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paracrine growth factor for aortic adventitia of rat
Regulatory Peptides 151 (2008) 88–94
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Urotensin II is an autocrine/paracrine growth factor for aortic adventitia of rat Yonggang Zhang a,⁎, Yuguang Li a, Ruihong Wei b, Zhijian Wang c, Dingfang Bu c, Jing Zhao c, Yongzheng Pang c, Chaoshu Tang c a b c
Department of Cardiovascular Diseases, First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515041, China Internal Medicine, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China Institute of Cardiovascular Research, Peking University First Hospital, Beijing 100034, China
a r t i c l e
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Article history: Received 9 February 2008 Received in revised form 7 September 2008 Accepted 1 October 2008 Available online 14 October 2008 Keywords: Adventitial fibroblasts Peptide GPR14 Receptor Collagen Vascular remodeling Proliferation
a b s t r a c t Urotensin II (UII) is a potent vasoconstrictive peptide; however, its significance in vascular adventitia has not been clearly elucidated. In this study, rat aortic adventitia showed mRNA expression and immunoreactivity of UII and its receptor (UT). Moreover, radioligand-binding assay showed that maximum binding capacity (Bmax) of [125I]UII was higher in adventitia than in media (28.60 ± 1.94 vs. 20.21 ± 1.11 fmol/mg, P b 0.01), with no difference in binding affinity (dissociation constant [Kd] 4.27 ± 0.49 vs. 4.60 ± 0.40 nM, P N 0.05). Furthermore, in cultured adventitial fibroblasts, UII stimulated DNA synthesis, collagen synthesis and secretion in a concentrationdependent manner. These effects were inhibited by the UII receptor antagonist urantide (10− 6 mol/l), Ca2+ channel blocker nicardipine (10− 5 mol/l), protein kinase C inhibitor H7 (10− 6 mol/l), and mitogen-activated protein kinase inhibitor PD98059 (10− 6 mol/l) but not the phosphatidyl inositol-3 kinase inhibitor wortmannin (10− 7 mol/l). UII may act as an autocrine/paracrine factor through its receptor and the Ca2+ channel, protein kinase C, and mitogenactivated protein kinase signal transduction pathways, in the pathogenesis of vascular remodeling by activating vascular adventitia. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Urotensin II (UII) is a potent vasoconstrictive neuropeptide initially isolated from fish urophysis. Recently, human homologues of UII and its specific receptor GPR14, now known as UT receptor, were identified [1,2]. UII is predominately expressed in spinal cord and certain brain areas and in myocardium and vessels, among other tissues [1–4]. UT receptor is mainly expressed in cardiovasculature, including myocardium, vascular smooth muscle cells (VSMCs), and endothelial cells [2,3,5]. UII induces potent vasoconstriction and was shown to be more potent than endothelin-1 [2]. In addition, UII can stimulate proliferation of VSMCs and induce hypertrophic responses in cardiomyocytes from neonatal rats [6–8]. In human monocyte-derived macrophages, UII accelerates foam cell formation [9]. UII is also a new chemotactic factor for UT receptorexpressing monocytes [4]. Moreover, UII and UT have been linked to vascular and cardiac remodeling. Human UII-like immunoreactivity was discovered in human coronary atheroma [2,5]. Increased human UII levels were associated with carotid atherosclerosis in essential hypertension [10]. In patients with coronary artery disease, increased plasma level of UII was significantly related to parameters of cardiac dysfunction [11]. ⁎ Corresponding author. Department of Cardiovascular Diseases, First Affiliated Hospital, Shantou University Medical College, No. 57, Changping Road, Shantou, Guangdong 515041, China. Tel.: +86 754 889 05337; fax: +86 754 882 59850. E-mail addresses: [email protected], [email protected] (Y. Zhang). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.10.004
Increased levels of UII, as well as greater density of binding sites for UII, were found in myocardium of patients with congestive heart failure [12]. We previously showed increased density of binding sites for UII in sarcolemma of myocardium and increased UII content in rat ventricles exposed to chronic hypoxia [13]. Recently, we found UII involved in the development of cardiac fibrosis and hypertrophy by synergistic effects with isoproterenol [14]. Increased expression of UII and UT receptor protein was observed in both infarcted and non-infarcted regions of the left ventricle in a rat model of heart failure after myocardial infarction [15]. In vitro experiments showed that UII stimulated proliferation and collagen synthesis of neonatal cardiac fibroblasts, as determined by 3Hthymidine and 3H-proline incorporation [6,15]. UII also increased the mRNA level of procollagens α1 (I) and α1 (III), and fibronectin in these cells [15]. Taken together, these data suggest that UII may be involved in the cardiovascular fibrogenesis through increased collagen synthesis and may target multiple cells in cardiovascular tissues, playing multiple functions in physiological and pathophysiological regulation. Recently, many studies have focused on the physiological and pathophysiological significance of tunica adventitia, a major effector site for neuro-humoral factors, such as hormones, cytokines, growth factors, inflammatory factors and active peptides [16,17]. The adventitia also secretes vasoactive factors such as nitric oxide, bioactive peptides, growth factors and cytokines under physiological or pathological conditions [16–19], thereby participating in the regulation of arterial structure and function. Therefore, the adventitia has paracrine/ autocrine actions, just like the intima [16]. In our previous study, we
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demonstrated that UII activates the L-arginine/nitric oxide pathway in isolated rat aortic adventitia [18]. However, the significance of UII and UT receptor in the adventitia is not yet elucidated. In this study, we determined the mRNA expression and immunoreactivity of UII and UT in rat aortic adventitia. In addition, we determined the binding capacity of UT in adventitia by radioligandbinding assay. Furthermore, in cultured adventitial fibroblasts, we evaluated the effects of UII on proliferation, collagen synthesis and secretion and its molecular mechanisms by 3H-proline and 3H-thymidine incorporation methods. 2. Materials and methods 2.1. Materials Male Sprague–Dawley rats weighing 180–200 g were supplied by the Animal Center, Health Science Center, Peking University. Animal care and experimental protocols complied with the Animal Management Rules of China (documentation number 55, 2001, Ministry of Health, China) and the Guide for Care and Use of Laboratory Animals, Peking University First Hospital. Rat UII (pEHGTAPECFWKYCI) and rat [125I]-UII [2200 Ci (81.4TBq)/ mmol] were purchased from Phoenix Pharmaceutical (Belmont, CA, USA). Rabbit anti-UII (rat) antibody and goat-anti rabbit-IgG were purchased from Boster Biological Technology (Wuhan, China). Rabbit anti-UT polyclonal antibody was purchased from Chemicon International (Temecula, CA, USA). A UII ISH kit was purchased from Haoyang Biotechnology Company (Tianjin, China). Nicardipine, wortmannin, bovine serum albumin (BSA), bacitracin, trypsin and Dulbecco modified Eagle medium (DMEM) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum was from Hyclone (Logan, UT, USA). 3H-thymidine and 3H-proline were from Amersham Pharmacia Biotech (Freiburg, Germany); TRIzol and dNTP were from Clontech Laboratories (Palo Alto, CA, USA). Taq DNA polymerase and oligo (dT)15 primers were from Promega (Madison, WI, USA). Oligonucleotides were synthesized by Sai Baisheng Biotechnology (Beijing, China). The sequences of oligonucleotide primers were rUT-S, 5′GCATCTTCACCCTGACCATAA-3′, and rUT-A, 5′-CCCAGAAGAGAAGGACGATACC-3′, used for the amplification of UT cDNA; and β-actin-S, 5′ATCTGGCACCACACCTTC-3′, and β-actin-A, 5′-AGCCAGGTCCAGACGCA-3′, for the amplification of β-actin for calibration of sample loading. PD98059 and H7 were from Calbiochem (Darmstadt, Germany). Urantide was from Peptides International (Louisville, KY, USA). Other chemicals and reagents were of analytical grade. 2.2. Isolation of adventitial and medial tissues of rat aorta The adventitial and medial tissues of rat aorta were prepared according to the method of Faber et al. [20] with some modification. Briefly, rats were anesthetized with use of isoflurane and rapidly decapitated. The full-length thoraco-abdominal aorta was isolated under a pool of 4 °C phosphate-buffered saline, after gently opening the parietal pleura, separating away loose connective tissue and collateral vessels, and transecting the segmental arteries at their origins. Under magnification, the adventitial and medial layers could be distinguished at both ends of the aorta. Endothelial cells were removed by gently rubbing the lumen with the blunt side of dissecting scissors, and the medial layer was peeled off with use of two forceps. Both the adventitial and medial layer were collected and quickly frozen in liquid nitrogen and stored at −80 °C until use. 2.3. In situ hybridization The full-length aorta was isolated as described above. Tissue sections fixed with 10% buffered formalin and embedded in paraffin were pretreated and underwent in situ hybridization for UII mRNA according to the manufacturer's instructions. The following probes labeled at the 5-end with digoxin were used: 5′-CTACGAAGA GCAGGCAGCAGAAGGG-
