- Email: [email protected]
Expression and localization of C-type natriuretic peptide in human vascular smooth muscle cells
Vascular Pharmacology 45 (2006) 368 – 373 www.elsevier.com/locate/vph
Expression and localization of C-type natriuretic peptide in human vascular smooth muscle cells Christopher J. Kelsall a,⁎, Adrian H. Chester a , Padmini Sarathchandra a , Donald R.J. Singer b a
b
Heart Science Centre, Harefield Hospital, Harefield, Middlesex, UK Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry, UK Received 3 March 2006; received in revised form 8 May 2006; accepted 2 June 2006
Abstract Objectives: C-type natriuretic peptide (CNP) released by vascular endothelium relaxes smooth muscle and is important in the maintenance of vascular tone. Since it is not known whether other human vascular cell types produce CNP, we investigated its expression in human vascular smooth muscle. Methods: CNP expression was examined by RT-PCR in vascular smooth muscle cells (SMC) cultured from human saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA), and CNP protein was probed using immunostaining, in tissue sections and in SMCs cultured from these vessels, respectively. Results: PCR for CNP produced a 334 bp product in all SMC cultures, as expressed in endothelial cells, although the band intensity was markedly less in SMCs. Myocardium from CNP-knockout mouse did not express CNP, while there was expression in wild-type mouse. CNP protein was detected by immunostaining in 100% of SMC cultures. By immunostaining of tissue sections, CNP was detected throughout the medial layer, but not adventitia, of all vessel types. Conclusions: Expression of CNP at gene and protein level by human vascular SMCs suggests that CNP may have the capacity to regulate vascular tone independently of the endothelium. © 2006 Published by Elsevier Inc. Keywords: C-type natriuretic peptide; Vascular smooth muscle; Gene expression; Immunostaining
1. Introduction The natriuretic peptide family includes atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide (CNP). This family of peptides is considered to have an important role in countering the influences of the renin–angiotensin system and in regulation of vascular tone. The natriuretic peptides bring about their biological actions via guanylate cyclase (GC)-coupled, membrane-spanning natriuretic peptide receptors (NPR) on the cell surface. Vascular smooth muscle cells express predominantly NPRB receptors, which have a very high affinity for CNP. Through activation of GC, CNP brings about an increase in intracellular cGMP, which mediates relaxation of ⁎ Corresponding author. Pharmacology, Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK. Tel.: +44 1895 828 508; fax: +44 1895 828 900. E-mail address: [email protected] (C.J. Kelsall). 1537-1891/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.vph.2006.06.011
vascular smooth muscle and inhibition of smooth muscle cell proliferation. A paracrine route for these effects of CNP from endothelium to vascular smooth muscle has been established. CNP expression in endothelial cells was first demonstrated by Suga et al. (1992). Since then, endothelial expression and release of CNP has been shown in a variety of studies on bovine endothelial cells (Doi et al., 1996; Komatsu et al., 1996; Chun et al., 2000). CNP expression has also been detected in diverse cell types such as cardiac fibroblasts, chondrocytes, glomerular cells, glial cells and macrophages in humans and in animal models (Horio et al., 2003; Suda et al., 2002; Middendorff et al., 1997; Yeung et al., 1996; Vollmar and Schulz, 1995), which implies that CNP has non-vascular roles. For example, CNP may be important in normal skeletal development (Chusho et al., 2001). However, the most significant source of CNP is considered to be the endothelium, as part of the complex array of factors that regulate vascular tone. A current view is that CNP is released by the
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
endothelium in an ablumenal direction to bring about relaxation of vascular smooth muscle, in opposition to contractile influences, such as those of the renin angiotensin system, endothelin and vasopressin. However, the potential for other cells within the vascular wall to serve as a source of CNP has not been adequately explored. One study has reported CNP expression of RNA and protein in cultured rat cerebral vascular and aortic smooth muscle cells (Woodard et al., 2002); in contrast, in intact rat carotid artery denuded of endothelium, Brown et al. (1997) failed to detect CNP immunoreactivity in the medial layer (predominantly comprising smooth muscle cells). Thus, it remains unclear as to whether the expression of CNP in cultured cells is a phenomenon seen in intact vessels and, importantly, if this effect is seen in man. The possibility that CNP could be involved in physiological maintenance of vascular tone, and other biologically important actions, independently of the endothelium in humans, has not been adequately explored. The demonstration of expression of CNP in human vascular smooth muscle cells, in vitro and in situ, would provide evidence for a pathophysiologically relevant autocrine pathway for CNP activity in the absence of a functional or intact endothelium. Therefore, the aims of this study were to determine whether CNP is expressed in human vascular smooth muscle and to detect the post-translational biologically active form of CNP in these cells. 2. Methods 2.1. Vascular tissue This study conforms to the principles outlined in the Declaration of Helsinki. Local ethical committee approval was given for use of human tissue. All tissue was obtained from coronary artery bypass patients whom had given written informed consent. Saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA) were collected in sterile Hank's solution containing 25 mM HEPES buffer and dissected free of surrounding tissue. Specimens designated for RNA extraction (3–5 mm long) were immersed in RNA Later (Sigma) and stored at − 80 °C. Other specimens (3–4 mm long) were mounted with Cryo-M-Bed (Bright Instrument Co., Huntingdon, Cambs, UK) on cork, snap-frozen and stored in liquid nitrogen for sectioning by cryostat. Specimens used for explanting were used within 24 h of collection. 2.2. Explants Specimens of vascular tissue were prepared for explanting by removal of endothelium and adventitia. The remaining medial layer was cut up into 2–3 mm pieces and laid out in a 6-well plate containing sterile growth medium (DMEM containing L-glutamine, penicillin [100 U/ml]/streptomycin [100 μg/ml] and 15% fetal calf serum), to allow smooth muscle cell migration and proliferation. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air and supplied with fresh medium every 3–4 days. Upon reaching confluence, cells were removed by trypsin/EDTA solution (Sigma) and seeded into tissue culture
369
