C-type Natriuretic Peptide Gene Research Articles

Mechanism of quercetin on the improvement of ovulation disorder and regulation of ovarian CNP/NPR2 in PCOS model rats

To investigate the effects of quercetin on ovulation disorder and expression of androgen receptor (AR) and C-type natriuretic peptide (CNP) / Natriuretic Peptide Receptor 2 (NPR2) in dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) rat model. DHEA was used to construct the PCOS rat model. After intervention with quercetin, metformin, and AR, the estrous cycle, ovarian and uterine weight of rats were measured. The morphological changes of ovarian and uterine were detected by hematoxylin-eosin staining (HE staining). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by Enzyme linked immunosorbent assay (ELISA). Immunohistochemical detection of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), B-cell lymphoma-2 (BCL-2), BCL2-Associated X (Bax) and AR expression in ovarian. Determination of the expression of CNP and NPR2 mRNA by qRT-PCR. Chromatin immunocoprecipitation (ChIP) was used to detect the ability of AR to bind to CNP or NPR2 promoter. The results showed that quercetin could significantly reduce the expression of Testosterone (T) , Estradiol (E2) , LH, Bax, IL-1β, IL-6 and TNF-α, increase the expression of FSH and Bcl-2, inhibit the expression of AR, regulate the expression of CNP / NPR2 gene and protein by affecting the combination of AR with the specific sequence of CNP and NPR2 gene promoters, restore the maturation of oocyte and ovulation. The results suggest that quercetin can alleviate the hormone, metabolic and ovulatory aberrations caused by PCOS, and provide experimental basis for the clinical application of quercetin in PCOS.

Tracking the recruitment and evolution of snake toxins using the evolutionary context provided by the Bothrops jararaca genome

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option.

Integration of C-type natriuretic peptide gene-modified bone marrow mesenchymal stem cells with chitosan/silk fibroin scaffolds as a promising strategy for articular cartilage regeneration.

The treatment of articular cartilage defects has become a major clinical concern. Currently, additional efforts are necessary to develop effective methods to cure this disease. In this work, we combined gene therapy with tissue engineering methods to test their effect on cartilage repair. In in vitro experiments, we obtained C-type natriuretic peptide (CNP) gene-modified bone marrow-derived mesenchymal stem cells (BMSCs) by transfection with recombinant adenovirus containing the CNP gene and revealed that CNP gene-modified BMSCs had good chondrogenic differentiation ability. By the freeze-drying method, we successfully synthesized a chitosan/silk fibroin (CS/SF) porous scaffold, which had a suitable aperture size for chondrogenesis. Then, we loaded CNP gene-modified BMSCs onto CS/SF scaffolds and tested their effect on repairing full-thickness cartilage defects in rat joints. The gross morphology and histology examination results showed that the composite of the CNP gene-modified BMSCs and CS/SF scaffolds had better repair effects than those of the other three groups at each time point. Additionally, compared to the group with BMSCs and scaffolds, we found that there was more cartilage matrix in the CNP gene-modified BMSCs and CS/SF scaffolds group. Data obtained in the present study suggest that the composite of CNP gene-modified BMSCs and CS/SF scaffolds represent promising strategies for repairing focal cartilage lesions.

Dexamethasone increases production of C-type natriuretic peptide in the sheep brain.

Although C-type natriuretic peptide (CNP) has high abundance in brain tissues and cerebrospinal fluid (CSF), the source and possible factors regulating its secretion within the central nervous system (CNS) are unknown. Here we report the dynamic effects of a single IV bolus of dexamethasone or saline solution on plasma, CSF, CNS and pituitary tissue content of CNP products in adult sheep, along with changes in CNP gene expression in selected tissues. Both CNP and NTproCNP (the amino-terminal product of proCNP) in plasma and CSF showed dose-responsive increases lasting 12-16 h after dexamethasone, whereas other natriuretic peptides were unaffected. CNS tissue concentrations of CNP and NTproCNP were increased by dexamethasone in all of the 12 regions examined. Abundance was highest in limbic tissues, pons and medulla oblongata. Relative to controls, CNP gene expression (NPPC) was upregulated by dexamethasone in 5 of 7 brain tissues examined. Patterns of responses differed in pituitary tissue. Whereas the abundance of CNP in both lobes of the pituitary gland greatly exceeded that of brain tissues, neither CNP nor NTproCNP concentration was affected by dexamethasone, despite an increase in NPPC expression. This is the first report of enhanced production and secretion of CNP in brain tissues in response to a corticosteroid. Activation of CNP secretion within CNS tissues by dexamethasone, not exhibited by other natriuretic peptides, suggests an important role for CNP in settings of acute stress. Differential findings in pituitary tissues likely relate to altered processing of proCNP storage and secretion.