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CAC-3′; 5′-TTCCCTCTGCTTCTGTGCCCACGGTCTG-3′. Visualization of the in situ hybrids was enabled by 3, 3′-diaminobenzidine, with dark brown spots in the tissue sections representing the UII mRNA-positive signals. Negative controls represented the absence of probes. 2.4. RT-PCR assay The expression of UT mRNA in adventitia and media was assessed by RT-PCR as described [21]. Total RNA extracted from the tissue was quantified by use of an ultraviolet spectrophotometer (UV2100, Shimadzu, Japan). Reverse transcription to cDNA was accomplished by priming 2 µg total RNA with oligo (dT)15 primers with use of moloney murine leukemia virus transcriptase. Products were then used for the following PCR amplification: 2.5 mmol/l/each dNTP 1 µl, 10 × PCR buffer (100 mmol/l Tris–HCI, pH 8.3, 15 mmol/l MgCl2, 500 mmol/l KCl) 2.5 µl, cDNA 1 µl, 5 µmol/l/each of UT-S and UT-A primers 1 µl and 1.25 unit of Taq DNA polymerase, in a total volume of 25 µl. After denaturing at 95 °C for 5 min, PCR cycles were run at 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, for 35 cycles, then 72 °C for 5 min. As an internal control for each PCR reaction, β-actin cDNA was amplified for each sample under the same conditions. PCR products were separated in 1.5% agarose gel and visualized by ethidium bromide staining. The intensity of the PCR product bands under UV light was measured by use of a gel image analyzer. Results were expressed as the ratio of UT PCR product (399 bp) to β-actin PCR product (291 bp) for each sample. All experiments were repeated three times. 2.5. Immunohistochemistry Immunoreactive UII and UT were assessed by a streptavidin–biotin peroxidase method on snap-frozen tissue sections postfixed with 4% paraformaldehyde, as reported previously [22]. Sections of rat thoracic aorta were incubated overnight at 4 °C with rabbit anti-rat UII antibody and rabbit anti-UII receptor (UT) antibody. UII antibody did not crossreact with a range of peptide ligands such as endothelin-1 (human, rat, mouse), adrenomedullin (1–50) (rat), or angiotensin II (human). The yellow-brown granules in the sections were considered positive signals. 2.6. Radio-ligand binding assay [125I]-U-II binding assay was performed as described [23,24] with some modifications. Briefly, minced adventitia and media were homogenized in ice-cold SEGT buffer [250 mmol/l sucrose, 1 mmol/l ethyleneglycol-bis-(2-aminoethyl ether)-N, N′-tetraacetic acid, 20 mmol/l Tris, pH 7.2]. After centrifugation, the pellet was re-suspended in SEGT buffer and filtered through four layers of cheesecloth. Protein content in the filter was determined by the Bradford method and adjusted to 1 mg/ml. The activity of Na+/K+ ATPase as the marker enzyme of sarcolemma was determined [25]. Ligand-binding assay was carried out in a polypropylene tube containing 50 µg membrane fraction and 0.2 ml binding assay buffer [20 mmol/l Tris–HCl, pH 7.4, 2 mmol/l MgCl2, 0.25% bovine serum albumin, 0.25 mg/ml bacitracin, and [125I]-UII (0.5,1, 2, 4, 8, 16 or 32 nmol/l)]. The mixture was incubated at 25 °C for 40 min, terminated by the addition of 2 ml ice-cold binding assay buffer, and filtered through acetate cellulose filters (0.45 mm). After washing the filters with ice-cold binding assay buffer three times, the radioactivity on filters was counted by use of a Wallac 1470 Wizard automatic gamma counter (Switzerland). Nonspecific binding of [125I]-UII was obtained in the presence of 20 µmol/l unlabeled rat UII. Dissociation constant (Kd) and maximal binding capacity (Bmax) were calculated by Scatchard plot analysis of saturation binding isotherms. 2.7. Culture of adventitial fibroblasts [20] The rat adventitial fibroblasts were cultured according to the method of Faber et al. [20] with some modification. Sterile-isolated adventitia of
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supernatants were harvested for the measurement of collagen synthesis and secretion, respectively. Collagen secretion was measured according to the method of Li [27]. Briefly, 100 µl of pepsin assay buffer (mixed with 25 µl of 5 mol/l acetic acid, pH 2–3, 25 µl of 1 mg/ ml pepsin solution in 0.5 mol/l acetic acid, and 50 µl of 10 mg/ml proline) was added to 1 ml of the supernatant from a well and kept for 3 h at 4 °C. To precipitate protein fractions, 250 µl of 1.2 mol/l cold trichloroacetic acid was added to the samples and incubated on ice for 2 h. Precipitates were applied onto filter units, washed with trichloroacetic acid and ethanol, and counted in a scintillation counter. Fig. 1. In situ hybridization for UII mRNA in rat aorta. A, negative control. B, expression of UII mRNA in rat aortic arteries, especially endothelial cells and adventitial cells. Magnification is ×400. AD, adventitia; ME, media; EC, endothelium.
thoracic aorta was minced into 1-mm2 pieces and incubated for 30 min at 37 °C without shaking in 2.4 units/ml neutral protease II. After gentle trituration, cells were placed in DMEM culture media with 20% fetal bovine serum (FBS) on ice to arrest protease activity. After repeating this dispersion procedure three times, pooled cells were gently resuspended in DMEM plus FBS and sieved (38 µmol/l) to separate the smaller adventitial fibroblasts from occasional non-fibroblasts present in adventitia (SMC-like cells, mast cells, macrophages, and adipocytes). Adventitial fibroblasts (20,000 cells/cm2) were grown in DMEM with 10% FBS, 200 mg/l L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin passaged at ~90% confluence with 0.125% trypsin/EDTA every 3 days. The morphology and growth characteristics of the cells were typical of fibroblasts and were distinguished from SMCs by the absence of a “hill-and-valley” growth pattern and lack of smooth muscle α-actin staining. Cells between passages 4–6 were used. To examine DNA synthesis, collagen synthesis and secretion of adventitial fibroblasts induced by UII, the cultured cells were incubated in DMEM with 10% FBS, then incubated with serum-deprived medium (containing 0.5% FBS) for 24 h. After synchronization of adventitial fibroblasts, the medium was changed to DMEM without serum. Thereafter, these cells were incubated in the presence or absence of UII. To examine the molecular basis of the action of UII, some inhibitors, including the UII receptor antagonist urantide (10− 6 mol/l), Ca2+ channel blocker nicardipine (10− 5 mol/l), phosphatidyl inositol-3 kinase (PI3K) inhibitor wortmannin (10− 7 mol/l), protein kinase C (PKC) inhibitor H7 (10− 6 mol/l), and mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10− 6 mol/l), were used. According to the experimental design, cells were divided into groups for treatment: control, serum-free DMEM only; 10− 10, 10− 9, 10− 8, or 10− 7 UII added to the medium; 10% FBSDMEM with or without UII (10− 8 mol/l); and pretreatment in serumfree DMEM for 0.5 h with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l) or wortmannin (10− 7 mol/l) then UII (10− 8 mol/l). The cells and media were collected after 24 h of incubation. Each experiment was repeated 6 times.
2.10. Statistical analysis Results are shown as mean±SD. Comparisons involved use of unpaired Student's t test and one-way ANOVA, followed by the Newman–Keuls multiple comparison test. A value of Pb 0.05 was considered statistically significant. 3. Results 3.1. Expression of UII and UT mRNA in the rat aortic adventitia In situ hybridization revealed UII mRNA expression in rat aorta, abundant in the adventitial layer and endothelium and to a lesser degree in media (Fig. 1). RT-PCR showed higher UT mRNA expression in both adventitia and media than that in the medulla oblongata, a positive control tissue (Fig. 2). UT mRNA level was slightly higher, but not significantly, in the adventitia than in the media (P N 0.05). 3.2. UII and UT immunoreactivity in rat adventitia Histological examination revealed distinct immunoreactivity for UII and UT in the aorta, which matched the mRNA expression results from in situ hybridization. As well, staining was abundant in the adventitial layer and endothelium but less so in the media (Fig. 3).
2.8. Determination of cell proliferation DNA synthesis was determined by 3H-thymidine incorporation as described previously [26]. The adventitial fibroblasts were incubated with different concentrations of UII and/or inhibitors or serum for 24 h, then exposed to 3H-thymidine at 1 µCi/well for the last 8 of the 24-h incubation. Then, cells were washed with ice-cold PBS and 10% trichloroacetic acid. Acid-insoluble 3H-thymidine was collected on glass fiber filters (Whatman, Kent, UK) and determined by use of a liquid scintillation counter (LS 6500, Beckman, Fullerton, CA, USA). 2.9. Determination of collagen synthesis and secretion Collagen synthesis was examined by measuring 3H-proline incorporation into cells [27]. At the end of the experiment, cells and
Fig. 2. Estimation of UT mRNA expression in adventitia and media of rat aorta by RTPCR. A. electrophoresis of PCR products; Lane 1: medulla oblongata; lane 2: media of rat aorta; lane 3: adventitia of rat aorta. B. ratio of UT to beta-actin products (mean ± SD, n = 3). ⁎P b 0.05 vs. MO; AD, adventitia; ME, media; MO, medulla oblongata as a positive control.
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Fig. 3. Immunohistochemical localization of UII and UT protein in thoracic aorta of healthy rats. A, staining for UII; B: staining for UT. (Magnification is ×400). AD, adventitia; ME, media; EC, endothelium.