flasks for propagation. Cultured human smooth muscle cells from SV, IMA or RA exhibited the characteristic ‘hill and valley’ morphology at confluence, under the microscope. Vascular smooth muscle cells were 100% pure and free of endothelial cells as assessed by positive immunostaining for smooth muscle α-actin and negatively for CD31, an endothelial cell marker. Cells used were passages 3–8. Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase treatment and propagated in flasks. Endothelial cells were grown in M199 containing 10% FCS, Lglutamine (2 mmol/l), penicillin (100 μg/ml)/streptomycin (100 U/ml), heparin (200 U/ml) and ECGS (all Sigma). HUVEC cultures grown in this way were 100% pure as assessed by anti-CD31 and anti-smooth muscle α-actin immunostaining. Cells used were passages 3–8. 2.3. RT-PCR Total RNA was extracted from tissue sections or from cultured cells using RNeasy extraction columns (Qiagen, Crawley, UK) according to manufacturer's instructions. Extracted RNA was eluted in 40 μl RNase-free water and stored at −80 °C. cDNA was synthesized by combination of 3 μl of eluted RNA with 1μl random decamer (TAG Newcastle, UK) per sample, heated at 70 °C for 10 min. The samples were cooled in a methanol-ice bath. A master mix containing (per sample) 3 μl 1st Strand Buffer (5×), 1.5 μl DTT (0.1 M), 1.5 μl dNTPs (10 mM each), 0.75 μl Superscript II (2000U/μl) and 0.75μl RNA Guard (31, 400U/μl) (all Invitrogen) was made and 7.5 μl of the mix was added to each sample. The cDNAs were heated at 37 °C for 1 h, then at 95 °C for 5 min. cDNAs were stored at − 80 °C. PCR reactions were carried out with 1 μl of cDNA in a total volume of 50 μl per reaction. The reaction mix for each sample contained (final concentration) Q-solution (× 1; Invitrogen), 10% glycerol, PCR buffer containing 15 mM MgCl2 (× 1; Invitrogen), dNTPs (0.2 mM each), primer pairs for C-type natriuretic peptide or α-3-actin (50 pmol/l) and Hotstar Taq polymerase (0.03 U/μl; Invitrogen). For CNP, a ‘nested’ PCR protocol was used, in which the PCR reaction mix was the same for stage 2 with the exception of the CNP primer sequence and that cDNA was substituted with stage 1 PCR product (diluted 1:20 with RNase-free water). A drop of mineral oil was added to each reaction tube, and the following conditions were applied: CNP-denatured at 95 °C for 15 min, 58 °C annealing temperature, 40 cycles (35 cycles for stage 2), extended at 72 °C for 30 s; α-3-actin-denaturing as for CNP, 52 °C annealing temperature, 35 cycles, extended at 72 °C for 30 s. All primers were synthesized by TAG Newcastle (Newcastle, UK); the CNP primers were used in a two-stage ‘nested’ PCR protocol, defined as generating (at end stage) a PCR product derived from a reaction using a PCR product as the template. The primers for the second stage of the reaction encompass the positions of the primer pair used in the first stage. Human CNP: sense primer 5′CTG CTC ACG CTG CTC TCC-3′, stage-1 antisense primer 5′-GTC GGG TGG GCC GTA CTC-3′. The expected stage 1 product size was 388 bp in length. Stage-2 antisense primer 5′CGC TCA TGG AGC CGA TTC-3′. The expected stage 2
370
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
Fig. 1. Gene expression of CNP in cultured vascular SMC derived from saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA). Panel A: comparison of CNP expression in EC (HUVEC) and SMC. Panel B: CNP expression in EC and two SMC cultures vs. acidic riboprotein (ARP) expression. Panel C: CNP and α-3-actin expression in wild type (WT) vs. CNP-knockout (KO) mouse myocardium.
product size was 334 bp in length. Human α-3-actin: sense primer 5′-GGC TCC ATC CTG GCC TCT-3′, antisense primer 5′-AAA AAC ACA TAG GTA ACG AGT-3′. The expected product size was 223 bp in length. Human acidic riboprotein: sense primer 5′ -CGA CCT GGA AGT CCA ACT AC-3′, antisense primer 5′-CGG GCA GAT GCA GCA GAT-3′. The expected product size was 110 bp in length. The ‘nested’ PCR procedure for CNP was validated using positive and negative control samples (myocardial RNA from wild-type and CNP-KO mice, respectively), which were gifted
by Dr. Hideki Chusho, Kyoto University, Japan; the primer sequences were modified, as the primers for human CNP were not able to amplify mouse CNP. Mouse CNP: sense primer 5′TGC TGC TCG CGC TAC TCT CG-3′, antisense primer 5′CGC TCA TGG AGC CGA TCC-3′. The expected product size was 332 bp in length. PCR products were run on a 1% agarose gel containing ethidium bromide and visualized under UV illumination. Product size was measured against size markers from 1 kb DNA Ladder (Invitrogen, Paisley, UK).
Fig. 2. Immunoperoxidase staining profiles in transverse sections of human SV, IMA and RA, respectively; (A) rabbit anti-human CNP, (B) negative control (no primary antibody). L indicates vessel lumen, M indicates medial layer. Magnification: all panels ×180. Blue staining (DAPI: 4′,6-diamidino-2-phenylindole), most notable in control sections, indicates cell nuclei.
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
2.4. Immunostaining of tissue sections Sections of human SV, IMA and RA (5 μm thick), mounted on glass slides, were washed and then fixed in ice-cold acetone for 10 min. To inhibit endogenous peroxidases, the slides were incubated with peroxidase block (DAKO) for 5 min, then washed in PBS. Background staining was blocked by incubation with diluted normal goat serum (1:30), in 1% BSA in PBS, for 30 min. Slides were then blotted and incubated with rabbit anti-human CNP antibody (1:400 in 1% BSA in PBS) for 1 h, washed in PBS, then incubated with biotinylated goat antirabbit IgG (1:200 in 1% BSA in PBS) for 1 h. The slides were washed again and incubated with ABC Complex for 1 h. Subsequently, sections were washed in PBS and immersed in 3,3′diaminobenzidine (DAB; Sigma) substrate, counterstained with Mayer's haematoxylin, dehydrated, cleared and mounted in DPX (distrene, dibutyl phthalate, xylene). These procedures were carried out at room temperature. Control slides were stained with anti-SM α-actin or without primary antibody. 2.5. Immunostaining of cultured cells Vascular smooth muscle cells (VSMC) cultivated from human saphenous vein, internal mammary artery and radial artery were propagated to around 80% confluence on glass coverslips, in 6well plates. Cells were washed in PBS and fixed in ice-cold acetone for 5 min, while antibodies were prepared in PBS containing 1% BSA to prevent non-specific binding. Cells were washed again and incubated with anti-human smooth muscle α-
371
actin (Sigma), anti-human CD31 (DAKO) or anti-human C-type natriuretic peptide (Peninsula) for 1 h. Residual antibody was washed off in PBS and the cells were exposed to Alexa Fluor secondary antibodies (also with 1% BSA) (Molecular Probes) for 1 h. The antibody for cells previously exposed to anti-human αsmooth muscle actin also included Phalloidin Green (Molecular Probes) to stain actin non-specifically. The secondary antibodies were washed off in PBS and coverslips were mounted on slides with Permafluor (Immunatech, France). These procedures were carried out at room temperature. Some coverslips were treated without primary antibody to assess non-specific binding of fluorescent secondary antibodies. Slides were visualized under a fluorescence microscope at wavelengths of 594 nm (red) or 488 nm (green) to detect cell staining. 3. Results 3.1. Expression of CNP by PCR Using primers designed to amplify a 340 bp length of the gene sequence coding for C-type natriuretic peptide in humans, CNP was found to be expressed in VSMC cultured from human saphenous vein, internal mammary artery and radial artery, with a product size of between 300 and 400 bp, as compared against 1 kb DNA ladder (Fig. 1). Due to low signal expression obtained by a single PCR protocol, a nested protocol was used to reliably detect CNP in VSMC. The protocol was validated by use of wild-type and CNP-knockout mouse myocardium extracts; the wild-type
Fig. 3. Immunofluorescent staining. EC (HUVEC) and vascular SMCs derived from RA. Left panels: smooth muscle α-actin staining (upper) and CD31 staining (lower) in SMCs; centre panels: positive CNP staining (upper) and CNP negative control (no primary antibody) in SMCs; right panels: CD31 staining (upper) and CNP staining (lower) in HUVECs. Magnification in SMCs ×720 and in HUVECs ×1440.