Impact of local CNP/GC-B system in growth plates on endochondral bone growth

Background C-type natriuretic peptide (CNP), which exerts its biological actions by binding a subtype of receptor, guanylyl cyclase-B (GC-B), is implicated in the endochondral bone growth and the gene disruption of CNP or GC-B results in marked dwarfism due to the defect of the endochondral bone formation in both animals and humans. Meanwhile, the physiological role of the CNP/ GC-B system in the growth plate cartilage is still unclear, because the CNP/GC-B system is widely distributed not only in the growth plate of the bone but also in the central and peripheral nervous system, gonad, vascular endothelial cells, macrophages and cardiac fibroblasts. Results In order to further elucidate the physiological role of the local CNP/GC-B system in the growth plate cartilage, we have performed targeted depletion of CNP or GC-B gene in the growth plate cartilage, using the Cre-loxP system. The growth of bones formed through the endochondral ossification was severely impaired in cartilage-specific CNP knockout mice, resulting in a prominent short-stature phenotype. The severity of impairment of endochondral bone growth observed in cartilage-specific CNP knockout mice was almost the same as in the systemic CNP knockout mice. The bone phenotype of cartilage specific GC-B knockout mice was essentially the same as that of CNP knockout mice, however, the shortening of bones was severer in cartilage specific GC-B knockout mice than that in cartilage specific CNP knockout mice. Histological examination revealed that the hypertrophic chondrocyte layer of the growth plate was drastically reduced and the thickness of the proliferative chondrocyte layer, along with the proliferation of chondrocytes in that region, was moderately reduced in both cartilage specific knockouts. On pulverized feed, the survival rate of cartilage specific CNP and GC-B knockout mice was comparable to that of systemic CNP knockout mice. Conclusion These results demonstrate that the local CNP/GC-B system in growth plates is responsible for the physiological endochondral bone growth.

High concentration of C-type natriuretic peptide promotes VEGF-dependent vasculogenesis in the remodeled region of infarcted swine heart with preserved left ventricular ejection fraction

Vasculogenesis is a hallmark of myocardial restoration. Post-ischemic late remodeling is associated with pathology and function worsening. At the same time, neo-vasculogenesis helps function improving and requires the release of vascular endothelial growth factor type A (VEGF-A). The vasculogenic role of C-type natriuretic peptide (CNP), a cardiac paracrine hormone, is unknown in infarcted hearts with preserved left ventricular (LV) ejection fraction (EF). We explored whether myocardial VEGF-dependent vasculogenesis is affected by CNP. To this end, infarcted swine hearts were investigated by magnetic resonance imaging (MRI), histological and molecular assays. At the fourth week, MRI showed that transmural myocardial infarction (MI) affected approximately 13% of the LV wall mass without impairing global function (LVEF>50%, n=9). Increased fibrosis, metalloproteases and capillary density were localized to the infarct border zone (BZ), and were associated with increased expression of CNP (p=0.03 vs. remote zone (RZ)), VEGF-A (p<0.001 vs. RZ), BNP, a marker of myocardial dysfunction (p<0.01 vs. RZ) and the endothelial marker, factor VIII-related antigen (p<0.01 vs. RZ). In vitro, CNP 1000 nM promoted VEGF-dependent vasculogenesis without affecting the cell growth and survival, although CNP 100 nM or a high concentration of VEGF-A halted vascular growth. CNP expression is locally increased in infarct remodeled myocardium in the presence of dense capillary network. The vasculogenic response requires the co-exposure to high concentration of CNP and VEGF-A. Our data will be helpful to develop combined myocardial delivery of CNP and VEGF-A genes in order to reverse the remodeling process.