3.3. Ligand-binding assay The activities of Na+/K+ ATPase (a marker enzyme of plasma membrane) were 6.8 ± 0.7 and 6.1 ± 0.5 µmol/mg protein/h in membrane preparations from adventitia and media, respectively, and were 0.97 ± 0.1 and 0.94 ± 0.1 µmol/mg protein/h in homogenates from the two tissues, respectively. The activities of Na+/K+ ATPase in the membrane preparations from adventitia and media were 6.0 and 5.5 times higher, respectively, than that in homogenates, which indicates that the membrane preparations were mainly plasma membrane. Radioligand binding studies showed that the non-specific binding of [125I]-UII (in the presence of 20 µmol/l unlabeled UII) was about 25% that of the total binding of the labeled peptide. Fig. 4 shows the characteristics of [125I]-UII binding to the membrane preparation of rat aortic adventitia and media. Scatchard analysis of the data demonstrated that the calculated maximal number of specific binding sites was 28.60 ± 1.94 fmol/mg protein in adventitia, which was 42% higher than that in the media (20.21 ± 1.11 fmol/mg protein, P b 0.01). However, adventitia and media did not differ in the Kd (4.27 ± 0.49 vs. 4.60 ± 0.40 nM, P N 0.05). 3.4. Effect of UII on adventitial fibroblasts proliferation Fig. 5 shows that UII stimulated the proliferation of cultured adventitial fibroblasts, as assessed by 3H-thymidine incorporation, in a dose-dependent manner; 3H-thymidine incorporation was increased by 39%, 90%, 103% and 171% with 10− 10, 10− 9, 10− 8 and 10− 7 mol/l UII treatment, respectively (P b 0.01). The incorporation was increased by 121% and 189% with serum and UII + serum treatment, respectively (P b 0.01), as compared with the control group (1360 ± 42 cpm/well). In
Fig. 4. [125I]-UII binding to the plasma membrane of adventitia and media of rat aorta (mean ± SD, n = 4). A, saturation binding curves; B, Scatchard plot.
Fig. 5. Effect of UII on 3H-thymidine incorporation in cultured adventitial fibroblasts (mean ± SD, n = 6). Adventitial fibroblasts were incubated with different concentrations of UII and/or serum for 16 h, then also exposed to 3H-thymidine at 1 µCi/well for 8 h, then 3H-thymidine incorporation was measured. Values are compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ⁎⁎P b 0.01 vs. Control; ##P b 0.01 vs. 10− 8 mol/l UII group or serum group.
the serum + UII (10− 8 mol/l) group, the incorporation was increased by 29% (P b 0.01) as compared with the serum-alone group and by 41% (P b 0.01) as compared with the UII (10− 8 mol/l)-alone group (P b 0.01). 3 H-thymidine incorporation stimulated by UII (10− 8 mol/l) was inhibited by the UII receptor antagonist urantide (10− 6 mol/l), MAPK inhibitor PD98059 (10− 6 mol/l), L-type voltage-dependent Ca2+ channel blocker nicardipine (10− 5 mol/l), and PKC inhibitor H7 (10− 6 mol/l) but not by the PI3K inhibitor wortmannin (10− 7 mol/l) (Fig. 6), which suggests that UT, Ca2+, PKC, and MAPK but not PI3K might be involved in the proliferation of adventitial fibroblasts induced by UII. 3.5. The effect of UII on collagen synthesis and secretion of adventitial fibroblasts Fig. 7 shows that UII stimulated 3H-proline incorporation in a concentration-dependent manner. 3H-proline incorporation with UII
Fig. 6. Effect of different kinds of inhibitors on UII-induced 3H-thymidine incorporation in cultured adventitial fibroblasts. Cells were preincubated with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l) or wortmannin (10− 7 mol/l) for 30 min, then incubated with UII (10− 8 mol/l) and the inhibitors for 16 h, then also exposed to 3H-thymidine at 1 µCi/well for 8 h, then 3H-thymidine incorporation was measured. Values were compared by one-way ANOVA and then by Newman–Keuls multiple comparison test. ##P b 0.01 vs. control; ⁎P b 0.05; ⁎⁎P b 0.01 vs. UII group. UII: urotensin II; Ura, urantide; Wor, wortmannin; PD, PD98059; Ni, nicardipine.
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Fig. 7. Effect of UII on 3H-proline incorporation in cultured adventitial fibroblasts (mean± SD, n = 6). Adventitial fibroblasts were incubated with different concentrations of UII and/or serum for 16 h, then also exposed to 3H-proline at 1 µCi/well for 8 h, then 3Hproline incorporation was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ⁎⁎P b 0.01 vs. control; ##P b 0.01 vs.10− 8 mol/l UII group or serum group.
Fig. 9. Effect of UII on collagen secretion from cultured adventitial fibroblasts (mean ± SD, n = 6). Adventitial fibroblasts were incubated with different concentrations of UII (10− 10, 10− 9, 10− 8 and 10− 7 mol/l) and/or serum for 16 h, then also exposed to 3Hproline at 1 µCi/well for 8 h, then collagen secretion was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ⁎⁎P b 0.01 vs. control; ##P b 0.01 vs. 10− 8 mol/l UII group or serum group.
concentrations of 10− 10, 10− 9, 10− 8 and 10− 7 mol/l was higher, by 24%, 42%, 56% and 57%, respectively (all P b 0.01), than with control treatment (1484 ± 69 cpm/well). In the 10% FBS group, 3H-proline incorporation was higher, by 27% (P b 0.01) than with control treatment. Moreover, in the serum + UII (10− 8 mol/l) group, 3H-proline incorporation was higher, by 58% (P b 0.01), than in the serum-alone group, and higher by 29% (P b 0.01), than in the UII (10− 8 mol/l)alone group. This effect was inhibited by pretreatment with urantide (10− 6 mol/l, P b 0.01) and PD98059 (10− 6 mol/l, P b 0.01) and partially suppressed by pretreatment with nicardipine (10− 5 mol/l, P b 0.05) and H7 (10− 6 mol/l, P b 0.05) but not suppressed by wortmannin (Fig. 8), which suggests that UT, MAPK, Ca2+, and PKC might be involved in collagen synthesis induced by UII. UII stimulated collagen secretion from cultured adventitial fibroblasts in a dose-dependent manner (Fig. 9). With UII at 10− 10–10− 7 mol/l, colla-
gen secretion was higher, by 41%–76% (Pb 0.01), than with control treatment. Collagen secretion was increased by 63% with 10% FBS treatment than with control treatment (Pb 0.01). In the serum+UII (10− 8 mol/l) group, collagen secretion was higher, by 19% (Pb 0.01), than in the serumalone group and higher, by 16% (Pb 0.01), than in the 10− 8 mol/l UII-alone group. This effect induced by UII was completely blocked by urantide (10− 6 mol/l) and PD98059 (10− 6 mol/l) and partially blocked by nicardipine (10− 5 mol/l) and H7 (10− 6 mol/l) but not blocked by wortmannin (10− 7 mol/l) (Fig. 10). These data suggest that UII induced collagen secretion in adventitial fibroblasts via UT, Ca2+ channel, PKC, and MAPK but not PI3K. 4. Discussion A growing body of evidence suggests that UII exerts a broad spectrum of physiological and pharmacological actions. In addition to
Fig. 8. Effect of different kinds of inhibitors on UII-induced 3H-proline incorporation in cultured adventitial fibroblasts. After preincubation with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l), wortmannin (10− 7 mol/l) for 30 min, rat adventitial fibroblasts were incubated with UII (10− 8 mol/l) and the inhibitors for 16 h, then also exposed to 3H-proline at 1 µCi/well for 8 h, then 3H-proline incorporation was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ##P b 0.01 vs. Control; ⁎P b 0.05; ⁎⁎P b 0.01 vs. UII group. UII: urotensin II; Ura, urantide; Wor, wortmannin; PD, PD 98059; Ni, nicardipine.
Fig. 10. Effect of different kinds of inhibitors on UII-induced collagen secretion in cultured adventitial fibroblasts. After preincubation with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l) or wortmannin (10− 7 mol/l) for 30 min, rat adventitial fibroblasts were incubated with UII (10− 8 mol/l) and the inhibitors for 16 h, then also exposed to 3H-proline at 1 µCi/well for 8 h, then collagen secretion was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ##P b 0.01 vs. Control; ⁎P b 0.05; ⁎⁎P b 0.01 vs. UII group. UII: urotensin II; Ura, urantide; Wor, wortmannin; PD, PD 98059; Ni, nicardipine.