372
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
showed positive expression from a nested protocol, while the CNP-knockout was negative (Fig. 1). In contrast, both extracts expressed α-3-actin. 3.2. Detection of CNP by immunostaining in vascular sections Sections of SV, IMA and RA (n = 3 for each), which were sectioned and stained for CNP, showed intense positive staining in endothelium and throughout the medial (smooth muscle) layer, although the smooth muscle staining was less intense than in the endothelium. Medial staining was not diffuse, but appeared in banded patterns (Fig. 2). The location and pattern of staining are consistent with it being correlated with the smooth muscle layer. CNP staining was also apparent in the vasa vasorum. The negative control sections showed no comparable staining. 3.3. Detection of CNP by immunostaining in cultured cells HUVECs, which stained positively for CD31 and negatively for smooth muscle α-actin, were positively stained for CNP, and were regarded as a positive control (Fig. 3). Vascular smooth muscle cells derived from human SV, IMA and RA were all positively stained for CNP, having been established as smooth muscle cells by positive staining for smooth muscle α-actin and negative staining for CD31 (Fig. 3). The intensity of CNP staining varied from one culture to another, but 100% of cells were positively stained. 4. Discussion The current work represents a novel investigation of CNP expression in human vascular smooth muscle. The major findings of this study are that the gene for C-type natriuretic peptide is present within the smooth muscle of the intact vascular wall in humans, and that CNP protein is constitutively expressed in these cells. The determination of expression of CNP in vascular tissue by nested PCR, with intron-spanning primers, is validated by the use of positive and negative controls, in the form of mouse wildtype and CNP-knockout myocardium; since the wild-type expressed CNP but the CNP-knockout did not, the knockout mouse clearly represents a negative control for the PCR reaction. While our PCR demonstrates CNP gene expression in smooth muscle, it does not signify protein synthesis. However, CNPpositive staining of isolated cultures of VSMC demonstrates that CNP protein is present in these cells; although positive staining in vascular sections could be due to sequestration or cell surface binding of CNP released from the endothelium. However, the presence of CNP staining in cultured cell populations in which there are no endothelial cells rules out this explanation, and supports the interpretation that human vascular smooth muscle cells synthesize CNP endogenously. It has been shown previously (Casco et al., 2002; Naruko et al., 2005) that CNP is expressed in tissue sections from atherosclerotic human arteries, similarly to staining of vascular sections by immunostaining in the current study. However, these studies did not establish conclusively the cellular source of the CNP; tissue sections are not guaranteed to be free of endothelium, which may act as a
source for the dissemination of CNP throughout the vascular section, from the vessel surface and from arterioles within the vessel wall. In our studies, staining of vascular sections was complemented by analysis of pure cultures of smooth muscle cells. We have attempted to measure the release of CNP peptide from both intact vessel segments (denuded of endothelium) and cultured vascular smooth muscle cells. However, in our experiments, levels were consistently indistinguishable from the detection limit of the radioimmunoassay used (data not shown). This may be explained by the potential for autosequestration of any CNP released by smooth muscle cells via the NPRC receptor population. Alternatively, more sensitive detection methods (e.g. mass spectrometry) may provide data in humans that reflect the results of measured CNP release from rat VSMC (Woodard et al., 2002). The levels of CNP reported to be released by VSMC in that study were consistently very low, and were derived from substantial numbers of cultured cells (control measurements of <2 pg CNP/106 cells). A limitation of our experiments is that it is difficult to grow such substantial numbers (at low passage) of VSMC cultured from human blood vessels, since typically the age and ill health of the patients from which the specimens are derived tends to render these cells senescent after relatively few passages (Ruiz-Torres et al., 1999). While CNP is traditionally considered as a candidate for counteracting the effects of the renin–angiotensin system on vascular tone under normal conditions, CNP may also fulfill an important role in counteracting the effects of endothelin, a potent vasoconstrictor, in cardiovascular disease. Endothelin is implicated in exacerbating the degradation of vascular function pathophysiologically (Ihling et al., 2004), it was originally discovered in endothelial cells (Yanagisawa et al., 1988), and later confirmed to be present in vascular smooth muscle (Bousette and Giaid, 2003), as CNP has been in this study. Natriuretic peptides have been shown to inhibit release of endothelin from endothelial cells (Kohno et al., 1992) and cardiac non-myocytes (Tokudome et al., 2004). Furthermore, CNP is a recognized vascular relaxing factor (Protter et al., 1996; Wiley and Davenport, 2001). Therefore, the presence of CNP in smooth muscle, and its presumed paracrine action, may serve to protect, at least in part, the vessel wall from damage caused by endothelin and other powerful local vasoconstrictors and cell growth-promoting agents. The presence of intact machinery for CNP to exert its biological effects is potentially very important. It has been demonstrated in a rabbit carotid artery balloon injury model that increased levels of CNP in the arterial wall (by adenoviral transfection) substantially reduce neointimal plaque formation (Qian et al., 2002). Transfection of CNP, again in rabbits, also demonstrated increased re-endothelialization of denuded artery, as well as suppression of thrombosis and neointimal hyperplasia (Ohno et al., 2002), all of which are consequences of chronic vascular disease. These results are supported in rats by the application of CNP via intravenous infusion (Takeuchi et al., 2003). Therefore, data that support the notion of a CNP delivery system that remains intact in the presence of a dysfunctional endothelium offer hope for improved long-term therapy for a range of vascular diseases in the future.
