cortical and medullary CNP mRNA levels were markedly enhanced in diabetic rats from the 14th day and remained enhanced with an accompanying elevation of urinary CNP excretion rates for the entire 60-day study period. All increases of renal CNP mRNA and urinary CNP excretion rates in diabetic rats were attenuated in low salt diet-treated diabetic rats as well as insulin-treated diabetic rats. These results demonstrate that renal CNP synthesis is enhanced in diabetic rats and the increase of renal CNP mRNA is ameliorated by salt restriction and insulin treatment. These results imply that renal-synthesized CNP is responsive to the alteration of water and electrolyte homeostasis in diabetic rats.

Shear Stress Augments Expression of C-Type Natriuretic Peptide and Adrenomedullin

Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls: these effects have been ascribed to shear stress-induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium-derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does. To elucidate the possible role of these EDRPs under shear stress, we examined the effect of physiological shear stress on CNP mRNA expression in endothelial cells derived from the human umbilical vein (HUVECs), bovine aorta (BAECs), and murine lymph nodes (MLECs) as well as adrenomedullin mRNA expression in HUVECs. CNP mRNA was stimulated prominently in HUVECs under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, sixfold increase compared with that in the static condition; 24 hours, 30-fold increase). Similar results were obtained in BAECs (4 hours, twofold increase; 24 hours, threefold increase) and MLECs (4 hours, threefold increase; 24 hours, 10-fold increase). Augmentation of CNP mRNA expression that was dependent on shear stress intensity was also observed (5 dyne/cm2, 2.5-fold increase of static; 15 dyne/cm2, 4.5-fold increase). Increased CNP secretion was also confirmed by the specific radioimmunoassay for CNP. Adrenomedullin mRNA expression in HUVECs increased under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, 1.2-fold increase of static: 24 hours, threefold increase) and shear stress intensity-dependent manner (15 dyne/cm2, threefold increase compared with that at 5 dyne/cm2). These results suggest that the coordinated augmentation of mRNA expression of these novel EDRPs may constitute shear stress-dependent vasodilator and antiproliferative effects.

Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells. Role of the vascular natriuretic peptide system in vascular growth inhibition.

Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-beta (TGF-beta) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF-beta abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-beta. Furthermore, the conditioned medium from ECs stimulated by TGF-beta was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti-CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs.

Vascular Endothelial Growth Factor Suppresses C-Type Natriuretic Peptide Secretion

Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. Vascular endothelial growth factor (VEGF) is now thought to be an endothelium-specific and potent angiogenic factor. We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from porcine brain, is produced by endothelial cells and proposed that CNP can exert control over vascular tone and growth as a local vascular regulator. In the present study, we examined the effect of VEGF on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP we developed. VEGF (1 to 100 ng/mL) dose-dependently suppressed CNP secretion from cultured bovine endothelial cells, and 100 ng/mL VEGF suppressed endothelial CNP secretion to 28% of control levels (31.7 +/- 5.5 versus 8.9 +/- 0.8 fmol/mL, vehicle versus VEGF). VEGF also suppressed CNP mRNA expression in endothelial cells 9 hours after administration. In contrast, basic fibroblast growth factor (20 ng/mL), an endothelium-nonspecific angiogenic factor, significantly stimulated CNP secretion by 290%. These results indicate that VEGF can regulate vascular tone and growth in the process of angiogenesis through suppression of endothelial secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide.

Shear stress induces expression of CNP gene in human endothelial cells

To elucidate the effect of blood flow on gene transcription of C-type natriuretic peptide (CNP), human umbilical vein endothelial cells were exposed to shear stress in a cone-plate viscometer. Expression of CNP mRNA, evaluated by reverse transcription-polymerase chain reaction, was markedly increased by exposure to shear stress of 24 dyne/cm 2 at 3 h. The CNP mRNA level was maintained until 12 h. Thus, the present study demonstrated for the first time that shear stress induces expression of CNP gene in human endothelial cells.

Localization of C-type natriuretic peptide mRNA in rat hypothalamus.

Central or peripheral administration of C-type natriuretic peptide (CNP) affects numerous neuroendocrine systems, including the hypothalamo-pituitary-gonadal, hypothalamo-pituitary-adrenocortical and hypothalamo-neurohypophysial axes. The present report characterizes the distribution of CNP mRNA in hypothalamus, providing the first definition of CNP-containing neuroendocrine circuits. In situ hybridization histochemical analysis revealed high expression of CNP mRNA in the anteroventral periventricular nucleus (AVPv) and in hypothalamic arcuate nucleus (ARC). Hybridization signals of significantly lower intensity were seen in the medial, median and periventricular preoptic area, the supraoptic, dorsomedial, ventral premammillary and lateral mammillary nuclei and in the posterior hypothalamic area. A few scattered CNP mRNA containing cells were visualized in the medial parvocellular paraventricular nucleus, posterior magnocellular paraventricular nucleus and lateral hypothalamic area. In the AVPv and ARC the pattern of CNP mRNA distribution paralleled that of ANP mRNA. The results indicate a distribution of CNP mRNA associated with key neuroendocrine systems, and underscores the potential importance of this novel natriuretic peptide in neuroendocrine regulation.