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having direct vasoconstrictive activity in smooth muscle receptors, UII stimulates proliferation of VSMCs, cardiac fibroblasts, glomerular mesangial cells, endothelial cells and tumor cells [6,28] and promotes hypertrophy of cardiac myocytes [8] and collagen synthesis of cardiac fibroblasts [6,15]. UII also accelerates foam cell formation in human monocyte-derived macrophages, induces chemotactic activity in monocytes, and inhibits insulin secretion in perfused rat pancreas [29]. Intracerebroventricular administration of UII in conscious sheep induced large, prolonged increases in plasma epinephrine, adrenocorticotropic hormone, cardiac output and arterial pressure [30]. These data demonstrate broad targets for UII actions. Importantly, recent studies have demonstrated that the vascular adventitia is an essential layer for the metabolism and function of the vascular wall and a target for actions of many vasoactive peptides [31]. However, the significance of the UII/UT system in activation of adventitial fibroblasts and vascular remodeling has still not been clarified completely. In this study, using in situ hybridization, we detected UII mRNA expression in rat aorta, especially in adventitia and endothelium. We also found large amount of UT mRNA expressed in the adventitia. Immunohistochemistry showed both UII and UT immunoreactivity in the aorta, with the adventitial layer exhibiting protein expression of UII and UT, which matched our mRNA expression results, as was found previously [32]. Moreover, we also determined that [125I]-UII bound to the adventitia with a high-affinity and saturable character and the number of binding sites of UII was higher in the adventitia than in the media, with no difference in Kd values between the tissues. Furthermore, we investigated the effects of UII on DNA synthesis and collagen production of adventitial fibroblasts in vitro by 3H-thymidine and 3H-proline incorporation, respectively, and demonstrated that UII could stimulate proliferation, collagen synthesis and secretion in a concentration-dependent manner. Urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l), inhibitors/ blocker of UII receptor, MAPK, Ca2+ channel and PKC, respectively, could inhibit UII-induced DNA synthesis and collagen production, but wortmannin (10− 7 mol/l), the inhibitor of PI3K, did not. Thus, UT, MAPK, Ca2+, and PKC pathways might be involved in DNA synthesis and collagen production of adventitial fibroblasts induced by UII. However, these UII-stimulated effects were not inhibited by PI3K inhibitor wortmannin, which excludes the involvement of PI3K in UII-induced DNA synthesis and collagen production. These results in adventitial fibroblasts are consistent with those in VSMCs [6,7,28]. We have reported that Ca2+ channel, PKC, MAPK and CaM-PK pathways are involved in VSMC proliferation [33]. Others reported that the activation of phospholipase C, small G-protein RhoA and Rho-Kinase, extracellular signal regulated protein kinases and PKC are involved in vasoconstriction and VSMC proliferation [2,28,8,34]. In addition, UII increased the expression of PAI-1 and enhanced SMC proliferation in an NADPH oxidase- and kinase-dependent manner [35]. UII accelerates the formation of human macrophage-derived foam cells by up-regulating the acyl-coenzyme A: cholesterol acyltransferase-1, which is mediated via the UT receptor/Gq protein/c-Src/PKC/ERK and RhoA/ROCK pathways [9]. In our previous study, we showed that rat UII may stimulate the L-arginine/NOS/NO pathway in rat aortic adventitia [18]. More recently, we found that UII could stimulate adventitial fibroblast phenotypic conversion and migration through the PKC, MAPK, calcineurin, Rho kinase, and/or Ca2+ signal transduction pathways [36]. The present results indicate that UII may also stimulate adventitial fibroblast proliferation and collagen synthesis and secretion through the UT, PKC, MAPK, and Ca2+ pathways. A growing body of evidence has documented that fibroblasts from adventitia play an important part in vascular remodeling through phenotypic conversion, proliferation, apoptosis, and migration [37]. Evidence also supports the activation of adventitial fibroblasts as the key regulator of vascular function and structure from the “outside in”, and contributes to the development of atherosclerotic lesions [16,38]. Numerous cytokines and growth factors, such as tumor necrosis
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factors, transforming growth factor-β, granulocyte-macrophage colony-stimulating factor, endothelin-1 and angiotensin II, may influence the proliferation and collagen synthesis of fibroblasts as well as their transition to myofibroblasts [16,37–40]. Recently, a study supported that hypoxia-induced stimulation of the hypoxia-inducible factor 1α axis trans-activates the ACE–AngII–AT1 system in pulmonary artery adventitial fibroblasts, which shows a possible role in autocrine and paracrine regulation of vascular function [41]. The present study identified that not only UII but also UT had highly specific UII binding sites within the adventitia, and UII could stimulate proliferation and collagen synthesis and secretion through the UT, Ca2+, PKC and MAPK signal transduction pathways. Therefore, UII may act mainly in an autocrine/paracrine manner in the activation of adventitial fibroblasts, thus contributing to the vascular remodeling. In conclusion, our results verify the abundant expression of UII and UT mRNA in the adventitia of rat aorta. UII and UT immunoreactivity was also apparent in the adventitia. Moreover, adventitia showed more UII binding sites than did the media. Furthermore, UII promotes proliferation and collagen synthesis and secretion in adventitial fibroblasts in vitro in a concentration-dependent manner. Intracellular signal transduction pathways, including UT, Ca2+, PKC, and MAPK, might be involved in these processes. The vascular adventitia may be a new target for UII action, and UII may act mainly in an autocrine/paracrine manner in the activation of adventitial fibroblasts in the development of vascular remodeling and contribute to the pathogenesis of cardiovascular diseases, the mechanisms of which are worthy of further investigation. Acknowledgments We thank Hongzhi Wang for technical help. This project was supported by the National Natural Science Foundation of China (No. 30470730), China Postdoctoral Science Foundation (No. 2003033439), and the Medical Science Foundation of Guangdong Province Health Department (No. A2007425), and Major State Basic Research Development Program of China (No. G2000056905). The experiment was completed with the assistance from the Institute of Cardiovascular Research, Peking University First Hospital. References [1] Coulouarn Y, Lihrmann I, Jegou S, Anouar Y, Tostivint H, Beauvillain JC, et al. Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci U S A 1998;95:15803–8. [2] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999;401:282–6. [3] Balat A, Karakök M, Yilmaz K, Kibar Y. Urotensin-II immunoreactivity in children with chronic glomerulonephritis. Ren Fail 2007;29:573–8. [4] Segain JP, Rolli-Derkinderen M, Gervois N, Raingeard de la Blétière D, Loirand G, Pacaud P. Urotensin II is a new chemotactic factor for UT receptor-expressing monocytes. J Immunol 2007;179:901–9. [5] Bousette N, Patel L, Douglas SA, Ohlstein EH, Giaid A. Increased expression of urotensin II and its cognate receptor GPR14 in atherosclerotic lesions of the human aorta. Atherosclerosis 2004;176:117–23. [6] Zhang YG, Chen YH, Ma CY, Qi YF, Pang YZ, Tang CS. Mitogenic effect of urotensin II on cells. Chin J Arterioscler 2001;9:14–6. [7] Watanabe T, Takahashi K, Kanome T, Hongo S, Miyazaki A, Koba S, et al. Human urotensin-II potentiates the mitogenic effect of mildly oxidized low-density lipoprotein on vascular smooth muscle cells: comparison with other vasoactive agents and hydrogen peroxide. Hypertens Res 2006;29:821–31. [8] Onan D, Pipolo L, Yang E, Hannan RD, Thomas WG. Urotensin-II promotes hypertrophy of cardiac myocytes via mitogen-activated protein kinases. Mol Endocrinol 2004;18:2344–54. [9] Watanabe T, Suguro T, Kanome T, Sakamoto Y, Kodate S, Hagiwara T, et al. Human urotensin II accelerates foam cell formation in human monocyte-derived macrophages. Hypertension 2005;46:738–44. [10] Suguro T, Watanabe T, Ban Y, Kodate S, Misaki A, Hirano T, et al. Increased human urotensin II levels are correlated with carotid atherosclerosis in essential hypertension. Am J Hypertens 2007;20:211–7. [11] Heringlake M, Kox T, Uzun O, Will B, Bahlmann L, Klaus S, et al. The relationship between urotensin II plasma immunoreactivity and left ventricular filling pressures in coronary artery disease. Regul Pept 2004;121:129–36.
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[12] Douglas SA, Tayara L, Ohlstein EH, Halawa N, Giaid A. Congestive heart failure and expression of myocardial urotensin II. Lancet 2002;359:1990–7. [13] Zhang YG, Li JX, Cao J, Chen J, Yang J, Zhang ZK, et al. Effect of chronic hypoxia on contents of urotensin II and its functional receptors in rat myocardium. Heart Vessels 2002;16:64–8. [14] Zhang YG, Li YG, Liu BG, Wei RH, Wang DM, Tan XR, et al. Urotensin II accelerates cardiac fibrosis and hypertrophy of rats induced by isoproterenol. Acta Pharmacol Sin 2007;28:36–43. [15] Tzanidis A, Hannan RD, Thomas WG, Onan D, Autelitano DJ, See F, et al. Direct actions of urotensin II on the heart: implications for cardiac fibrosis and hypertrophy. Circ Res 2003;93:246–53. [16] Sartore S, Chiavegato A, Faggin E, Franch R, Puato M, Ausoni S, et al. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res 2001;89:1111–21. [17] An SJ, Boyd R, Zhu M, Chapman A, Pimentel DR, Wang HD. NADPH oxidase mediates angiotensin II-induced endothelin-1 expression in vascular adventitial fibroblasts. Cardiovasc Res 2007;75:702–9. [18] Lin L, Ding WH, Jiang W, Zhang YG, Qi YF, Yuan WJ, et al. Urotensin-II activates l-arginine/ nitric oxide pathway in isolated rat aortic adventitia. Peptides 2004;25:1977–84. [19] Haurani MJ, Pagano PJ. Adventitial fibroblast reactive oxygen species as autacrine and paracrine mediators of remodeling: bellwether for vascular disease? Cardiovasc Res 2007;75:679–89. [20] Faber JE, Yang N, Xin X. Expression of alpha-adrenoceptor subtypes by smooth muscle cells and dventitial fibroblasts in rat aorta and in cell culture. J Pharmacol Exp Ther 2001;298:441–52. [21] Jiang W, Jiang HF, Cai DY, Pan CS, Qi YF, Pang YZ, et al. Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock. Regul Pept 2004;118:199–208. [22] Qi JG, Du JB, Tang XY, Li J, Wei B, Tang CS. The upregulation of endothelial nitric oxide synthase and urotensin-II is associated with pulmonary hypertension and vascular diseases in rats produced by aortocaval shunting. Heart Vessels 2004;19:81–8. [23] Itoh H, McMaster D, Lederis K. Functional receptors for fish neuropeptide urotensin II in major rat arteries. Eur J Pharmacol 1988;149:61–6. [24] Wolff AA, Hines DK, Karliner JS. Refined membrane preparations mask ischemic fall in myocardial beta-receptor density. Am J Physiol 1989;257:H1032–1036. [25] Jones LR, Besch Jr HR. Isolation of canine cardiac sarcolemmal vesicles. In: Schwartz A, editor. Methods in pharmacology. New York: Plenum Publishing Corporation; 1984. p. 1–12. [26] Yu SM, Tsai SY, Guh JH, Ko FN, Teng CM, Ou JT. Mechanism of catecholamineinduced proliferation of vascular smooth muscle cells. Circulation 1996;94:547–54. [27] Li YR. Biochemistry of extracellular mesenchyma and study methods. First editionBeijing: People Health Publishing; 1988. p. 222–3.