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
In conclusion, our findings show that CNP is expressed by human vascular smooth muscle at both gene and peptide level. This provides a potential alternative pathway by which local endogenous CNP may remain available to modulate development of vascular pathology despite impaired endothelial function. Acknowledgments This work was supported by the British Heart Foundation (PG 2001058) and the Magdi Yacoub Institute. We are grateful to the surgeons of Harefield Hospital Cardiac Theatre for provision of human vascular specimens. The mouse myocardial RNA was a kind gift from Dr. Hideki Chusho of Kyoto University, Japan. References Bousette, N., Giaid, A., 2003. Endothelin-1 in atherosclerosis and other vasculopathies. Can. J. Physiol. Pharmacol. 81, 578–587. Brown, J., Chen, Q., Hong, G., 1997. An autocrine system for C-type natriuretic peptide within rat carotid neointima during arterial repair. Am. J. Physiol. 272, H2919–H2931. Casco, V.H., Veinot, J.P., de Bold, M.L., Masters, R.G., Stevenson, M.M., de Bold, A.J., 2002. Natriuretic peptide system gene expression in human coronary arteries. J. Histochem. Cytochem. 50, 799–809. Chun, T.H., Itoh, H., Saito, T., Yamahara, K., Doi, K., Mori, Y., Ogawa, Y., Yamashita, J., Tanaka, T., Inoue, M., Masatsugu, K., Sawada, N., Fukunaga, Y., Nakao, K., 2000. Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin. J. Hypertens. 18, 575–580. Chusho, H., Tamura, N., Ogawa, Y., Yasoda, A., Suda, M., Miyazawa, T., Nakamura, K., Nakao, K., Kurihara, T., Komatsu, Y., Itoh, H., Tanaka, K., Saito, Y., Katsuki, M., Nakao, K., 2001. Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc. Natl. Acad. Sci. U. S. A. 98, 4016–4021. Doi, K., Itoh, H., Komatsu, Y., Igaki, T., Chun, T.H., Takaya, K., Yamashita, J., Inoue, M., Yoshimasa, T., Nakao, K., 1996. Vascular endothelial growth factor suppresses C-type natriuretic peptide secretion. Hypertens 27, 811–815. Horio, T., Tokudome, T., Maki, T., Yoshihara, F., Suga, S., Nishikimi, T., Kojima, M., Kawano, Y., Kangawa, K., 2003. Gene expression, secretion, and autocrine action of C-type natriuretic peptide in cultured adult rat cardiac fibroblasts. Endocrinology 144, 2279–2284. Ihling, C., Bohrmann, B., Schaefer, H.E., Technau-Ihling, K., Loeffler, B.M., 2004. Endothelin-1 and endothelin converting enzyme-1 in human atherosclerosis- novel targets for pharmacotherapy in atherosclerosis. Curr. Vasc. Pharmacol. 2, 249–258. Kohno, M., Yokokawa, K., Horio, T., Yasunari, K., Murakawa, K., Takeda, T., 1992. Atrial and brain natriuretic peptides inhibit the endothelin-1 secretory response to angiotensin II in porcine aorta. Circ. Res. 70, 241–247. Komatsu, Y., Itoh, H., Suga, S., Ogawa, Y., Hama, N., Kishimoto, I., Nakagawa, O., Igaki, T., Doi, K., Yoshimasa, T., Nakao, K., 1996. 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. Circ. Res. 78, 606–614. Middendorff, R., Paust, H.J., Davidoff, M.S., Olcese, J., 1997. Synthesis of Ctype natriuretic peptide (CNP) by immortalized LHRH cells. J. Neuroendocrinol. 9, 177–182.
373
Naruko, T., Itoh, A., Haze, K., Ehara, S., Fukushima, H., Sugama, Y., Shirai, N., Ikura, Y., Ohsawa, M., Ueda, M., 2005. C-type natriuretic peptide and natriuretic peptide receptors are expressed by smooth muscle cells in the neointima after percutaneous coronary intervention. Atherosclerosis 181, 241–250. Ohno, N., Itoh, H., Ikeda, T., Ueyama, K., Yamahara, K., Doi, K., Yamashita, J., Inoue, M., Masatsugu, K., Sawada, N., Fukunaga, Y., Sakaguchi, S., Sone, M., Yurugi, T., Kook, H., Komeda, M., Nakao, K., 2002. Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation 105, 1623–1626. Protter, A.A., Wallace, A.M., Ferraris, V.A., Weishaar, R.E., 1996. Relaxant effect of human brain natriuretic peptide on human artery and vein tissue. Am. J. Hypertens. 9, 432–436. Qian, J.Y., Haruno, A., Asada, Y., Nishida, T., Saito, Y., Matsuda, T., Ueno, H., 2002. Local expression of C-type natriuretic peptide suppresses inflammation, eliminates shear stress-induced thrombosis, and prevents neointima formation through enhanced nitric oxide production in rabbit injured carotid arteries. Circ. Res. 91, 1063–1069. Ruiz-Torres, A., Gimeno, A., Melon, J., Mendez, L., Munoz, F.J., Macia, M., 1999. Age-related loss of proliferative activity of human vascular smooth muscle cells in culture. Mech. Ageing Dev. 110, 49–55. Suda, M., Tanaka, K., Yasoda, A., Komatsu, Y., Chusho, H., Miura, M., Tamura, N., Ogawa, Y., Nakao, K., 2002. C-type natriuretic peptide/guanylate cyclase B system in ATDC5 cells, a chondrogenic cell line. J. Bone Miner. Metab. 20, 136–141. Suga, S., Nakao, K., Itoh, H., Komatsu, Y., Ogawa, Y., Hama, N., Imura, H., 1992. Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-beta. Possible existence of “vascular natriuretic peptide system”. J. Clin. Invest. 90, 1145–1149. Takeuchi, H., Ohmori, K., Kondo, I., Oshita, A., Shinomiya, K., Yu, Y., Takagi, Y., Mizushige, K., Kangawa, K., Kohno, M., 2003. Potentiation of C-type natriuretic peptide with ultrasound and microbubbles to prevent neointimal formation after vascular injury in rats. Cardiovasc. Res. 58, 231–238. Tokudome, T., Horio, T., Soeki, T., Mori, K., Kishimoto, I., Suga, S., Yoshihara, F., Kawano, Y., Kohno, M., Kangawa, K., 2004. Inhibitory effect of C-type natriuretic peptide (CNP) on cultured cardiac myocyte hypertrophy: interference between CNP and endothelin-1 signaling pathways. Endocrinology 145, 2131–2140. Vollmar, A.M., Schulz, R., 1995. Expression and differential regulation of natriuretic peptides in mouse macrophages. J. Clin. Invest. 95, 2442–2450. Wiley, K.E., Davenport, A.P., 2001. Physiological antagonism of endothelin-1 in human conductance and resistance coronary artery. Br. J. Pharmacol. 133, 568–574. Woodard, G.E., Rosado, J.A., Brown, J., 2002. Expression and control of C-type natriuretic peptide in rat vascular smooth muscle cells. Am. J. Physiol. 282, R156–R165. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., Masaki, T., 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411–415. Yeung, V.T., Mak, A.S., Chui, Y.L., Ho, S.K., Lai, K.N., Nicholls, M.G., Cockram, C.S., 1996. Identification of C-type natriuretic peptide gene transcripts in glial cells. Neuroreport 7, 1709–1712.