Plasma C‐type natriuretic peptide forms and thyroid status in prepubertal children with acquired thyroid disease

C-type natriuretic peptide (CNP) and thyroid hormone (TH) are essential for normal skeletal growth. Plasma CNP peptides correlate with growth velocity, but the relationship between thyroid status and CNP production is unknown. This study examined the impact of restoring normal TH levels on CNP and height velocity (HV) in children with acquired hypo- and hyperthyroidism. We performed a prospective, observational study in prepubertal children with acquired hypothyroidism (n = 15) and hyperthyroidism (n = 12). Blood levels of CNP, amino-terminal proCNP (NTproCNP), bone-specific alkaline phosphatase (BSAP), IGF-I and TH levels were measured before and during the first 6 months of standard treatment for hypo- and hyperthyroidism, and correlations were determined. At baseline, HV, CNP, NTproCNP and BSAP were significantly higher in hyper- than in hypothyroid subjects. Changes in TH after treatment were closely coupled to change in CNP and NTproCNP in hyperthyroid, but not in hypothyroid, children. In addition, a positive association of HV with CNP peptides was found during treatment of hyperthyroidism. Normalizing TH did not correlate with changes in BSAP or IGF-I in either group. Plasma CNP peptides are higher in children with hyperthyroidism than in those with hypothyroidism at diagnosis and, in hyperthyroid children, change concordantly with TH and HV during treatment. Differential responses of CNP in the two groups suggest CNP production is dependent on growth plate activity and not a direct effect of TH on CNP gene expression. Our findings suggest novel mechanisms underlying changes in skeletal response during treatment in children with acquired thyroid disease.

The trophoblast binucleate cell is the source of maternal circulating C-type natriuretic peptide during ovine pregnancy

Maternal plasma concentrations of C-type natriuretic peptide (CNP) and a co-secreted bioinactive amino-terminal fragment (NTproCNP) are elevated during ovine pregnancy. Although the uteroplacental unit has been implicated as a likely source of CNP, the relative contributions of specific uterine and placental tissues, and identity of the cellular site/s of production remain unknown. Therefore, we measured CNP and NTproCNP in intercaruncular uterine tissue and maternal (caruncle) and fetal (cotyledon) placental tissues throughout gestation. Concentrations of CNP forms in placental tissues greatly exceeded those in intercaruncular uterine tissue throughout pregnancy (P < 0.05). Mean caruncular concentrations (CNP 32 ± 4, NTproCNP 56 ± 6 pmol g−1) peaked at day 60 whereas in the cotyledon there was a progressive increase in CNP forms to peak values (CNP 66 ± 6, NTproCNP 134 ± 9 pmol g−1) at day 100–135 followed by a sharp decline just prior to term (day 143). At term CNP gene expression was 6-fold greater in placental tissue compared with intercaruncular uterine tissue. Changes in maternal plasma concentration of CNP forms closely followed those in cotyledonary tissue whereas fetal plasma levels fell progressively throughout gestation. Immunohistochemistry revealed staining in binucleate cells (BNC) and around placental blood vessels.CNP’s localization to the BNC suggests a novel endocrine role during pregnancy, in addition to its paracrine actions within the placental vasculature. The function of CNP in maternal circulation remains to be determined, but as proposed for other BNC products, may involve manipulation of maternal physiology and placental function to favour fetal growth.