[28] Matsushita M, Shichiri M, Fukai N, Ozawa N, Yoshimoto T, Takasu N, Hirata Y. Urotensin II is an autocrine/paracrine growth factor for the porcine renal epithelial cell line, LLCPK1. Endocrinology 2003;144:1825–31. [29] Marco J, Egido EM, Hernández R, Silvestre RA. Evidence for endogenous urotensinII as an inhibitor of insulin secretion. Study in the perfused rat pancreas. Peptides 2008;29:852–8. [30] Watson AM, Lambert GW, Smith KJ, May CN. Urotensin II acts centrally to increase epinephrine and ACTH release and cause potent inotropic and chronotropic actions. Hypertension 2003;42:373–9. [31] Xu C, Zarins CK, Pannaraj PS, Bassiouny HS, Glagov S. Hypercholesterolemia superimposed by experimental hypertension induces differential distribution of collagen and elastin. Arterioscler Thromb Vasc Biol 2000;20:2566–72. [32] Rakowski E, Hassan GS, Dhanak D, Ohlstein EH, Douglas SA, Giaid A. A role for urotensin II in restenosis following balloon angioplasty: use of a selective UT receptor blocker. J Mol Cell Cardiol 2005;39:785–91. [33] Zhang YG, Qi YF, Xia CF, Pang YZ, Yang J, Zhang ZK, Tang CS. Stimulating proliferation of aorta smooth muscle cells of rat by rat urotensin II. Chin Pharmacol Bull 2001;17:155–7. [34] Maguire JJ, Kuc RE, Wiley KE, Kleinz MJ, Davenport AP. Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries. Peptides 2004;25:1767–74. [35] Djordjevic T, BelAiba RS, Bonello S, Pfeilschifter J, Hess J, Gorlach A. Human urotensin II is a novel activator of NADPH oxidase in human pulmonary artery smooth muscle cells. Arterioscler Thromb Vasc Biol 2005;25:519–25. [36] Zhang YG, Li J, Li YG, Wei RH. Urotensin II induces phenotypic differentiation, migration, and collagen synthesis of adventitial fibroblasts from rat aorta. J Hypertens 2008;26:1119–26. [37] Maiellaro K, Taylor WR. The role of the adventitia in vascular inflammation. Cardiovasc Res 2007;75:640–8. [38] Xu F, Ji J, Li L, Chen R, Hu W. Activation of adventitial fibroblasts contributes to the early development of atherosclerosis: a novel hypothesis that complements the “response-to-injury hypothesis” and the “inflammation hypothesis”. Med Hypotheses 2007;69:908–12. [39] Li L, Terry CM, Blumenthal DK, Kuji T, Masaki T, Kwan BC, Zhuplatov I, Leypoldt JK, Cheung AK. Cellular and morphological changes during neointimal hyperplasia development in a porcine arteriovenous graft model. Nephrol Dial Transplant 2007;22:3139–46. [40] Miller FJ. Adventitial fibroblasts. Backstage journeymen. Arterioscler Thromb Vasc Biol 2001;21:722–3. [41] Krick S, Hänze J, Eul B, Savai R, Seay U, Grimminger F, et al. Hypoxiadriven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF1alpha and an autocrine angiotensin system. FASEB J 2005;19:857–9.
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Urotensin II is an autocrine/paracrine growth factor for aortic adventitia of rat Yonggang Zhang a,⁎, Yuguang Li a, Ruihong Wei b, Zhijian Wang c, Dingfang Bu c, Jing Zhao c, Yongzheng Pang c, Chaoshu Tang c a b c
Department of Cardiovascular Diseases, First Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong 515041, China Internal Medicine, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China Institute of Cardiovascular Research, Peking University First Hospital, Beijing 100034, China
a r t i c l e
i n f o
Article history: Received 9 February 2008 Received in revised form 7 September 2008 Accepted 1 October 2008 Available online 14 October 2008 Keywords: Adventitial fibroblasts Peptide GPR14 Receptor Collagen Vascular remodeling Proliferation
a b s t r a c t Urotensin II (UII) is a potent vasoconstrictive peptide; however, its significance in vascular adventitia has not been clearly elucidated. In this study, rat aortic adventitia showed mRNA expression and immunoreactivity of UII and its receptor (UT). Moreover, radioligand-binding assay showed that maximum binding capacity (Bmax) of [125I]UII was higher in adventitia than in media (28.60 ± 1.94 vs. 20.21 ± 1.11 fmol/mg, P b 0.01), with no difference in binding affinity (dissociation constant [Kd] 4.27 ± 0.49 vs. 4.60 ± 0.40 nM, P N 0.05). Furthermore, in cultured adventitial fibroblasts, UII stimulated DNA synthesis, collagen synthesis and secretion in a concentrationdependent manner. These effects were inhibited by the UII receptor antagonist urantide (10− 6 mol/l), Ca2+ channel blocker nicardipine (10− 5 mol/l), protein kinase C inhibitor H7 (10− 6 mol/l), and mitogen-activated protein kinase inhibitor PD98059 (10− 6 mol/l) but not the phosphatidyl inositol-3 kinase inhibitor wortmannin (10− 7 mol/l). UII may act as an autocrine/paracrine factor through its receptor and the Ca2+ channel, protein kinase C, and mitogenactivated protein kinase signal transduction pathways, in the pathogenesis of vascular remodeling by activating vascular adventitia. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Urotensin II (UII) is a potent vasoconstrictive neuropeptide initially isolated from fish urophysis. Recently, human homologues of UII and its specific receptor GPR14, now known as UT receptor, were identified [1,2]. UII is predominately expressed in spinal cord and certain brain areas and in myocardium and vessels, among other tissues [1–4]. UT receptor is mainly expressed in cardiovasculature, including myocardium, vascular smooth muscle cells (VSMCs), and endothelial cells [2,3,5]. UII induces potent vasoconstriction and was shown to be more potent than endothelin-1 [2]. In addition, UII can stimulate proliferation of VSMCs and induce hypertrophic responses in cardiomyocytes from neonatal rats [6–8]. In human monocyte-derived macrophages, UII accelerates foam cell formation [9]. UII is also a new chemotactic factor for UT receptorexpressing monocytes [4]. Moreover, UII and UT have been linked to vascular and cardiac remodeling. Human UII-like immunoreactivity was discovered in human coronary atheroma [2,5]. Increased human UII levels were associated with carotid atherosclerosis in essential hypertension [10]. In patients with coronary artery disease, increased plasma level of UII was significantly related to parameters of cardiac dysfunction [11]. ⁎ Corresponding author. Department of Cardiovascular Diseases, First Affiliated Hospital, Shantou University Medical College, No. 57, Changping Road, Shantou, Guangdong 515041, China. Tel.: +86 754 889 05337; fax: +86 754 882 59850. E-mail addresses: [email protected], [email protected] (Y. Zhang). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.10.004
Increased levels of UII, as well as greater density of binding sites for UII, were found in myocardium of patients with congestive heart failure [12]. We previously showed increased density of binding sites for UII in sarcolemma of myocardium and increased UII content in rat ventricles exposed to chronic hypoxia [13]. Recently, we found UII involved in the development of cardiac fibrosis and hypertrophy by synergistic effects with isoproterenol [14]. Increased expression of UII and UT receptor protein was observed in both infarcted and non-infarcted regions of the left ventricle in a rat model of heart failure after myocardial infarction [15]. In vitro experiments showed that UII stimulated proliferation and collagen synthesis of neonatal cardiac fibroblasts, as determined by 3Hthymidine and 3H-proline incorporation [6,15]. UII also increased the mRNA level of procollagens α1 (I) and α1 (III), and fibronectin in these cells [15]. Taken together, these data suggest that UII may be involved in the cardiovascular fibrogenesis through increased collagen synthesis and may target multiple cells in cardiovascular tissues, playing multiple functions in physiological and pathophysiological regulation. Recently, many studies have focused on the physiological and pathophysiological significance of tunica adventitia, a major effector site for neuro-humoral factors, such as hormones, cytokines, growth factors, inflammatory factors and active peptides [16,17]. The adventitia also secretes vasoactive factors such as nitric oxide, bioactive peptides, growth factors and cytokines under physiological or pathological conditions [16–19], thereby participating in the regulation of arterial structure and function. Therefore, the adventitia has paracrine/ autocrine actions, just like the intima [16]. In our previous study, we