Expression and localization of C-type natriuretic peptide in human vascular smooth muscle cells Christopher J. Kelsall a,⁎, Adrian H. Chester a , Padmini Sarathchandra a , Donald R.J. Singer b a
b
Heart Science Centre, Harefield Hospital, Harefield, Middlesex, UK Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry, UK Received 3 March 2006; received in revised form 8 May 2006; accepted 2 June 2006
Abstract Objectives: C-type natriuretic peptide (CNP) released by vascular endothelium relaxes smooth muscle and is important in the maintenance of vascular tone. Since it is not known whether other human vascular cell types produce CNP, we investigated its expression in human vascular smooth muscle. Methods: CNP expression was examined by RT-PCR in vascular smooth muscle cells (SMC) cultured from human saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA), and CNP protein was probed using immunostaining, in tissue sections and in SMCs cultured from these vessels, respectively. Results: PCR for CNP produced a 334 bp product in all SMC cultures, as expressed in endothelial cells, although the band intensity was markedly less in SMCs. Myocardium from CNP-knockout mouse did not express CNP, while there was expression in wild-type mouse. CNP protein was detected by immunostaining in 100% of SMC cultures. By immunostaining of tissue sections, CNP was detected throughout the medial layer, but not adventitia, of all vessel types. Conclusions: Expression of CNP at gene and protein level by human vascular SMCs suggests that CNP may have the capacity to regulate vascular tone independently of the endothelium. © 2006 Published by Elsevier Inc. Keywords: C-type natriuretic peptide; Vascular smooth muscle; Gene expression; Immunostaining
1. Introduction The natriuretic peptide family includes atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide (CNP). This family of peptides is considered to have an important role in countering the influences of the renin–angiotensin system and in regulation of vascular tone. The natriuretic peptides bring about their biological actions via guanylate cyclase (GC)-coupled, membrane-spanning natriuretic peptide receptors (NPR) on the cell surface. Vascular smooth muscle cells express predominantly NPRB receptors, which have a very high affinity for CNP. Through activation of GC, CNP brings about an increase in intracellular cGMP, which mediates relaxation of ⁎ Corresponding author. Pharmacology, Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK. Tel.: +44 1895 828 508; fax: +44 1895 828 900. E-mail address: [email protected] (C.J. Kelsall). 1537-1891/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.vph.2006.06.011
vascular smooth muscle and inhibition of smooth muscle cell proliferation. A paracrine route for these effects of CNP from endothelium to vascular smooth muscle has been established. CNP expression in endothelial cells was first demonstrated by Suga et al. (1992). Since then, endothelial expression and release of CNP has been shown in a variety of studies on bovine endothelial cells (Doi et al., 1996; Komatsu et al., 1996; Chun et al., 2000). CNP expression has also been detected in diverse cell types such as cardiac fibroblasts, chondrocytes, glomerular cells, glial cells and macrophages in humans and in animal models (Horio et al., 2003; Suda et al., 2002; Middendorff et al., 1997; Yeung et al., 1996; Vollmar and Schulz, 1995), which implies that CNP has non-vascular roles. For example, CNP may be important in normal skeletal development (Chusho et al., 2001). However, the most significant source of CNP is considered to be the endothelium, as part of the complex array of factors that regulate vascular tone. A current view is that CNP is released by the
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
endothelium in an ablumenal direction to bring about relaxation of vascular smooth muscle, in opposition to contractile influences, such as those of the renin angiotensin system, endothelin and vasopressin. However, the potential for other cells within the vascular wall to serve as a source of CNP has not been adequately explored. One study has reported CNP expression of RNA and protein in cultured rat cerebral vascular and aortic smooth muscle cells (Woodard et al., 2002); in contrast, in intact rat carotid artery denuded of endothelium, Brown et al. (1997) failed to detect CNP immunoreactivity in the medial layer (predominantly comprising smooth muscle cells). Thus, it remains unclear as to whether the expression of CNP in cultured cells is a phenomenon seen in intact vessels and, importantly, if this effect is seen in man. The possibility that CNP could be involved in physiological maintenance of vascular tone, and other biologically important actions, independently of the endothelium in humans, has not been adequately explored. The demonstration of expression of CNP in human vascular smooth muscle cells, in vitro and in situ, would provide evidence for a pathophysiologically relevant autocrine pathway for CNP activity in the absence of a functional or intact endothelium. Therefore, the aims of this study were to determine whether CNP is expressed in human vascular smooth muscle and to detect the post-translational biologically active form of CNP in these cells. 2. Methods 2.1. Vascular tissue This study conforms to the principles outlined in the Declaration of Helsinki. Local ethical committee approval was given for use of human tissue. All tissue was obtained from coronary artery bypass patients whom had given written informed consent. Saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA) were collected in sterile Hank's solution containing 25 mM HEPES buffer and dissected free of surrounding tissue. Specimens designated for RNA extraction (3–5 mm long) were immersed in RNA Later (Sigma) and stored at − 80 °C. Other specimens (3–4 mm long) were mounted with Cryo-M-Bed (Bright Instrument Co., Huntingdon, Cambs, UK) on cork, snap-frozen and stored in liquid nitrogen for sectioning by cryostat. Specimens used for explanting were used within 24 h of collection. 2.2. Explants Specimens of vascular tissue were prepared for explanting by removal of endothelium and adventitia. The remaining medial layer was cut up into 2–3 mm pieces and laid out in a 6-well plate containing sterile growth medium (DMEM containing L-glutamine, penicillin [100 U/ml]/streptomycin [100 μg/ml] and 15% fetal calf serum), to allow smooth muscle cell migration and proliferation. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air and supplied with fresh medium every 3–4 days. Upon reaching confluence, cells were removed by trypsin/EDTA solution (Sigma) and seeded into tissue culture
369