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

Central and peripheral forms of C-type natriuretic peptide (CNP): Evidence for differential regulation in plasma and cerebrospinal fluid

Aminoterminal proCNP (NTproCNP), a stable product of CNP gene expression and readily measured in human plasma, provides a new approach to studies of CNP which is rapidly degraded at source. CNP is detectable in human CSF but the presence and proportions of NTproCNP in CSF are unknown. Since CNP is widely expressed throughout the CNS, we hypothesized that the ratio of NTproCNP to CNP in CSF is greatly increased when compared to plasma and that CSF CNP peptides may contribute to their concentrations in the systemic circulation. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anesthesia for arranged orthopedic procedures. Elevated concentrations of NTproCNP (1045 ± 359 pmol/L), characterized by HPLC-RIA, were found in CSF and greatly exceeded those of CNP (7.9 ± 3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145 ± 55) was much higher than in plasma (31 ± 27). A significant inverse relation was found between plasma and CSF CNP concentrations ( r = −0.29, p < 0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. We conclude that CNP is differentially regulated across the brain in normal health. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in the diagnosis and monitoring of subjects with central neural disorders.

The role of C-type natriuretic peptide in rat testes during spermatogenesis

C-type natriuretic peptide (CNP) is a 22-amino acid peptide and act as a local paracrine or autocrine regulator. There is growing evidence that CNP is involved in male reproductive processes. To investigate the role of CNP during spermatogenesis, we measured the mRNA expression of CNP and its specific membrane-bound natriuretic peptide receptor-B (NPR-B) using real-time RT-PCR in the testes of normal rats on different postnatal days. After that spermatogenesis dysfunction model induced by ornidazole was established with the aim to study the correlation of CNP with spermatogenic dysfunction. Then, Sertoli cells from 18- to 22-day-old healthy male rats were cultured in the presence of different CNP concentrations (1×10(-6), 1×10(-7) and 1×10(-8) mol l(-1)), and the mRNA expression levels of androgen-binding protein, inhibin B and transferrin were examined at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. During the postnatal development of rat testes, the highest mRNA expression levels of CNP and NPR-B were found at postnatal D(0), and the levels then declined gradually, with a second CNP peak at postnatal D(35). In the ornidazole-induced infertile rat testes, CNP gene expression was lower than in the uninduced rats (P<0.05), while NPR-B gene expression was greater (P<0.05). In cultured Sertoli cells, supplementation with CNP stimulated the gene expression of androgen-binding protein/inhibin B/transferrin, particularly at 12 h, and 1×10(-7) mol l(-1) CNP had the highest upregulation effect. The gene expression levels of CNP/NPR-B in rat testes at different postnatal stages and in infertile rat testes indicated that CNP may participate in the physiology and/or pathology related to spermatogenesis. Moreover, CNP regulated endocrine function in Sertoli cells. Taken together, these results showed that CNP is closely tied to spermatogenesis.

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, αT3-1 and LβT2 gonadotroph cells

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

C‐type natriuretic peptide in prostate cancer

C-type natriuretic peptide (CNP) is expressed in the male reproductive organs in pigs. To examine whether the human prostate also expresses the CNP gene, we measured CNP and N-terminal proCNP in prostate cancer tissue extracts and performed immunohistochemical biopsy staining. Additionally, proCNP-derived peptides were quantitated in plasma from patients with prostate cancer. Blood was collected from healthy controls and patients before surgery for localized prostate cancer. Tissue extracts were prepared from tissue biopsies obtained from radical prostatectomy surgery. N-terminal proCNP, proCNP (1-50) and CNP were measured in plasma and tissue extracts. Biopsies were stained for CNP-22 and N-terminal proCNP. Tissue extracts from human prostate cancer contained mostly N-terminal proCNP [median 5.3 pmol/g tissue (range 1.0-12.9)] and less CNP [0.14 pmol/g tissue (0.01-1.34)]. Immunohistochemistry demonstrated the presence of the peptides in prostatic epithelial cells. The N-terminal proCNP concentrations in plasma were marginally lower in patients with localized prostate cancer compared with control subjects [13.8 pmol/l (11.0-17.2) vs. 15.1 pmol/l (10.4-23.2), p=0.002] but not enough to justify the use of N-terminal proCNP as a cancer marker. Further research is needed to establish whether measurement of proCNP-derived peptides may offer clinical information.