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demonstrated that UII activates the L-arginine/nitric oxide pathway in isolated rat aortic adventitia [18]. However, the significance of UII and UT receptor in the adventitia is not yet elucidated. In this study, we determined the mRNA expression and immunoreactivity of UII and UT in rat aortic adventitia. In addition, we determined the binding capacity of UT in adventitia by radioligandbinding assay. Furthermore, in cultured adventitial fibroblasts, we evaluated the effects of UII on proliferation, collagen synthesis and secretion and its molecular mechanisms by 3H-proline and 3H-thymidine incorporation methods. 2. Materials and methods 2.1. Materials Male Sprague–Dawley rats weighing 180–200 g were supplied by the Animal Center, Health Science Center, Peking University. Animal care and experimental protocols complied with the Animal Management Rules of China (documentation number 55, 2001, Ministry of Health, China) and the Guide for Care and Use of Laboratory Animals, Peking University First Hospital. Rat UII (pEHGTAPECFWKYCI) and rat [125I]-UII [2200 Ci (81.4TBq)/ mmol] were purchased from Phoenix Pharmaceutical (Belmont, CA, USA). Rabbit anti-UII (rat) antibody and goat-anti rabbit-IgG were purchased from Boster Biological Technology (Wuhan, China). Rabbit anti-UT polyclonal antibody was purchased from Chemicon International (Temecula, CA, USA). A UII ISH kit was purchased from Haoyang Biotechnology Company (Tianjin, China). Nicardipine, wortmannin, bovine serum albumin (BSA), bacitracin, trypsin and Dulbecco modified Eagle medium (DMEM) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum was from Hyclone (Logan, UT, USA). 3H-thymidine and 3H-proline were from Amersham Pharmacia Biotech (Freiburg, Germany); TRIzol and dNTP were from Clontech Laboratories (Palo Alto, CA, USA). Taq DNA polymerase and oligo (dT)15 primers were from Promega (Madison, WI, USA). Oligonucleotides were synthesized by Sai Baisheng Biotechnology (Beijing, China). The sequences of oligonucleotide primers were rUT-S, 5′GCATCTTCACCCTGACCATAA-3′, and rUT-A, 5′-CCCAGAAGAGAAGGACGATACC-3′, used for the amplification of UT cDNA; and β-actin-S, 5′ATCTGGCACCACACCTTC-3′, and β-actin-A, 5′-AGCCAGGTCCAGACGCA-3′, for the amplification of β-actin for calibration of sample loading. PD98059 and H7 were from Calbiochem (Darmstadt, Germany). Urantide was from Peptides International (Louisville, KY, USA). Other chemicals and reagents were of analytical grade. 2.2. Isolation of adventitial and medial tissues of rat aorta The adventitial and medial tissues of rat aorta were prepared according to the method of Faber et al. [20] with some modification. Briefly, rats were anesthetized with use of isoflurane and rapidly decapitated. The full-length thoraco-abdominal aorta was isolated under a pool of 4 °C phosphate-buffered saline, after gently opening the parietal pleura, separating away loose connective tissue and collateral vessels, and transecting the segmental arteries at their origins. Under magnification, the adventitial and medial layers could be distinguished at both ends of the aorta. Endothelial cells were removed by gently rubbing the lumen with the blunt side of dissecting scissors, and the medial layer was peeled off with use of two forceps. Both the adventitial and medial layer were collected and quickly frozen in liquid nitrogen and stored at −80 °C until use. 2.3. In situ hybridization The full-length aorta was isolated as described above. Tissue sections fixed with 10% buffered formalin and embedded in paraffin were pretreated and underwent in situ hybridization for UII mRNA according to the manufacturer's instructions. The following probes labeled at the 5-end with digoxin were used: 5′-CTACGAAGA GCAGGCAGCAGAAGGG-
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CAC-3′; 5′-TTCCCTCTGCTTCTGTGCCCACGGTCTG-3′. Visualization of the in situ hybrids was enabled by 3, 3′-diaminobenzidine, with dark brown spots in the tissue sections representing the UII mRNA-positive signals. Negative controls represented the absence of probes. 2.4. RT-PCR assay The expression of UT mRNA in adventitia and media was assessed by RT-PCR as described [21]. Total RNA extracted from the tissue was quantified by use of an ultraviolet spectrophotometer (UV2100, Shimadzu, Japan). Reverse transcription to cDNA was accomplished by priming 2 µg total RNA with oligo (dT)15 primers with use of moloney murine leukemia virus transcriptase. Products were then used for the following PCR amplification: 2.5 mmol/l/each dNTP 1 µl, 10 × PCR buffer (100 mmol/l Tris–HCI, pH 8.3, 15 mmol/l MgCl2, 500 mmol/l KCl) 2.5 µl, cDNA 1 µl, 5 µmol/l/each of UT-S and UT-A primers 1 µl and 1.25 unit of Taq DNA polymerase, in a total volume of 25 µl. After denaturing at 95 °C for 5 min, PCR cycles were run at 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, for 35 cycles, then 72 °C for 5 min. As an internal control for each PCR reaction, β-actin cDNA was amplified for each sample under the same conditions. PCR products were separated in 1.5% agarose gel and visualized by ethidium bromide staining. The intensity of the PCR product bands under UV light was measured by use of a gel image analyzer. Results were expressed as the ratio of UT PCR product (399 bp) to β-actin PCR product (291 bp) for each sample. All experiments were repeated three times. 2.5. Immunohistochemistry Immunoreactive UII and UT were assessed by a streptavidin–biotin peroxidase method on snap-frozen tissue sections postfixed with 4% paraformaldehyde, as reported previously [22]. Sections of rat thoracic aorta were incubated overnight at 4 °C with rabbit anti-rat UII antibody and rabbit anti-UII receptor (UT) antibody. UII antibody did not crossreact with a range of peptide ligands such as endothelin-1 (human, rat, mouse), adrenomedullin (1–50) (rat), or angiotensin II (human). The yellow-brown granules in the sections were considered positive signals. 2.6. Radio-ligand binding assay [125I]-U-II binding assay was performed as described [23,24] with some modifications. Briefly, minced adventitia and media were homogenized in ice-cold SEGT buffer [250 mmol/l sucrose, 1 mmol/l ethyleneglycol-bis-(2-aminoethyl ether)-N, N′-tetraacetic acid, 20 mmol/l Tris, pH 7.2]. After centrifugation, the pellet was re-suspended in SEGT buffer and filtered through four layers of cheesecloth. Protein content in the filter was determined by the Bradford method and adjusted to 1 mg/ml. The activity of Na+/K+ ATPase as the marker enzyme of sarcolemma was determined [25]. Ligand-binding assay was carried out in a polypropylene tube containing 50 µg membrane fraction and 0.2 ml binding assay buffer [20 mmol/l Tris–HCl, pH 7.4, 2 mmol/l MgCl2, 0.25% bovine serum albumin, 0.25 mg/ml bacitracin, and [125I]-UII (0.5,1, 2, 4, 8, 16 or 32 nmol/l)]. The mixture was incubated at 25 °C for 40 min, terminated by the addition of 2 ml ice-cold binding assay buffer, and filtered through acetate cellulose filters (0.45 mm). After washing the filters with ice-cold binding assay buffer three times, the radioactivity on filters was counted by use of a Wallac 1470 Wizard automatic gamma counter (Switzerland). Nonspecific binding of [125I]-UII was obtained in the presence of 20 µmol/l unlabeled rat UII. Dissociation constant (Kd) and maximal binding capacity (Bmax) were calculated by Scatchard plot analysis of saturation binding isotherms. 2.7. Culture of adventitial fibroblasts [20] The rat adventitial fibroblasts were cultured according to the method of Faber et al. [20] with some modification. Sterile-isolated adventitia of
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supernatants were harvested for the measurement of collagen synthesis and secretion, respectively. Collagen secretion was measured according to the method of Li [27]. Briefly, 100 µl of pepsin assay buffer (mixed with 25 µl of 5 mol/l acetic acid, pH 2–3, 25 µl of 1 mg/ ml pepsin solution in 0.5 mol/l acetic acid, and 50 µl of 10 mg/ml proline) was added to 1 ml of the supernatant from a well and kept for 3 h at 4 °C. To precipitate protein fractions, 250 µl of 1.2 mol/l cold trichloroacetic acid was added to the samples and incubated on ice for 2 h. Precipitates were applied onto filter units, washed with trichloroacetic acid and ethanol, and counted in a scintillation counter. Fig. 1. In situ hybridization for UII mRNA in rat aorta. A, negative control. B, expression of UII mRNA in rat aortic arteries, especially endothelial cells and adventitial cells. Magnification is ×400. AD, adventitia; ME, media; EC, endothelium.
thoracic aorta was minced into 1-mm2 pieces and incubated for 30 min at 37 °C without shaking in 2.4 units/ml neutral protease II. After gentle trituration, cells were placed in DMEM culture media with 20% fetal bovine serum (FBS) on ice to arrest protease activity. After repeating this dispersion procedure three times, pooled cells were gently resuspended in DMEM plus FBS and sieved (38 µmol/l) to separate the smaller adventitial fibroblasts from occasional non-fibroblasts present in adventitia (SMC-like cells, mast cells, macrophages, and adipocytes). Adventitial fibroblasts (20,000 cells/cm2) were grown in DMEM with 10% FBS, 200 mg/l L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin passaged at ~90% confluence with 0.125% trypsin/EDTA every 3 days. The morphology and growth characteristics of the cells were typical of fibroblasts and were distinguished from SMCs by the absence of a “hill-and-valley” growth pattern and lack of smooth muscle α-actin staining. Cells between passages 4–6 were used. To examine DNA synthesis, collagen synthesis and secretion of adventitial fibroblasts induced by UII, the cultured cells were incubated in DMEM with 10% FBS, then incubated with serum-deprived medium (containing 0.5% FBS) for 24 h. After synchronization of adventitial fibroblasts, the medium was changed to DMEM without serum. Thereafter, these cells were incubated in the presence or absence of UII. To examine the molecular basis of the action of UII, some inhibitors, including the UII receptor antagonist urantide (10− 6 mol/l), Ca2+ channel blocker nicardipine (10− 5 mol/l), phosphatidyl inositol-3 kinase (PI3K) inhibitor wortmannin (10− 7 mol/l), protein kinase C (PKC) inhibitor H7 (10− 6 mol/l), and mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10− 6 mol/l), were used. According to the experimental design, cells were divided into groups for treatment: control, serum-free DMEM only; 10− 10, 10− 9, 10− 8, or 10− 7 UII added to the medium; 10% FBSDMEM with or without UII (10− 8 mol/l); and pretreatment in serumfree DMEM for 0.5 h with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l) or wortmannin (10− 7 mol/l) then UII (10− 8 mol/l). The cells and media were collected after 24 h of incubation. Each experiment was repeated 6 times.