flasks for propagation. Cultured human smooth muscle cells from SV, IMA or RA exhibited the characteristic ‘hill and valley’ morphology at confluence, under the microscope. Vascular smooth muscle cells were 100% pure and free of endothelial cells as assessed by positive immunostaining for smooth muscle α-actin and negatively for CD31, an endothelial cell marker. Cells used were passages 3–8. Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase treatment and propagated in flasks. Endothelial cells were grown in M199 containing 10% FCS, Lglutamine (2 mmol/l), penicillin (100 μg/ml)/streptomycin (100 U/ml), heparin (200 U/ml) and ECGS (all Sigma). HUVEC cultures grown in this way were 100% pure as assessed by anti-CD31 and anti-smooth muscle α-actin immunostaining. Cells used were passages 3–8. 2.3. RT-PCR Total RNA was extracted from tissue sections or from cultured cells using RNeasy extraction columns (Qiagen, Crawley, UK) according to manufacturer's instructions. Extracted RNA was eluted in 40 μl RNase-free water and stored at −80 °C. cDNA was synthesized by combination of 3 μl of eluted RNA with 1μl random decamer (TAG Newcastle, UK) per sample, heated at 70 °C for 10 min. The samples were cooled in a methanol-ice bath. A master mix containing (per sample) 3 μl 1st Strand Buffer (5×), 1.5 μl DTT (0.1 M), 1.5 μl dNTPs (10 mM each), 0.75 μl Superscript II (2000U/μl) and 0.75μl RNA Guard (31, 400U/μl) (all Invitrogen) was made and 7.5 μl of the mix was added to each sample. The cDNAs were heated at 37 °C for 1 h, then at 95 °C for 5 min. cDNAs were stored at − 80 °C. PCR reactions were carried out with 1 μl of cDNA in a total volume of 50 μl per reaction. The reaction mix for each sample contained (final concentration) Q-solution (× 1; Invitrogen), 10% glycerol, PCR buffer containing 15 mM MgCl2 (× 1; Invitrogen), dNTPs (0.2 mM each), primer pairs for C-type natriuretic peptide or α-3-actin (50 pmol/l) and Hotstar Taq polymerase (0.03 U/μl; Invitrogen). For CNP, a ‘nested’ PCR protocol was used, in which the PCR reaction mix was the same for stage 2 with the exception of the CNP primer sequence and that cDNA was substituted with stage 1 PCR product (diluted 1:20 with RNase-free water). A drop of mineral oil was added to each reaction tube, and the following conditions were applied: CNP-denatured at 95 °C for 15 min, 58 °C annealing temperature, 40 cycles (35 cycles for stage 2), extended at 72 °C for 30 s; α-3-actin-denaturing as for CNP, 52 °C annealing temperature, 35 cycles, extended at 72 °C for 30 s. All primers were synthesized by TAG Newcastle (Newcastle, UK); the CNP primers were used in a two-stage ‘nested’ PCR protocol, defined as generating (at end stage) a PCR product derived from a reaction using a PCR product as the template. The primers for the second stage of the reaction encompass the positions of the primer pair used in the first stage. Human CNP: sense primer 5′CTG CTC ACG CTG CTC TCC-3′, stage-1 antisense primer 5′-GTC GGG TGG GCC GTA CTC-3′. The expected stage 1 product size was 388 bp in length. Stage-2 antisense primer 5′CGC TCA TGG AGC CGA TTC-3′. The expected stage 2
370
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
Fig. 1. Gene expression of CNP in cultured vascular SMC derived from saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA). Panel A: comparison of CNP expression in EC (HUVEC) and SMC. Panel B: CNP expression in EC and two SMC cultures vs. acidic riboprotein (ARP) expression. Panel C: CNP and α-3-actin expression in wild type (WT) vs. CNP-knockout (KO) mouse myocardium.
product size was 334 bp in length. Human α-3-actin: sense primer 5′-GGC TCC ATC CTG GCC TCT-3′, antisense primer 5′-AAA AAC ACA TAG GTA ACG AGT-3′. The expected product size was 223 bp in length. Human acidic riboprotein: sense primer 5′ -CGA CCT GGA AGT CCA ACT AC-3′, antisense primer 5′-CGG GCA GAT GCA GCA GAT-3′. The expected product size was 110 bp in length. The ‘nested’ PCR procedure for CNP was validated using positive and negative control samples (myocardial RNA from wild-type and CNP-KO mice, respectively), which were gifted
by Dr. Hideki Chusho, Kyoto University, Japan; the primer sequences were modified, as the primers for human CNP were not able to amplify mouse CNP. Mouse CNP: sense primer 5′TGC TGC TCG CGC TAC TCT CG-3′, antisense primer 5′CGC TCA TGG AGC CGA TCC-3′. The expected product size was 332 bp in length. PCR products were run on a 1% agarose gel containing ethidium bromide and visualized under UV illumination. Product size was measured against size markers from 1 kb DNA Ladder (Invitrogen, Paisley, UK).
Fig. 2. Immunoperoxidase staining profiles in transverse sections of human SV, IMA and RA, respectively; (A) rabbit anti-human CNP, (B) negative control (no primary antibody). L indicates vessel lumen, M indicates medial layer. Magnification: all panels ×180. Blue staining (DAPI: 4′,6-diamidino-2-phenylindole), most notable in control sections, indicates cell nuclei.
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
2.4. Immunostaining of tissue sections Sections of human SV, IMA and RA (5 μm thick), mounted on glass slides, were washed and then fixed in ice-cold acetone for 10 min. To inhibit endogenous peroxidases, the slides were incubated with peroxidase block (DAKO) for 5 min, then washed in PBS. Background staining was blocked by incubation with diluted normal goat serum (1:30), in 1% BSA in PBS, for 30 min. Slides were then blotted and incubated with rabbit anti-human CNP antibody (1:400 in 1% BSA in PBS) for 1 h, washed in PBS, then incubated with biotinylated goat antirabbit IgG (1:200 in 1% BSA in PBS) for 1 h. The slides were washed again and incubated with ABC Complex for 1 h. Subsequently, sections were washed in PBS and immersed in 3,3′diaminobenzidine (DAB; Sigma) substrate, counterstained with Mayer's haematoxylin, dehydrated, cleared and mounted in DPX (distrene, dibutyl phthalate, xylene). These procedures were carried out at room temperature. Control slides were stained with anti-SM α-actin or without primary antibody. 2.5. Immunostaining of cultured cells Vascular smooth muscle cells (VSMC) cultivated from human saphenous vein, internal mammary artery and radial artery were propagated to around 80% confluence on glass coverslips, in 6well plates. Cells were washed in PBS and fixed in ice-cold acetone for 5 min, while antibodies were prepared in PBS containing 1% BSA to prevent non-specific binding. Cells were washed again and incubated with anti-human smooth muscle α-
371
actin (Sigma), anti-human CD31 (DAKO) or anti-human C-type natriuretic peptide (Peninsula) for 1 h. Residual antibody was washed off in PBS and the cells were exposed to Alexa Fluor secondary antibodies (also with 1% BSA) (Molecular Probes) for 1 h. The antibody for cells previously exposed to anti-human αsmooth muscle actin also included Phalloidin Green (Molecular Probes) to stain actin non-specifically. The secondary antibodies were washed off in PBS and coverslips were mounted on slides with Permafluor (Immunatech, France). These procedures were carried out at room temperature. Some coverslips were treated without primary antibody to assess non-specific binding of fluorescent secondary antibodies. Slides were visualized under a fluorescence microscope at wavelengths of 594 nm (red) or 488 nm (green) to detect cell staining. 3. Results 3.1. Expression of CNP by PCR Using primers designed to amplify a 340 bp length of the gene sequence coding for C-type natriuretic peptide in humans, CNP was found to be expressed in VSMC cultured from human saphenous vein, internal mammary artery and radial artery, with a product size of between 300 and 400 bp, as compared against 1 kb DNA ladder (Fig. 1). Due to low signal expression obtained by a single PCR protocol, a nested protocol was used to reliably detect CNP in VSMC. The protocol was validated by use of wild-type and CNP-knockout mouse myocardium extracts; the wild-type
Fig. 3. Immunofluorescent staining. EC (HUVEC) and vascular SMCs derived from RA. Left panels: smooth muscle α-actin staining (upper) and CD31 staining (lower) in SMCs; centre panels: positive CNP staining (upper) and CNP negative control (no primary antibody) in SMCs; right panels: CD31 staining (upper) and CNP staining (lower) in HUVECs. Magnification in SMCs ×720 and in HUVECs ×1440.