C‐Type Natriuretic Peptide is a Schwann Cell‐Derived Factor For Development and Function of Sensory Neurones

Cyclic GMP (cGMP) is known to play important roles for neuronal development and neurite pathfinding. However, the regulatory mechanism that governs the synthesis of cGMP in the nervous system is not well defined. In the present study, we examined the role of C-type natriuretic peptide (CNP), which increases intracellular cGMP upon binding to its receptor, guanylyl cyclase (GC)-B, in the peripheral nervous system. Immunohistochemistry revealed that CNP is demonstrated in Schwann cells, whereas GC-B mRNA is highly expressed in dorsal root ganglion (DRG) neurones. In cultured DRG neurones, GC-B was demonstrated in dendrites of TrkA-positive cells, where it co-exists with cGMP-dependent protein kinase I (cGKI), the major intracellular mediator of cGMP actions. Addition of CNP in the culture medium increased the density of fine neurites, which was accompanied by the increase in phosphorylation of vasodilator-stimulated phosphoprotein, a cGKI substrate. Furthermore, in mice deficient for the CNP gene (CNP-KO), the numbers of TrkA-positive DRG neurones were diminished. Likewise, there were much less cGKI-positive neurones in DRG and cGKI-positive fibres in the dorsal spinal cord of CNP-KO than wild-type mice. Finally, the bone deformity-rescued CNP-KO mice displayed a decreased response to formalin-induced pain compared to wild-type. Taken together, these results suggest that CNP is derived from Schwann cells and plays an important role for the development and function of nociceptive sensory neurones.

Expression of C-type natriuretic peptide and of its receptor NPR-B in normal and failing heart

C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n = 5) and with pacing-induced HF ( n = 5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9 ± 10.4 pg/ml vs.16.7 ± 1.1, p = 0.013, mean ± S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7 ± 1.9 pg/mg protein; LA: 8.7 ± 3.8; RV: 1.07 ± 0.33; LV: 0.93 ± 0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8 ± 9.9 pg/mg protein vs. 0.9 ± 0.17, p = 0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab−/− mice

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality ( lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP lbab was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP lbab to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP lbab than wild-type CNP was required to compete with [ 125I][Tyr 0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab −/− mice.

Inhibition of Restenosis Formation Without Compromising Reendothelialization as a Potential Solution to Thrombosis Following Angioplasty?

Stent thrombosis remains an important problem after the implantation of different stent types. A potential solution to this problem may be vasoactive agents with dual effects on different cell types like C-type natriuretic peptide (CNP). Therefore, in vitro and in vivo effects of CNP were investigated in a porcine restenotic model. Gene transfer of CNP in cultures of porcine vascular cells revealed up to 30% reduction of growth of smooth muscle cells (p<.05), but no suppression of endothelial growth using CNP. Applied in vivo, angiography revealed a trend of reduced restenosis formation in balloon-injured porcine arteries treated with CNP gene or beta-galactosidase (beta-Gal) control gene after three months (2.59 +/- 2.04-fold reduction, p = n.s.). Histologically, morphometry revealed significantly reduced neointima formation after treatment with CNP plasmid (7.26 +/- 1.44-fold reduction, p < .05). Evans blue staining demonstrated complete endothelial repair already 3 weeks after intervention using CNP. Transfer of CNP gene resulted in a significant inhibition of neointima formation without compromising endothelial repair. Therefore, use of the CNP gene may offer a solution to suppress restenosis formation while preventing subacute or late thrombosis.

In vitro effects of homologous natriuretic peptides on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus

C-type natriuretic peptide (CNP) cDNA was cloned from the tilapia brain and its inferred mature sequence was chemically synthesized together with previously cloned tilapia A-type and B-type natriuretic peptides (ANP and BNP). The cloned CNP belongs to the CNP-1 type of teleosts. Reverse-transcription polymerase chain reaction showed that the ANP and BNP genes were hardly expressed in the tilapia brain and pituitary, whereas the CNP gene was expressed strongly in the brain and slightly in the pituitary. Effects of homologous natriuretic peptides (100 nM each) on growth hormone (GH) and prolactin (PRL) release were examined using dispersed tilapia pituitary cells. Tilapia ANP and BNP stimulated GH and PRL release during 4–8, and 8–24 h of incubation. BNP appeared to be more potent than ANP, also stimulating GH and PRL release during 0–4 h of incubation. CNP stimulated GH release only during 4–8 h of incubation; CNP was without effect on PRL release. All three NPs stimulated GH and PRL mRNA expression in dispersed pituitary cells following 24 h of incubation. ANP and BNP significantly elevated intracellular cGMP accumulation in dispersed pituitary cells after 15 min of exposure, whereas no effect of CNP was observed. These results indicate a long-lasting stimulation of GH and PRL release by ANP and BNP that is mediated, at least in part, by the guanylyl cyclase-linked NP receptor.

C-Type Natriuretic Peptide Inhibits Constrictive Remodeling Without Compromising Re-Endothelialization in Balloon-Dilated Renal Arteries

To investigate the long-term effect of local, liposome-mediated gene transfer of C-type natriuretic peptide (CNP) plasmid versus CNP protein on restenosis in porcine renal arteries following balloon angioplasty. The renal arteries of 15 pigs were dilated and the adventitia at the site of balloon injury injected with CNP protein, pCR3.1 plasmid encoding CNP, or the beta-galactosidase gene (control) via a needle injection catheter. Five animals receiving the CNP and control genes in dilated arteries were sacrificed after 3 weeks to analyze re-endothelialization, proliferation, and early CNP expression. Ten animals designated for the long-term experiments (3 months) were treated with the CNP gene versus CNP protein (n=3), the CNP gene versus the control gene (n=3), and the CNP protein versus the control gene (n=3). One animal served as a dilated non-treated control. Transfection and expression of CNP and beta-galactosidase were measured by polymerase chain reaction (PCR) and reverse transcription PCR. Renal arterial lumen narrowing was measured with angiography and histology. Endothelialization was assessed using Evans blue stain; vWF, CD31, factor VIII, and Ki67 were markers for immunohistochemical analysis. An intact endothelial layer was seen at 3 weeks following angioplasty in all transfected arteries. Three months following treatment, computer-assisted morphometric analysis revealed significant enlargement of the arterial cross-sectional areas in CNP plasmid- treated vessels compared to dilated but untreated arteries (CNP plasmid +34.8%+/-13.9% versus CNP protein -1.75%+/-19.9% versus beta-galactosidase -47.0%+/-13.9%, p<0.01). Angiographic analysis showed significant enlargement of the arterial diameter compared to dilated, untreated arteries (CNP plasmid +20.8%+/-6.8% versus CNP protein +5.7%+/-6.0% versus beta-galactosidase -24.5%+/-10.2%, p<0.01). Local application of CNP plasmid proved superior to CNP protein in producing rapid re-endothelialization and significantly enlarging the renal arterial lumen following dilation.

CNP gene expression is activated by Wnt signaling and correlates with Wnt4 expression during renal injury.

C-type natriuretic peptide (CNP) regulates salt excretion, vascular tone, and fibroblast proliferation and activation. CNP inhibits fibroblast activation in vitro and fibrosis in vivo, but endogenous CNP gene (Nppc) expression during tissue fibrosis has not been reported. We determined that Nppc is induced in renal tubular epithelia and then in interstitial myofibroblasts after unilateral ureteral obstruction (UUO). Induction of Nppc occurred in identical cell populations to those in which Wnt4 is induced after renal injury. In addition, Nppc was activated in Wnt4-expressing cells during nephrogenesis. Wnt signaling components beta-catenin and T cell factor/lymphoid enhancer binding factor (TCF/LEF) specifically bound to cognate elements in the Nppc proximal promoter. Wnt-4, beta-catenin, and LEF-1 activated an Nppc transgene in cultured cells, and transgene activation by Wnt-4 and LEF-1 was dependent on the presence of intact cognate elements. These findings suggest that Wnt-4 stimulates Nppc in a TCF/LEF-dependent manner after renal injury and thus may contribute to limiting renal fibrosis.