2.10. Statistical analysis Results are shown as mean±SD. Comparisons involved use of unpaired Student's t test and one-way ANOVA, followed by the Newman–Keuls multiple comparison test. A value of Pb 0.05 was considered statistically significant. 3. Results 3.1. Expression of UII and UT mRNA in the rat aortic adventitia In situ hybridization revealed UII mRNA expression in rat aorta, abundant in the adventitial layer and endothelium and to a lesser degree in media (Fig. 1). RT-PCR showed higher UT mRNA expression in both adventitia and media than that in the medulla oblongata, a positive control tissue (Fig. 2). UT mRNA level was slightly higher, but not significantly, in the adventitia than in the media (P N 0.05). 3.2. UII and UT immunoreactivity in rat adventitia Histological examination revealed distinct immunoreactivity for UII and UT in the aorta, which matched the mRNA expression results from in situ hybridization. As well, staining was abundant in the adventitial layer and endothelium but less so in the media (Fig. 3).
2.8. Determination of cell proliferation DNA synthesis was determined by 3H-thymidine incorporation as described previously [26]. The adventitial fibroblasts were incubated with different concentrations of UII and/or inhibitors or serum for 24 h, then exposed to 3H-thymidine at 1 µCi/well for the last 8 of the 24-h incubation. Then, cells were washed with ice-cold PBS and 10% trichloroacetic acid. Acid-insoluble 3H-thymidine was collected on glass fiber filters (Whatman, Kent, UK) and determined by use of a liquid scintillation counter (LS 6500, Beckman, Fullerton, CA, USA). 2.9. Determination of collagen synthesis and secretion Collagen synthesis was examined by measuring 3H-proline incorporation into cells [27]. At the end of the experiment, cells and
Fig. 2. Estimation of UT mRNA expression in adventitia and media of rat aorta by RTPCR. A. electrophoresis of PCR products; Lane 1: medulla oblongata; lane 2: media of rat aorta; lane 3: adventitia of rat aorta. B. ratio of UT to beta-actin products (mean ± SD, n = 3). ⁎P b 0.05 vs. MO; AD, adventitia; ME, media; MO, medulla oblongata as a positive control.
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Fig. 3. Immunohistochemical localization of UII and UT protein in thoracic aorta of healthy rats. A, staining for UII; B: staining for UT. (Magnification is ×400). AD, adventitia; ME, media; EC, endothelium.
3.3. Ligand-binding assay The activities of Na+/K+ ATPase (a marker enzyme of plasma membrane) were 6.8 ± 0.7 and 6.1 ± 0.5 µmol/mg protein/h in membrane preparations from adventitia and media, respectively, and were 0.97 ± 0.1 and 0.94 ± 0.1 µmol/mg protein/h in homogenates from the two tissues, respectively. The activities of Na+/K+ ATPase in the membrane preparations from adventitia and media were 6.0 and 5.5 times higher, respectively, than that in homogenates, which indicates that the membrane preparations were mainly plasma membrane. Radioligand binding studies showed that the non-specific binding of [125I]-UII (in the presence of 20 µmol/l unlabeled UII) was about 25% that of the total binding of the labeled peptide. Fig. 4 shows the characteristics of [125I]-UII binding to the membrane preparation of rat aortic adventitia and media. Scatchard analysis of the data demonstrated that the calculated maximal number of specific binding sites was 28.60 ± 1.94 fmol/mg protein in adventitia, which was 42% higher than that in the media (20.21 ± 1.11 fmol/mg protein, P b 0.01). However, adventitia and media did not differ in the Kd (4.27 ± 0.49 vs. 4.60 ± 0.40 nM, P N 0.05). 3.4. Effect of UII on adventitial fibroblasts proliferation Fig. 5 shows that UII stimulated the proliferation of cultured adventitial fibroblasts, as assessed by 3H-thymidine incorporation, in a dose-dependent manner; 3H-thymidine incorporation was increased by 39%, 90%, 103% and 171% with 10− 10, 10− 9, 10− 8 and 10− 7 mol/l UII treatment, respectively (P b 0.01). The incorporation was increased by 121% and 189% with serum and UII + serum treatment, respectively (P b 0.01), as compared with the control group (1360 ± 42 cpm/well). In
Fig. 4. [125I]-UII binding to the plasma membrane of adventitia and media of rat aorta (mean ± SD, n = 4). A, saturation binding curves; B, Scatchard plot.
Fig. 5. Effect of UII on 3H-thymidine incorporation in cultured adventitial fibroblasts (mean ± SD, n = 6). Adventitial fibroblasts were incubated with different concentrations of UII and/or serum for 16 h, then also exposed to 3H-thymidine at 1 µCi/well for 8 h, then 3H-thymidine incorporation was measured. Values are compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ⁎⁎P b 0.01 vs. Control; ##P b 0.01 vs. 10− 8 mol/l UII group or serum group.
the serum + UII (10− 8 mol/l) group, the incorporation was increased by 29% (P b 0.01) as compared with the serum-alone group and by 41% (P b 0.01) as compared with the UII (10− 8 mol/l)-alone group (P b 0.01). 3 H-thymidine incorporation stimulated by UII (10− 8 mol/l) was inhibited by the UII receptor antagonist urantide (10− 6 mol/l), MAPK inhibitor PD98059 (10− 6 mol/l), L-type voltage-dependent Ca2+ channel blocker nicardipine (10− 5 mol/l), and PKC inhibitor H7 (10− 6 mol/l) but not by the PI3K inhibitor wortmannin (10− 7 mol/l) (Fig. 6), which suggests that UT, Ca2+, PKC, and MAPK but not PI3K might be involved in the proliferation of adventitial fibroblasts induced by UII. 3.5. The effect of UII on collagen synthesis and secretion of adventitial fibroblasts Fig. 7 shows that UII stimulated 3H-proline incorporation in a concentration-dependent manner. 3H-proline incorporation with UII
Fig. 6. Effect of different kinds of inhibitors on UII-induced 3H-thymidine incorporation in cultured adventitial fibroblasts. Cells were preincubated with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l) or wortmannin (10− 7 mol/l) for 30 min, then incubated with UII (10− 8 mol/l) and the inhibitors for 16 h, then also exposed to 3H-thymidine at 1 µCi/well for 8 h, then 3H-thymidine incorporation was measured. Values were compared by one-way ANOVA and then by Newman–Keuls multiple comparison test. ##P b 0.01 vs. control; ⁎P b 0.05; ⁎⁎P b 0.01 vs. UII group. UII: urotensin II; Ura, urantide; Wor, wortmannin; PD, PD98059; Ni, nicardipine.
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Fig. 7. Effect of UII on 3H-proline incorporation in cultured adventitial fibroblasts (mean± SD, n = 6). Adventitial fibroblasts were incubated with different concentrations of UII and/or serum for 16 h, then also exposed to 3H-proline at 1 µCi/well for 8 h, then 3Hproline incorporation was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ⁎⁎P b 0.01 vs. control; ##P b 0.01 vs.10− 8 mol/l UII group or serum group.
Fig. 9. Effect of UII on collagen secretion from cultured adventitial fibroblasts (mean ± SD, n = 6). Adventitial fibroblasts were incubated with different concentrations of UII (10− 10, 10− 9, 10− 8 and 10− 7 mol/l) and/or serum for 16 h, then also exposed to 3Hproline at 1 µCi/well for 8 h, then collagen secretion was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ⁎⁎P b 0.01 vs. control; ##P b 0.01 vs. 10− 8 mol/l UII group or serum group.
concentrations of 10− 10, 10− 9, 10− 8 and 10− 7 mol/l was higher, by 24%, 42%, 56% and 57%, respectively (all P b 0.01), than with control treatment (1484 ± 69 cpm/well). In the 10% FBS group, 3H-proline incorporation was higher, by 27% (P b 0.01) than with control treatment. Moreover, in the serum + UII (10− 8 mol/l) group, 3H-proline incorporation was higher, by 58% (P b 0.01), than in the serum-alone group, and higher by 29% (P b 0.01), than in the UII (10− 8 mol/l)alone group. This effect was inhibited by pretreatment with urantide (10− 6 mol/l, P b 0.01) and PD98059 (10− 6 mol/l, P b 0.01) and partially suppressed by pretreatment with nicardipine (10− 5 mol/l, P b 0.05) and H7 (10− 6 mol/l, P b 0.05) but not suppressed by wortmannin (Fig. 8), which suggests that UT, MAPK, Ca2+, and PKC might be involved in collagen synthesis induced by UII. UII stimulated collagen secretion from cultured adventitial fibroblasts in a dose-dependent manner (Fig. 9). With UII at 10− 10–10− 7 mol/l, colla-
gen secretion was higher, by 41%–76% (Pb 0.01), than with control treatment. Collagen secretion was increased by 63% with 10% FBS treatment than with control treatment (Pb 0.01). In the serum+UII (10− 8 mol/l) group, collagen secretion was higher, by 19% (Pb 0.01), than in the serumalone group and higher, by 16% (Pb 0.01), than in the 10− 8 mol/l UII-alone group. This effect induced by UII was completely blocked by urantide (10− 6 mol/l) and PD98059 (10− 6 mol/l) and partially blocked by nicardipine (10− 5 mol/l) and H7 (10− 6 mol/l) but not blocked by wortmannin (10− 7 mol/l) (Fig. 10). These data suggest that UII induced collagen secretion in adventitial fibroblasts via UT, Ca2+ channel, PKC, and MAPK but not PI3K. 4. Discussion A growing body of evidence suggests that UII exerts a broad spectrum of physiological and pharmacological actions. In addition to
Fig. 8. Effect of different kinds of inhibitors on UII-induced 3H-proline incorporation in cultured adventitial fibroblasts. After preincubation with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l), wortmannin (10− 7 mol/l) for 30 min, rat adventitial fibroblasts were incubated with UII (10− 8 mol/l) and the inhibitors for 16 h, then also exposed to 3H-proline at 1 µCi/well for 8 h, then 3H-proline incorporation was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ##P b 0.01 vs. Control; ⁎P b 0.05; ⁎⁎P b 0.01 vs. UII group. UII: urotensin II; Ura, urantide; Wor, wortmannin; PD, PD 98059; Ni, nicardipine.