372
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
showed positive expression from a nested protocol, while the CNP-knockout was negative (Fig. 1). In contrast, both extracts expressed α-3-actin. 3.2. Detection of CNP by immunostaining in vascular sections Sections of SV, IMA and RA (n = 3 for each), which were sectioned and stained for CNP, showed intense positive staining in endothelium and throughout the medial (smooth muscle) layer, although the smooth muscle staining was less intense than in the endothelium. Medial staining was not diffuse, but appeared in banded patterns (Fig. 2). The location and pattern of staining are consistent with it being correlated with the smooth muscle layer. CNP staining was also apparent in the vasa vasorum. The negative control sections showed no comparable staining. 3.3. Detection of CNP by immunostaining in cultured cells HUVECs, which stained positively for CD31 and negatively for smooth muscle α-actin, were positively stained for CNP, and were regarded as a positive control (Fig. 3). Vascular smooth muscle cells derived from human SV, IMA and RA were all positively stained for CNP, having been established as smooth muscle cells by positive staining for smooth muscle α-actin and negative staining for CD31 (Fig. 3). The intensity of CNP staining varied from one culture to another, but 100% of cells were positively stained. 4. Discussion The current work represents a novel investigation of CNP expression in human vascular smooth muscle. The major findings of this study are that the gene for C-type natriuretic peptide is present within the smooth muscle of the intact vascular wall in humans, and that CNP protein is constitutively expressed in these cells. The determination of expression of CNP in vascular tissue by nested PCR, with intron-spanning primers, is validated by the use of positive and negative controls, in the form of mouse wildtype and CNP-knockout myocardium; since the wild-type expressed CNP but the CNP-knockout did not, the knockout mouse clearly represents a negative control for the PCR reaction. While our PCR demonstrates CNP gene expression in smooth muscle, it does not signify protein synthesis. However, CNPpositive staining of isolated cultures of VSMC demonstrates that CNP protein is present in these cells; although positive staining in vascular sections could be due to sequestration or cell surface binding of CNP released from the endothelium. However, the presence of CNP staining in cultured cell populations in which there are no endothelial cells rules out this explanation, and supports the interpretation that human vascular smooth muscle cells synthesize CNP endogenously. It has been shown previously (Casco et al., 2002; Naruko et al., 2005) that CNP is expressed in tissue sections from atherosclerotic human arteries, similarly to staining of vascular sections by immunostaining in the current study. However, these studies did not establish conclusively the cellular source of the CNP; tissue sections are not guaranteed to be free of endothelium, which may act as a
source for the dissemination of CNP throughout the vascular section, from the vessel surface and from arterioles within the vessel wall. In our studies, staining of vascular sections was complemented by analysis of pure cultures of smooth muscle cells. We have attempted to measure the release of CNP peptide from both intact vessel segments (denuded of endothelium) and cultured vascular smooth muscle cells. However, in our experiments, levels were consistently indistinguishable from the detection limit of the radioimmunoassay used (data not shown). This may be explained by the potential for autosequestration of any CNP released by smooth muscle cells via the NPRC receptor population. Alternatively, more sensitive detection methods (e.g. mass spectrometry) may provide data in humans that reflect the results of measured CNP release from rat VSMC (Woodard et al., 2002). The levels of CNP reported to be released by VSMC in that study were consistently very low, and were derived from substantial numbers of cultured cells (control measurements of <2 pg CNP/106 cells). A limitation of our experiments is that it is difficult to grow such substantial numbers (at low passage) of VSMC cultured from human blood vessels, since typically the age and ill health of the patients from which the specimens are derived tends to render these cells senescent after relatively few passages (Ruiz-Torres et al., 1999). While CNP is traditionally considered as a candidate for counteracting the effects of the renin–angiotensin system on vascular tone under normal conditions, CNP may also fulfill an important role in counteracting the effects of endothelin, a potent vasoconstrictor, in cardiovascular disease. Endothelin is implicated in exacerbating the degradation of vascular function pathophysiologically (Ihling et al., 2004), it was originally discovered in endothelial cells (Yanagisawa et al., 1988), and later confirmed to be present in vascular smooth muscle (Bousette and Giaid, 2003), as CNP has been in this study. Natriuretic peptides have been shown to inhibit release of endothelin from endothelial cells (Kohno et al., 1992) and cardiac non-myocytes (Tokudome et al., 2004). Furthermore, CNP is a recognized vascular relaxing factor (Protter et al., 1996; Wiley and Davenport, 2001). Therefore, the presence of CNP in smooth muscle, and its presumed paracrine action, may serve to protect, at least in part, the vessel wall from damage caused by endothelin and other powerful local vasoconstrictors and cell growth-promoting agents. The presence of intact machinery for CNP to exert its biological effects is potentially very important. It has been demonstrated in a rabbit carotid artery balloon injury model that increased levels of CNP in the arterial wall (by adenoviral transfection) substantially reduce neointimal plaque formation (Qian et al., 2002). Transfection of CNP, again in rabbits, also demonstrated increased re-endothelialization of denuded artery, as well as suppression of thrombosis and neointimal hyperplasia (Ohno et al., 2002), all of which are consequences of chronic vascular disease. These results are supported in rats by the application of CNP via intravenous infusion (Takeuchi et al., 2003). Therefore, data that support the notion of a CNP delivery system that remains intact in the presence of a dysfunctional endothelium offer hope for improved long-term therapy for a range of vascular diseases in the future.
C.J. Kelsall et al. / Vascular Pharmacology 45 (2006) 368–373
In conclusion, our findings show that CNP is expressed by human vascular smooth muscle at both gene and peptide level. This provides a potential alternative pathway by which local endogenous CNP may remain available to modulate development of vascular pathology despite impaired endothelial function. Acknowledgments This work was supported by the British Heart Foundation (PG 2001058) and the Magdi Yacoub Institute. We are grateful to the surgeons of Harefield Hospital Cardiac Theatre for provision of human vascular specimens. The mouse myocardial RNA was a kind gift from Dr. Hideki Chusho of Kyoto University, Japan. References Bousette, N., Giaid, A., 2003. Endothelin-1 in atherosclerosis and other vasculopathies. Can. J. Physiol. Pharmacol. 81, 578–587. Brown, J., Chen, Q., Hong, G., 1997. An autocrine system for C-type natriuretic peptide within rat carotid neointima during arterial repair. Am. J. Physiol. 272, H2919–H2931. Casco, V.H., Veinot, J.P., de Bold, M.L., Masters, R.G., Stevenson, M.M., de Bold, A.J., 2002. Natriuretic peptide system gene expression in human coronary arteries. J. Histochem. Cytochem. 50, 799–809. Chun, T.H., Itoh, H., Saito, T., Yamahara, K., Doi, K., Mori, Y., Ogawa, Y., Yamashita, J., Tanaka, T., Inoue, M., Masatsugu, K., Sawada, N., Fukunaga, Y., Nakao, K., 2000. Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin. J. Hypertens. 18, 575–580. Chusho, H., Tamura, N., Ogawa, Y., Yasoda, A., Suda, M., Miyazawa, T., Nakamura, K., Nakao, K., Kurihara, T., Komatsu, Y., Itoh, H., Tanaka, K., Saito, Y., Katsuki, M., Nakao, K., 2001. Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc. Natl. Acad. Sci. U. S. A. 98, 4016–4021. Doi, K., Itoh, H., Komatsu, Y., Igaki, T., Chun, T.H., Takaya, K., Yamashita, J., Inoue, M., Yoshimasa, T., Nakao, K., 1996. Vascular endothelial growth factor suppresses C-type natriuretic peptide secretion. Hypertens 27, 811–815. Horio, T., Tokudome, T., Maki, T., Yoshihara, F., Suga, S., Nishikimi, T., Kojima, M., Kawano, Y., Kangawa, K., 2003. Gene expression, secretion, and autocrine action of C-type natriuretic peptide in cultured adult rat cardiac fibroblasts. Endocrinology 144, 2279–2284. Ihling, C., Bohrmann, B., Schaefer, H.E., Technau-Ihling, K., Loeffler, B.M., 2004. Endothelin-1 and endothelin converting enzyme-1 in human atherosclerosis- novel targets for pharmacotherapy in atherosclerosis. Curr. Vasc. Pharmacol. 2, 249–258. Kohno, M., Yokokawa, K., Horio, T., Yasunari, K., Murakawa, K., Takeda, T., 1992. Atrial and brain natriuretic peptides inhibit the endothelin-1 secretory response to angiotensin II in porcine aorta. Circ. Res. 70, 241–247. Komatsu, Y., Itoh, H., Suga, S., Ogawa, Y., Hama, N., Kishimoto, I., Nakagawa, O., Igaki, T., Doi, K., Yoshimasa, T., Nakao, K., 1996. 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. Circ. Res. 78, 606–614. Middendorff, R., Paust, H.J., Davidoff, M.S., Olcese, J., 1997. Synthesis of Ctype natriuretic peptide (CNP) by immortalized LHRH cells. J. Neuroendocrinol. 9, 177–182.
373
Naruko, T., Itoh, A., Haze, K., Ehara, S., Fukushima, H., Sugama, Y., Shirai, N., Ikura, Y., Ohsawa, M., Ueda, M., 2005. C-type natriuretic peptide and natriuretic peptide receptors are expressed by smooth muscle cells in the neointima after percutaneous coronary intervention. Atherosclerosis 181, 241–250. Ohno, N., Itoh, H., Ikeda, T., Ueyama, K., Yamahara, K., Doi, K., Yamashita, J., Inoue, M., Masatsugu, K., Sawada, N., Fukunaga, Y., Sakaguchi, S., Sone, M., Yurugi, T., Kook, H., Komeda, M., Nakao, K., 2002. Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation 105, 1623–1626. Protter, A.A., Wallace, A.M., Ferraris, V.A., Weishaar, R.E., 1996. Relaxant effect of human brain natriuretic peptide on human artery and vein tissue. Am. J. Hypertens. 9, 432–436. Qian, J.Y., Haruno, A., Asada, Y., Nishida, T., Saito, Y., Matsuda, T., Ueno, H., 2002. Local expression of C-type natriuretic peptide suppresses inflammation, eliminates shear stress-induced thrombosis, and prevents neointima formation through enhanced nitric oxide production in rabbit injured carotid arteries. Circ. Res. 91, 1063–1069. Ruiz-Torres, A., Gimeno, A., Melon, J., Mendez, L., Munoz, F.J., Macia, M., 1999. Age-related loss of proliferative activity of human vascular smooth muscle cells in culture. Mech. Ageing Dev. 110, 49–55. Suda, M., Tanaka, K., Yasoda, A., Komatsu, Y., Chusho, H., Miura, M., Tamura, N., Ogawa, Y., Nakao, K., 2002. C-type natriuretic peptide/guanylate cyclase B system in ATDC5 cells, a chondrogenic cell line. J. Bone Miner. Metab. 20, 136–141. Suga, S., Nakao, K., Itoh, H., Komatsu, Y., Ogawa, Y., Hama, N., Imura, H., 1992. Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-beta. Possible existence of “vascular natriuretic peptide system”. J. Clin. Invest. 90, 1145–1149. Takeuchi, H., Ohmori, K., Kondo, I., Oshita, A., Shinomiya, K., Yu, Y., Takagi, Y., Mizushige, K., Kangawa, K., Kohno, M., 2003. Potentiation of C-type natriuretic peptide with ultrasound and microbubbles to prevent neointimal formation after vascular injury in rats. Cardiovasc. Res. 58, 231–238. Tokudome, T., Horio, T., Soeki, T., Mori, K., Kishimoto, I., Suga, S., Yoshihara, F., Kawano, Y., Kohno, M., Kangawa, K., 2004. Inhibitory effect of C-type natriuretic peptide (CNP) on cultured cardiac myocyte hypertrophy: interference between CNP and endothelin-1 signaling pathways. Endocrinology 145, 2131–2140. Vollmar, A.M., Schulz, R., 1995. Expression and differential regulation of natriuretic peptides in mouse macrophages. J. Clin. Invest. 95, 2442–2450. Wiley, K.E., Davenport, A.P., 2001. Physiological antagonism of endothelin-1 in human conductance and resistance coronary artery. Br. J. Pharmacol. 133, 568–574. Woodard, G.E., Rosado, J.A., Brown, J., 2002. Expression and control of C-type natriuretic peptide in rat vascular smooth muscle cells. Am. J. Physiol. 282, R156–R165. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., Masaki, T., 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411–415. Yeung, V.T., Mak, A.S., Chui, Y.L., Ho, S.K., Lai, K.N., Nicholls, M.G., Cockram, C.S., 1996. Identification of C-type natriuretic peptide gene transcripts in glial cells. Neuroreport 7, 1709–1712.