Fig. 10. Effect of different kinds of inhibitors on UII-induced collagen secretion in cultured adventitial fibroblasts. After preincubation with urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l) or wortmannin (10− 7 mol/l) for 30 min, rat adventitial fibroblasts were incubated with UII (10− 8 mol/l) and the inhibitors for 16 h, then also exposed to 3H-proline at 1 µCi/well for 8 h, then collagen secretion was measured. Values were compared by one-way ANOVA, then Newman–Keuls multiple comparison test. ##P b 0.01 vs. Control; ⁎P b 0.05; ⁎⁎P b 0.01 vs. UII group. UII: urotensin II; Ura, urantide; Wor, wortmannin; PD, PD 98059; Ni, nicardipine.
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having direct vasoconstrictive activity in smooth muscle receptors, UII stimulates proliferation of VSMCs, cardiac fibroblasts, glomerular mesangial cells, endothelial cells and tumor cells [6,28] and promotes hypertrophy of cardiac myocytes [8] and collagen synthesis of cardiac fibroblasts [6,15]. UII also accelerates foam cell formation in human monocyte-derived macrophages, induces chemotactic activity in monocytes, and inhibits insulin secretion in perfused rat pancreas [29]. Intracerebroventricular administration of UII in conscious sheep induced large, prolonged increases in plasma epinephrine, adrenocorticotropic hormone, cardiac output and arterial pressure [30]. These data demonstrate broad targets for UII actions. Importantly, recent studies have demonstrated that the vascular adventitia is an essential layer for the metabolism and function of the vascular wall and a target for actions of many vasoactive peptides [31]. However, the significance of the UII/UT system in activation of adventitial fibroblasts and vascular remodeling has still not been clarified completely. In this study, using in situ hybridization, we detected UII mRNA expression in rat aorta, especially in adventitia and endothelium. We also found large amount of UT mRNA expressed in the adventitia. Immunohistochemistry showed both UII and UT immunoreactivity in the aorta, with the adventitial layer exhibiting protein expression of UII and UT, which matched our mRNA expression results, as was found previously [32]. Moreover, we also determined that [125I]-UII bound to the adventitia with a high-affinity and saturable character and the number of binding sites of UII was higher in the adventitia than in the media, with no difference in Kd values between the tissues. Furthermore, we investigated the effects of UII on DNA synthesis and collagen production of adventitial fibroblasts in vitro by 3H-thymidine and 3H-proline incorporation, respectively, and demonstrated that UII could stimulate proliferation, collagen synthesis and secretion in a concentration-dependent manner. Urantide (10− 6 mol/l), PD98059 (10− 6 mol/l), nicardipine (10− 5 mol/l), H7 (10− 6 mol/l), inhibitors/ blocker of UII receptor, MAPK, Ca2+ channel and PKC, respectively, could inhibit UII-induced DNA synthesis and collagen production, but wortmannin (10− 7 mol/l), the inhibitor of PI3K, did not. Thus, UT, MAPK, Ca2+, and PKC pathways might be involved in DNA synthesis and collagen production of adventitial fibroblasts induced by UII. However, these UII-stimulated effects were not inhibited by PI3K inhibitor wortmannin, which excludes the involvement of PI3K in UII-induced DNA synthesis and collagen production. These results in adventitial fibroblasts are consistent with those in VSMCs [6,7,28]. We have reported that Ca2+ channel, PKC, MAPK and CaM-PK pathways are involved in VSMC proliferation [33]. Others reported that the activation of phospholipase C, small G-protein RhoA and Rho-Kinase, extracellular signal regulated protein kinases and PKC are involved in vasoconstriction and VSMC proliferation [2,28,8,34]. In addition, UII increased the expression of PAI-1 and enhanced SMC proliferation in an NADPH oxidase- and kinase-dependent manner [35]. UII accelerates the formation of human macrophage-derived foam cells by up-regulating the acyl-coenzyme A: cholesterol acyltransferase-1, which is mediated via the UT receptor/Gq protein/c-Src/PKC/ERK and RhoA/ROCK pathways [9]. In our previous study, we showed that rat UII may stimulate the L-arginine/NOS/NO pathway in rat aortic adventitia [18]. More recently, we found that UII could stimulate adventitial fibroblast phenotypic conversion and migration through the PKC, MAPK, calcineurin, Rho kinase, and/or Ca2+ signal transduction pathways [36]. The present results indicate that UII may also stimulate adventitial fibroblast proliferation and collagen synthesis and secretion through the UT, PKC, MAPK, and Ca2+ pathways. A growing body of evidence has documented that fibroblasts from adventitia play an important part in vascular remodeling through phenotypic conversion, proliferation, apoptosis, and migration [37]. Evidence also supports the activation of adventitial fibroblasts as the key regulator of vascular function and structure from the “outside in”, and contributes to the development of atherosclerotic lesions [16,38]. Numerous cytokines and growth factors, such as tumor necrosis
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factors, transforming growth factor-β, granulocyte-macrophage colony-stimulating factor, endothelin-1 and angiotensin II, may influence the proliferation and collagen synthesis of fibroblasts as well as their transition to myofibroblasts [16,37–40]. Recently, a study supported that hypoxia-induced stimulation of the hypoxia-inducible factor 1α axis trans-activates the ACE–AngII–AT1 system in pulmonary artery adventitial fibroblasts, which shows a possible role in autocrine and paracrine regulation of vascular function [41]. The present study identified that not only UII but also UT had highly specific UII binding sites within the adventitia, and UII could stimulate proliferation and collagen synthesis and secretion through the UT, Ca2+, PKC and MAPK signal transduction pathways. Therefore, UII may act mainly in an autocrine/paracrine manner in the activation of adventitial fibroblasts, thus contributing to the vascular remodeling. In conclusion, our results verify the abundant expression of UII and UT mRNA in the adventitia of rat aorta. UII and UT immunoreactivity was also apparent in the adventitia. Moreover, adventitia showed more UII binding sites than did the media. Furthermore, UII promotes proliferation and collagen synthesis and secretion in adventitial fibroblasts in vitro in a concentration-dependent manner. Intracellular signal transduction pathways, including UT, Ca2+, PKC, and MAPK, might be involved in these processes. The vascular adventitia may be a new target for UII action, and UII may act mainly in an autocrine/paracrine manner in the activation of adventitial fibroblasts in the development of vascular remodeling and contribute to the pathogenesis of cardiovascular diseases, the mechanisms of which are worthy of further investigation. Acknowledgments We thank Hongzhi Wang for technical help. This project was supported by the National Natural Science Foundation of China (No. 30470730), China Postdoctoral Science Foundation (No. 2003033439), and the Medical Science Foundation of Guangdong Province Health Department (No. A2007425), and Major State Basic Research Development Program of China (No. G2000056905). The experiment was completed with the assistance from the Institute of Cardiovascular Research, Peking University First Hospital. References [1] Coulouarn Y, Lihrmann I, Jegou S, Anouar Y, Tostivint H, Beauvillain JC, et al. Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci U S A 1998;95:15803–8. [2] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999;401:282–6. [3] Balat A, Karakök M, Yilmaz K, Kibar Y. Urotensin-II immunoreactivity in children with chronic glomerulonephritis. Ren Fail 2007;29:573–8. [4] Segain JP, Rolli-Derkinderen M, Gervois N, Raingeard de la Blétière D, Loirand G, Pacaud P. Urotensin II is a new chemotactic factor for UT receptor-expressing monocytes. J Immunol 2007;179:901–9. [5] Bousette N, Patel L, Douglas SA, Ohlstein EH, Giaid A. Increased expression of urotensin II and its cognate receptor GPR14 in atherosclerotic lesions of the human aorta. Atherosclerosis 2004;176:117–23. [6] Zhang YG, Chen YH, Ma CY, Qi YF, Pang YZ, Tang CS. Mitogenic effect of urotensin II on cells. Chin J Arterioscler 2001;9:14–6. [7] Watanabe T, Takahashi K, Kanome T, Hongo S, Miyazaki A, Koba S, et al. Human urotensin-II potentiates the mitogenic effect of mildly oxidized low-density lipoprotein on vascular smooth muscle cells: comparison with other vasoactive agents and hydrogen peroxide. Hypertens Res 2006;29:821–31. [8] Onan D, Pipolo L, Yang E, Hannan RD, Thomas WG. Urotensin-II promotes hypertrophy of cardiac myocytes via mitogen-activated protein kinases. Mol Endocrinol 2004;18:2344–54. [9] Watanabe T, Suguro T, Kanome T, Sakamoto Y, Kodate S, Hagiwara T, et al. Human urotensin II accelerates foam cell formation in human monocyte-derived macrophages. Hypertension 2005;46:738–44. [10] Suguro T, Watanabe T, Ban Y, Kodate S, Misaki A, Hirano T, et al. Increased human urotensin II levels are correlated with carotid atherosclerosis in essential hypertension. Am J Hypertens 2007;20:211–7. [11] Heringlake M, Kox T, Uzun O, Will B, Bahlmann L, Klaus S, et al. The relationship between urotensin II plasma immunoreactivity and left ventricular filling pressures in coronary artery disease. Regul Pept 2004;121:129–36.
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